Detailed Description
Definition of
As used herein, the following definitions shall apply unless otherwise indicated. For the purposes of the present invention, chemical elements are identified according to the periodic table of the elements, CAS version, Handbook of chemistry and Physics, 75 th edition. In addition, the general principle of Organic Chemistry is described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalitio: 1999 and "March's sadadvanced Organic Chemistry [ massa advanced Organic Chemistry ]", 5 th edition, editions: smith, m.b. and March, j., John Wiley press (John Wiley & Sons), new york: 2001.
Aliphatic: as used herein, "aliphatic" means a straight (i.e., unbranched) or branched substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is fully saturated or contains one or more units of unsaturation (but which is not aromatic), or a combination thereof. In some embodiments, the aliphatic group contains 1-50 aliphatic carbon atoms. In some embodiments, the aliphatic group contains 1-20 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-10 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-9 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-8 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-7 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in still other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof, such as (cycloalkyl) alkyl, (cycloalkenyl) alkyl or (cycloalkyl) alkenyl.
Alkenyl: the term "alkenyl" as used herein refers to an alkyl group as defined herein having one or more double bonds.
Alkyl groups: as used herein, the term "alkyl" is given its ordinary meaning in the art and may include saturated aliphatic groups including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl groups (alicyclic groups), alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In some embodiments, the alkyl group has 1-100 carbon atoms. In certain embodiments, the straight or branched chain alkyl group has from about 1 to 20 carbon atoms in the backbone (e.g., straight is C)1-C20The branch being C2-C20) And alternatively about 1 to 10 carbon atoms. In some embodiments, cycloalkyl rings are when such rings are monocyclic, bicyclic, or polycyclicHaving about 3-10 carbon atoms in the ring structure, and alternatively having about 5, 6 or 7 carbon atoms in the ring structure. In some embodiments, the alkyl group can be a lower alkyl group, wherein the lower alkyl group contains 1 to 4 carbon atoms (e.g., straight chain lower alkyl is C1-C4)。
Alkynyl: the term "alkynyl" as used herein refers to an alkyl group as defined herein having one or more triple bonds.
Animals: as used herein, the term "animal" refers to any member of the kingdom animalia. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and/or pig). In some embodiments, the animal includes, but is not limited to, a mammal, a bird, a reptile, an amphibian, a fish, and/or a worm. In some embodiments, the animal can be a transgenic animal, a genetically engineered animal, and/or a clone.
About: as used herein, the term "about" or "approximately" with respect to a number is generally intended to include numbers that fall within 5%, 10%, 15%, or 20% of the stated number in either direction (greater than or less than) unless otherwise indicated or otherwise evident from the context (except for such numbers that may be less than 0% or more than 100% of the possible values). In some embodiments, the term "about" used in reference to a dose means ± 5 mg/kg/day.
Aryl: as used herein, the term "aryl", used alone or as part of a larger moiety such as "aralkyl", "aralkoxy", or "aryloxyalkyl", refers to monocyclic, bicyclic, or polycyclic ring systems having a total of five to thirty ring members, wherein at least one ring in the system is aromatic. In some embodiments, the aryl group is a monocyclic, bicyclic, or polycyclic ring system having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members. In some embodiments, the aryl group is a biaryl group. The term "aryl" is used interchangeably with the term "aryl ring". In certain embodiments of the present disclosure, "aryl" refers to an aromatic ring system including, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracenyl, and the like, which may have one or more substituents. Also included within the scope of the term "aryl" as used herein are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthyridinyl, phenanthridinyl, or tetrahydronaphthyl, and the like.
Comparative: the term "comparable" is used herein to describe conditions or environments in which two (or more) groups are sufficiently similar to each other to allow comparison of results obtained or observed phenomena. In some embodiments, a set of comparable conditions or environments is characterized by a plurality of substantially identical features and one or a few varying features. One of ordinary skill in the art will appreciate that groups of conditions are comparable to one another when characterized by a sufficient number and type of substantially identical features to ensure a reasonable conclusion that differences in results or observed phenomena obtained under different groups of conditions or environments are caused or indicated by changes in those changing features.
A cycloaliphatic group: the terms "cycloaliphatic", "carbocycle", "carbocyclyl", "carbocyclic radical" and "carbocyclic ring" are used interchangeably and, as used herein, refer to a saturated or partially unsaturated but non-aromatic cycloaliphatic monocyclic, bicyclic or polycyclic ring system as described herein having from 3 to 30 ring members, unless otherwise specified. Cycloaliphatic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, the cycloaliphatic group has 3 to 6 carbon atoms. In some embodiments, the cycloaliphatic group is saturated and is cycloalkyl. The term "cycloaliphatic" may also include aliphatic rings fused to one or more aromatic or non-aromatic rings, e.g.Decahydronaphthyl or tetrahydronaphthyl. In some embodiments, the cycloaliphatic group is bicyclic. In some embodiments, the cycloaliphatic group is tricyclic. In some embodiments, the cycloaliphatic group is polycyclic. In some embodiments, "cycloaliphatic" refers to a C that is fully saturated or contains one or more units of unsaturation, but is not aromatic3-C6Monocyclic hydrocarbon or C8-C10Bicyclic or polycyclic hydrocarbons having a single point of attachment to the remainder of the molecule, or C which is fully saturated or contains one or more units of unsaturation, but which is not aromatic9-C16Polycyclic hydrocarbons having a single point of attachment to the rest of the molecule.
The administration scheme is as follows: as used herein, a "dosing regimen" or "treatment regimen" refers to a set of unit doses (typically more than one) that are administered individually, typically separated by a period of time, to an individual. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, the dosing regimen comprises multiple administrations, each of which are separated from each other by a period of the same length; in some embodiments, a dosing regimen comprises multiple doses and at least two different time periods separating the individual doses. In some embodiments, all administrations within a dosing regimen have the same unit dose. In some embodiments, different administrations within a dosing regimen have different amounts. In some embodiments, a dosing regimen comprises a first administration in a first administered amount followed by one or more additional administrations in a second administered amount different from the first administered amount. In some embodiments, the dosing regimen comprises a first administration in a first administered amount followed by one or more additional administrations in a second administered amount that is the same as the first administered amount.
Aliphatic hetero-ester: as used herein, the term "heteroaliphatic" is given its ordinary meaning in the art and refers to an aliphatic group as described herein in which one or more carbon atoms are independently replaced by one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). In some embodiments, selected from C, CH2And CH3One or more ofMultiple units are independently substituted with one or more heteroatoms (including oxidized and/or substituted forms thereof). In some embodiments, the heteroaliphatic group is a heteroalkyl group. In some embodiments, the heteroaliphatic group is a heteroalkenyl group.
Heteroalkyl group: as used herein, the term "heteroalkyl" is given its ordinary meaning in the art and refers to an alkyl group as described herein in which one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly (ethylene glycol) -, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, and the like.
Heteroaryl group: as used herein, the terms "heteroaryl" and "heteroar-" used alone or as part of a larger moiety such as "heteroaralkyl" or "heteroaralkoxy" refer to monocyclic, bicyclic, or polycyclic ring systems having a total of five to thirty ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In some embodiments, heteroaryl groups are groups having 5 to 10 ring atoms (i.e., monocyclic, bicyclic, or polycyclic), in some embodiments having 5, 6, 9, or 10 ring atoms. In some embodiments, heteroaryl groups have 6, 10, or 14 pi electrons shared in a cyclic array; and having one to five heteroatoms in addition to carbon atoms. Heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, heteroaryl is a heterobiaryl group, such as bipyridyl and the like. As used herein, the terms "heteroaryl" and "heteroaryl-" also include groups in which the heteroaryl ring is fused to one or more aryl, cycloaliphatic or heterocyclic rings, with the attachment group or point on the heteroaryl ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolyl, tetrahydroisoquinolyl, and pyrido [2, 3-b ] -1, 4-oxazin-3 (4H) -one. Heteroaryl groups may be monocyclic, bicyclic or polycyclic. The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring", "heteroaryl group" or "heteroaromatic", any of which terms includes an optionally substituted ring. The term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group, wherein the alkyl portion and the heteroaryl portion are independently optionally substituted.
Heteroatom: as used herein, the term "heteroatom" means an atom that is not carbon or hydrogen. In some embodiments, the heteroatom is boron, oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; quaternized forms of any basic nitrogen or heterocyclic substitutable nitrogen (e.g., N as in 3, 4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + (as in N-substituted pyrrolidinyl), etc.).
Heterocyclic ring: as used herein, the terms "heterocycle" (heterocyclic) "," heterocyclyl group "(heterocyclic)" and "heterocyclic ring" (heterocyclic ring) "are used interchangeably and refer to a monocyclic, bicyclic or polycyclic moiety (e.g., 3-30 membered) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, a heterocyclyl group is a stable 5-to 7-membered monocyclic or 7-to 10-membered bicyclic heterocyclic moiety that is saturated or partially unsaturated and has one or more, preferably one to four, heteroatoms as defined above in addition to carbon atoms. The term "nitrogen" when used in reference to a ring atom of a heterocyclic ring includes substituted nitrogens. For example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3, 4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or+NR (as in N-substituted pyrrolidinyl). The heterocyclic ring canTo be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any ring atom may be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazaOxygen nitrogen base, oxygen nitrogen heteroRadical, sulfur nitrogen heteroMesityl, morpholinyl and quinuclidinyl. The terms "heterocyclic", "heterocyclyl", "heterocyclic ring", "heterocyclic group", "heterocyclic moiety" and "heterocyclic" are used interchangeably herein and also include groups in which the heterocyclic ring is fused to one or more aryl, heteroaryl or cycloaliphatic rings, such as indolyl, 3H-indolyl, chromanyl, phenanthridinyl or tetrahydroquinolinyl. Heterocyclyl groups may be monocyclic, bicyclic or polycyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl, wherein the alkyl portion and the heterocyclyl portion are independently optionally substituted.
In vitro: as used herein, the term "in vitro" refers to an event that occurs in an artificial environment (e.g., in a test tube or reaction vessel, in cell culture, etc.) rather than within an organism (e.g., an animal, plant, and/or microorganism).
In vivo: as used herein, the term "in vivo" refers to an event that occurs within an organism (e.g., an animal, plant, and/or microorganism).
Optionally substituted: as described herein, a compound (e.g., an oligonucleotide) of the present disclosure can contain an optionally substituted moiety and/or a substituted moiety. Generally, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents at each position may be the same or different. In some embodiments, the optionally substituted group is unsubstituted. Combinations of substituents contemplated by the present disclosure are preferably combinations that result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that are not substantially altered when subjected to the conditions for their preparation, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
Suitable monovalent substituents on a substitutable atom (e.g., a suitable carbon atom) are independently halogen; - (CH)2)0-4Ro；-(CH2)0-4ORo；-O(CH2)0-4Ro、-O-(CH2)0-4C(O)ORo；-(CH2)0-4CH(ORo)2；-(CH2)0-4Ph, which may be via RoSubstitution; - (CH)2)0-4O(CH2)0-1Ph, which may be via RoSubstitution; -CH ═ CHPh, which may be substituted by RoSubstitution; - (CH)2)0-4O(CH2)0-1-pyridyl, which may be via RoSubstitution; -NO2；-CN；-N3；-(CH2)0-4N(Ro)2；-(CH2)0-4N(Ro)C(O)Ro；-N(Ro)C(S)Ro；-(CH2)0-4N(Ro)C(O)NRo2；-N(Ro)C(S)NRo2；-(CH2)0-4N(Ro)C(O)ORo；-N(Ro)N(Ro)C(O)Ro；-N(Ro)N(Ro)C(O)NRo2；-N(Ro)N(Ro)C(O)ORo；-(CH2)0-4C(O)Ro；-C(S)Ro；-(CH2)0-4C(O)ORo；-(CH2)0-4C(O)SRo；-(CH2)0-4C(O)OSiRo3；-(CH2)0-4OC(O)Ro；-OC(O)(CH2)0-4SR，-SC(S)SRo；-(CH2)0-4SC(O)Ro；-(CH2)0-4C(O)NRo2；-C(S)NRo2；-C(S)SRo；-SC(S)SRo，-(CH2)0-4OC(O)NRo2；-C(O)N(ORo)Ro；-C(O)C(O)Ro；-C(O)CH2C(O)Ro；-C(NORo)Ro；-(CH2)0-4SSRo；-(CH2)0-4S(O)2Ro；-(CH2)0-4S(O)2ORo；-(CH2)0-4OS(O)2Ro；-S(O)2NRo2；-(CH2)0-4S(O)Ro；-N(Ro)S(O)2NRo2；-N(Ro)S(O)2Ro；-N(ORo)Ro；-C(NH)NRo2；-Si(Ro)3；-OSi(Ro)3；-B(Ro)2；-OB(Ro)2；-OB(ORo)2；-P(Ro)2；-P(ORo)2；-OP(Ro)2；-OP(ORo)2；-P(O)(Ro)2；-P(O)(ORo)2；-OP(O)(Ro)2；-OP(O)(ORo)2；-OP(O)(ORo)(SRo)；-SP(O)(Ro)2；-SP(O)(ORo)2；-N(Ro)P(O)(Ro)2；-N(Ro)P(O)(ORo)2；-P(Ro)2[B(Ro)3]；-P(ORo)2[B(Ro)3]；-OP(Ro)2[B(Ro)3]；-OP(ORo)2[B(Ro)3]；-(C1-4Straight or branched alkylene) O-N (R)o)2(ii) a Or- (C)1-4Straight or branched alkylene) C (O) O-N (R)o)2Wherein each RoMay be substituted as defined below and independently is hydrogen; c1-20An aliphatic group; c having 1 to 5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus1-20A heteroaliphatic group; -CH2-(C6-14Aryl groups); -O (CH)2)0-1(C6-14Aryl groups); -CH2- (5-to 14-membered heteroaryl ring); a 5-to 20-membered monocyclic, bicyclic, or polycyclic saturated, partially unsaturated, or aryl ring having 0 to 5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus; or, regardless of the above definition, two independently occurring RoTogether with one or more intervening atoms thereof, form a 5-to 20-membered monocyclic, bicyclic, or polycyclic saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus, which may be substituted as defined below.
Ro(or by two independently occurring RoA ring formed with the intervening atoms) are independently halogen, - (CH)2)0-2R·- (halogeno radical R)·)、-(CH2)0-2OH、-(CH2)0-2OR·、-(CH2)0-2CH(OR·)2-O (halo R)·)、-CN、-N3、-(CH2)0-2C(O)R·、-(CH2)0-2C(O)OH、-(CH2)0-2C(O)OR·、-(CH2)0-2SR·、-(CH2)0-2SH、-(CH2)0-2NH2、-(CH2)0-2NHR·、-(CH2)0-2NR·2、-NO2、-SiR·3、-OSiR·3、-C(O)SR·、-(C1-4Straight OR branched alkylene) C (O) OR·or-SSR·Wherein each R·Unsubstituted or substituted with only one or more halogen(s) if preceded by "halo", and is independently selected from C1-4Aliphatic, -CH2Ph、-O(CH2)0-1Ph or a 5-to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. At RoSuitable divalent substituents on the saturated carbon atom of (a) include ═ O and ═ S.
For example, suitable divalent substituents on suitable carbon atoms are independently the following: is one of O, S and NNR*2、＝NNHC(O)R*、＝NNHC(O)OR*、＝NNHS(O)2R*、＝NR*、＝NOR*、-O(C(R*2))2-3O-, or-S (C (R)*2))2-3S-, wherein each independently occurs R*Is selected from hydrogen, C which may be substituted as defined below1-6An aliphatic group, and an unsubstituted 5-to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents bonded to a substitutable carbon ortho to the "optionally substituted" group include: -O (CR)*2)2-3O-, in which each occurrence of R is independent*Is selected from hydrogen, C which may be substituted as defined below1-6An aliphatic group, and an unsubstituted 5-to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
R*Suitable substituents on the aliphatic radical of (A) are independently halogen, -R·- (halogeno radical R)·)、-OH、-OR·-O (halo R)·)、-CN、-C(O)OH、-C(O)OR·、-NH2、-NHR·、-NR·2or-NO2Wherein each R·Unsubstituted or substituted by one or more halogens only if preceded by "halo", and independently is C1-4Aliphatic, -CH2Ph、-O(CH2)0-1Ph or a 5-to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
Oral administration: the phrase "oral administration and administration" as used herein has the meaning understood in the art and refers to administration of a compound or composition by mouth.
And (3) parenteral administration: the phrase "parenteral administration and administered parentally" as used herein has its art-understood meaning and refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
Partially unsaturated: as used herein, the term "partially unsaturated" refers to a cyclic moiety that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties as defined herein.
The pharmaceutical composition comprises: as used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition can be specifically formulated for administration in solid or liquid form, including those suitable for use in: oral administration, e.g., drench (aqueous or non-aqueous solution or suspension), tablets (e.g., those directed to buccal, sublingual and systemic absorption), boluses, powders, granules, pastes (applied to the tongue); parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, as, e.g., a sterile solution or suspension or sustained release formulation; topical application, e.g., as a cream, ointment, or controlled release patch or spray, to the skin, lungs, or oral cavity; intravaginally or intrarectally, e.g., as a pessary, cream, or foam; under the tongue; an eye portion; transdermal; or nasal, pulmonary, and other mucosal surfaces.
Pharmaceutically acceptable: as used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
A pharmaceutically acceptable carrier: as used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ or portion of the body to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; a pH buffer solution; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.
Pharmaceutically acceptable salts: as used herein, the term "pharmaceutically acceptable salt" refers to salts of such compounds that are suitable for use in a pharmaceutical environment, i.e., salts that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M.Berge et al in J.pharmaceutical Sciences [ journal of pharmaceutical Sciences],66: pharmaceutically acceptable salts are described in detail in 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts, which are salts with amino groups formed using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or using organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, gluconates, hemisulfates (hemisulfates), heptanoates, hexanoates, hydroiodides, 2-hydroxy-ethanesulfonates, lactobionates, lactates, laurates, malates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoate, pectinates, persulfates, laurates, malates, malonates, methanesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamonates, pamoate, pectinates, persulfates, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate and the like. In some embodiments, provided compounds (e.g., oligonucleotides) comprise one or more acidic groups, and the pharmaceutically acceptable salt is an alkali metal salt, an alkaline earth metal salt, or an ammonium salt(e.g., N (R))3Wherein each R is independently defined and described in the present disclosure). Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. In some embodiments, the pharmaceutically acceptable salt is a sodium salt. In some embodiments, the pharmaceutically acceptable salt is a potassium salt. In some embodiments, the pharmaceutically acceptable salt is a calcium salt. In some embodiments, pharmaceutically acceptable salts suitably include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl groups having from 1 to 6 carbon atoms, sulfonates, and arylsulfonates. In some embodiments, provided compounds comprise more than one acidic group, e.g., provided oligonucleotides can comprise two or more acidic groups (e.g., natural phosphate linkages and/or modified internucleotide linkages). In some embodiments, a pharmaceutically acceptable salt (or, typically, a salt) of such a compound comprises two or more cations, which may be the same or different. In some embodiments, in the pharmaceutically acceptable salt (or, in general, salt), all of the ionizable hydrogens in the acidic group are replaced with cations. In some embodiments, the pharmaceutically acceptable salt is a sodium salt of the provided oligonucleotide. In some embodiments, the pharmaceutically acceptable salt is a sodium salt of the provided oligonucleotides, wherein each acidic phosphate group is present in salt form (all sodium salts).
As used herein, the term "protecting group" includes those well known in the art and includes protecting groups such as proctecting Groups in Organic Synthesis, N.W. Greene, P.M. Wuts, N.W. N.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.R.N.N.N.N.N.N.R.R.R.R.R.R.R.R.R.R.R.R.R.S.R.N.N.R.R.R.R.R.R.S.R.R.S.S.N.N.S.N.N.S.N.S.S.S.S.S.S.S.N.S.S.S.S.S.N.N.S.S.S.S.S.N.S.N.N.S.S.S.S.S.S.N.N.N.N.N.S.S.N.N.N.S.S.S.S.S.N.N.S.S.N.N.S.N.N.N.S.S.S.N.N.N.N.N.N.S.N.N.N.N.N.S.S.S.S.S.S.S.N.S.S.S.S.S.S.S.S.S.N.N.S.N.N.S.S.S.S.S.S.N.N.S.S.N.N.N.N.N.N.S.N.N.N.N.N.N.S.N.N.N.N.N.S.S.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.S.S.S.S.N.S.S.S.N.N.N.N.N.S.N.N.S.S.S.N.N.N.S.S.N.N.S.N.N.N.N.N.N.S.N.S.S.S.N.N.N.N.N.N.N.N.N.N.N.N.N.N.S.S.N.N.S.N.N.N.N.N.N.N.N.N.S.S.S.S.N.S.S.S.S.S.S.S.S.S.N.N.S.S.S.S.S.S.S.S.S.N.N.N.N.N.N.N.N.S.S.S.S.N.N.N.S.S.S.S.S.N.N.N.S.N.N.N.N.N.N.N.N.N.N.N.N.N.N.S.S.S.S.S.N.N.S.S.N.N.N.N.N.S.S.N.N.N.N.N.S.N.N.N.N.N.S.S.N.N.N.N.N.N.N.S.S.N.N.S.S.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.S.S.S.N.N.N.N.N.S.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.S.N.N.N.N.S.S.N.N.N.N.N.N.S.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.S.N.N.N.N.N.N.N.N.N.S.S.S.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.S.S.N.N.S.S.S.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.N.
Suitably protected carboxylic acids further include, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butylbiphenylsilyl, triisopropylsilyl, and the like. Examples of suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3, 4-dimethoxybenzyl, trityl, tert-butyl, tetrahydropyran-2-yl. Examples of suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl. Examples of suitable arylalkyl groups include optionally substituted benzyl (e.g., p-methoxybenzyl (MPM), 3, 4-dimethoxybenzyl, o-nitrobenzyl, p-halobenzyl, 2, 6-dichlorobenzyl, p-cyanobenzyl), and 2-and 4-picolyl.
Suitable hydroxy-protecting groups include methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyldimethylsilyl) methoxymethyl (SMOM), Benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy) methyl (p-AOM), Guaiacolmethyl (GUM), tert-butoxymethyl, 4-Pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2, 2, 2-trichloroethoxymethyl, bis (2-chloroethoxy) methyl, 2- (trimethylsilyl) ethoxymethyl (SEMOR), tetrahydropyranyl (P), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-Methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydropyranyl S, S-dioxide, 1- [ (2-chloro-4-methyl) phenyl ] -4-methoxypiperidin-4-yl (TMS), 1, 7-dimethyl-ethyl) acetal, 2-4-ethoxybenzyl, 2, 2-ethoxybenzyl, 2, 4-bis (N-ethoxybenzyl) methyl (2, 2, 4-ethoxybenzyl) methyl (N-ethyl) methacrylate, 2, 2-4-bis (TMS, 4-ethoxybenzyl) methyl) methacrylate, 2, 2, 2-4-ethoxybenzyl (N-ethyl) methyl) methacrylate, 2, 2, 2-4-ethoxybenzyl (N-ethoxymethyl) methyl (N-ethoxymethyl) methacrylate, 2, 2, 2-ethoxymethyl) methyl (7-ethyl) methyl (2, 2, 2-ethoxymethyl) methyl (2, 2, 2, 2-ethyl (2-ethoxymethyl) methyl (2, 2-ethyl (2-ethyl) methyl) methacrylate, 2-ethyl (2, 2-4-ethyl (2, 2-ethyl) methyl (2, 2, 2, 2-ethyl (2, 2, 2-4-ethyl (2-4-ethyl) methyl (2, 2, 2-ethyl (P, 2, 2, 2-ethyl (4, 2, 2-ethyl (2-ethyl) methyl (2, 2, 2, methyl) ethyl (2, 2, methyl) methyl (2, 2, methyl) methyl (2, 2, 2, methyl) methyl (2, methyl) ethyl (2, methyl) methyl (2, methyl) ethyl (2, methyl) ethyl (4, methyl) ethyl (2, methyl) ethyl (2, 2, methyl) ethyl (2, methyl) ethyl (4, methyl) ethyl (2, methyl) ethyl (P, methyl) ethyl (2, methyl) ethyl (2, methyl) ethyl (P, methyl) ethyl (2, methyl) ethyl (4, methyl) ethyl (2, methyl) ethyl (P, methyl) ethyl (2, methyl) ethyl (2, methyl) ethyl (2, methyl) ethyl (P, methyl) ethyl (2, methyl) ethyl (P, 2, methyl) ethyl (2, methyl) ethyl (2, methyl) ethyl (2, methyl) ethyl (4, methyl) ethyl (2, methyl) ethyl (7, methyl) ethyl (2, methyl) ethyl (4, methyl) ethyl (2, methyl) ethyl (4, methyl) ethyl (2, methyl) ethyl (4, methyl) ethyl (2, methyl) ethyl (.
In some embodiments, the hydroxyl protecting group is acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2, 4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2, 6-dichlorobenzyl, biphenylmethyl, p-nitrobenzyl, triphenylmethyl (trityl), 4' -dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butylbiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, methyl acetate, mesylate, tosylate, triflate, trityl, monomethoxytrityl (MMTr), 4 '-dimethoxytrityl (DMTr) and 4, 4' -trimethoxytrityl (TMTr), 2-cyanoethyl (CE or Cne), 2- (trimethylsilyl) ethyl (TSE), 2- (2-nitrophenyl) ethyl, 2- (4-cyanophenyl) ethyl, 2- (4-nitrophenyl) ethyl (NPE), 2- (4-nitrophenylsulfonyl) ethyl, 3, 5-dichlorophenyl, 2, 4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2, 4, 6-trimethylphenyl, 2- (2-nitrophenyl) ethyl, butylthiocarbonyl, 4, 4' -tris (benzoyloxy) trityl, biphenylcarbamoyl, levulinyl, 2- (dibromomethyl) benzoyl (Dbmb), 2- (isopropylthiomethoxymethyl) benzoyl (Ptmt), 9-phenylxanthen-9-yl (phenylxanthyl) or 9- (p-methoxyphenyl) xanth-9-yl (MOX). In some embodiments, each hydroxyl protecting group is independently selected from acetyl, benzyl, t-butyldimethylsilyl, t-butylbiphenylsilyl, and 4, 4' -dimethoxytrityl. In some embodiments, the hydroxyl protecting group is selected from the group consisting of: trityl, monomethoxytrityl and 4, 4' -dimethoxytrityl radicals. In some embodiments, a phosphorus linkage protecting group is a group that is attached to a phosphorus linkage (e.g., an internucleotide linkage) throughout oligonucleotide synthesis. In some embodiments, the protecting group is attached to the sulfur atom of the phosphorothioate group. In some embodiments, the protecting group is attached to the oxygen atom of the internucleotide phosphorothioate linkage. In some embodiments, the protecting group is attached to the oxygen atom of the internucleotide phosphate linkage. In some embodiments, the protecting group is 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o-nitrobenzyl, 2- (p-nitrophenyl) ethyl (NPE or Npe), 2-phenylethyl, 3- (N-tert-butylcarboxamido) -1-propyl, 4-oxopentyl, 4-methylthio-1-butyl, 2-cyano-1, 1-dimethylethyl, 4-N-methylaminobutyl, 3- (2-pyridyl) -1-propyl, 2- [ N-methyl-N- (2-pyridyl) ] aminoethyl, 2- (N-formyl, n-methyl) aminoethyl, or 4- [ N-methyl-N- (2, 2, 2-trifluoroacetyl) amino ] butyl.
Sample preparation: as used herein, a "sample" is a particular organism or material obtained therefrom. In some embodiments, the sample is a biological sample obtained or derived from a source of interest as described herein. In some embodiments, the source of interest comprises an organism, such as an animal or human. In some embodiments, the biological sample comprises a biological tissue or a bodily fluid. In some embodiments, the biological sample is or comprises: bone marrow; blood; blood cells; ascites fluid; tissue or fine needle biopsy samples; a cell-containing body fluid; free floating nucleic acids; sputum; saliva; (ii) urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluid (gynecologic fluid); a skin swab; a vaginal swab; a buccal swab; a nasal swab; wash or lavage fluids, such as ductal or bronchoalveolar lavage fluid; an aspirate; scraping scraps; a bone marrow sample; a tissue biopsy sample; a surgical sample; feces, other body fluids, secretions, and/or excretions; and/or cells derived therefrom, and the like. In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from a source of interest by any suitable method. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of: biopsies (e.g., fine needle aspiration or tissue biopsy), surgery, collecting bodily fluids (e.g., blood, lymph, stool, etc.), and the like. In some embodiments, as the context clearly indicates, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components of the primary sample and/or by adding one or more reagents to the primary sample). For example, filtration is performed using a semipermeable membrane. Such "processed sample" may comprise, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components. In some embodiments, the sample is an organism. In some embodiments, the sample is a plant. In some embodiments, the sample is an animal. In some embodiments, the sample is a human. In some embodiments, the sample is an organism other than a human.
Subject: as used herein, the term "subject" or "test subject" refers to any organism to which a provided compound or composition is administered according to the present disclosure, e.g., for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, the subject may be suffering from and/or susceptible to a disease, disorder, and/or condition.
Essentially: as used herein, the term "substantially" refers to a qualitative state exhibiting an overall or near overall extent or degree of a feature or characteristic of interest. The base sequence substantially complementary to the second sequence is not identical to the second sequence but is largely identical or almost identical to the second sequence. Furthermore, it will be understood by those of ordinary skill in the biological arts that biological and chemical phenomena, if any, are less likely to achieve completion and/or proceed to completion or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to obtain inherent completeness that is potentially lacking in many biological and/or chemical phenomena.
Has the following symptoms: an individual "suffering" from a disease, disorder, and/or condition has been diagnosed with and/or exhibits one or more symptoms of the disease, disorder, and/or condition.
Susceptible to: an individual "susceptible to" a disease, disorder, and/or condition is an individual at higher risk of developing the disease, disorder, and/or condition than a member of the general public. In some embodiments, an individual who is predisposed to a disease, disorder, and/or condition is predisposed to the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not be diagnosed as having the disease, disorder, and/or condition. In some embodiments, an individual who is predisposed to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is predisposed to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is predisposed to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is predisposed to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
Whole body: as used herein, the phrases "systemic administration," "systemically administering," "peripherally administering," and "peripherally administering" have art-understood meanings, and refer to the administration of a compound or composition into the system of a subject.
Therapeutic agents: as used herein, the phrase "therapeutic agent" refers to an agent that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, reduce, inhibit, prevent, delay the onset of, reduce the severity of, and/or reduce the incidence of one or more symptoms or features of a disease, disorder, and/or condition.
A therapeutically effective amount of: as used herein, the term "therapeutically effective amount" means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and/or delay the onset of a disease, disorder, and/or condition when administered to a subject suffering from or susceptible to the disease, disorder, and/or condition. As one of ordinary skill in the art will appreciate, the effective amount of a substance may vary depending on such factors as: such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, and the like. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and/or condition is an amount that alleviates, ameliorates, reduces, inhibits, prevents, delays the onset of, reduces the severity of, and/or reduces the incidence of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.
Treatment: as used herein, the term "treating" or "treatment" refers to any method for partially or completely alleviating, ameliorating, reducing, inhibiting, preventing, delaying the onset of, reducing the severity of, and/or reducing the incidence of one or more symptoms or features of a disease, disorder, and/or condition. The treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of a disease, disorder, and/or condition, e.g., for the purpose of reducing the risk of developing a pathology associated with the disease, disorder, and/or condition.
Unsaturated: as used herein, the term "unsaturated" means a moiety having one or more units of unsaturation.
Unit dose: as used herein, the expression "unit dose" refers to an amount administered as a single dose and/or in physically discrete units of a pharmaceutical composition. In many embodiments, the unit dose contains a predetermined amount of active agent. In some embodiments, a unit dose contains the entire single dose of the pharmaceutical agent. In some embodiments, more than one unit dose is administered to achieve a total single dose. In some embodiments, it is necessary or desirable to administer multiple unit doses to achieve the desired effect. A unit dose can be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined amount of one or more therapeutic agents, a predetermined amount of one or more therapeutic agents in solid form, a sustained release formulation or drug delivery device containing a predetermined amount of one or more therapeutic agents, and the like. It is to be understood that the unit dose may be present in a formulation that includes any of a variety of components in addition to the one or more therapeutic agents. For example, as described below, acceptable carriers (e.g., pharmaceutically acceptable carriers), diluents, stabilizers, buffers, preservatives, and the like can be included. One skilled in the art will appreciate that in many embodiments, the total appropriate daily dosage of a particular therapeutic agent may comprise a fraction or multiple unit doses and may be determined, for example, by an attending physician within the scope of sound medical judgment. In some embodiments, the specific effective dose level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the particular active compound employed; the particular composition employed; the age, weight, general health, sex, and diet of the subject; the time of administration, and the rate of excretion of the particular active compound employed; the duration of the treatment; drugs and/or other therapies used in combination or in concert with the particular compound employed, and similar factors well known in the medical arts.
Wild type: as used herein, the term "wild-type" has its art-understood meaning, which refers to an entity having a structure and/or activity as found in nature in a "normal" (as opposed to mutant, diseased, altered, etc.) state or context. One of ordinary skill in the art will appreciate that wild-type genes and polypeptides typically exist in a variety of different forms (e.g., alleles).
Nucleic acid (A): as used herein, the term "nucleic acid" includes any nucleotide and polymers thereof. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length (ribonucleotides (RNA) or Deoxyribonucleotides (DNA)). These terms refer to the primary structure of the molecule and include double-and single-stranded DNA, and double-and single-stranded RNA. These terms include, as equivalents, analogs of RNA or DNA made from modified nucleotides and/or modified polynucleotides (such as, but not limited to, methylated, protected, and/or capped nucleotides or polynucleotides). The term encompasses polyribonucleotides or oligoribonucleotides (RNA) and polydeoxyribonucleotides or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N-or C-glycosides of nucleobases and/or modified nucleobases; nucleic acids derived from sugars and/or modified sugars; and nucleic acids derived from phosphate bridges and/or modified internucleotide linkages. The term encompasses nucleic acids containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified internucleotide linkages. Examples include, and are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxyribose moieties, nucleic acids containing ribose moieties and modified ribose moieties. Unless otherwise indicated, the prefix "poly-" refers to a nucleic acid containing from 2 to about 10,000 nucleotide monomer units, and wherein the prefix "oligo-" refers to a nucleic acid containing from 2 to about 200 nucleotide monomer units.
Nucleotide: as used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide, which consists of a nucleobase, a sugar and one or more internucleotide linkages. Naturally occurring bases (guanine (G), adenine (a), cytosine (C), thymine (T), and uracil (U)) are derivatives of purines or pyrimidines, but it is understood that naturally occurring and non-naturally occurring base analogs are also included. Naturally occurring sugars are pentoses (five carbon sugars), i.e. deoxyribose (which forms DNA) or ribose (which forms RNA), but it is understood that naturally occurring and non-naturally occurring sugar analogs are also included. Nucleotides are linked via internucleotide linkages to form nucleic acids, or polynucleotides. Many internucleotide linkages are known in the art (such as but not limited to phosphate, phosphorothioate, boranophosphate, etc.). Artificial nucleic acids include PNA (peptide nucleic acid), phosphotriesters, phosphorothioates, H-phosphonates, phosphoramidates, boranophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates, and other variants of the phosphate backbone of natural nucleic acids, such as those described herein. In some embodiments, a natural nucleotide comprises a naturally occurring base, sugar, and internucleotide linkage. As used herein, the term "nucleotide" also encompasses structural analogs, such as modified nucleotides and nucleotide analogs, that are used in place of natural nucleotides or naturally occurring nucleotides.
Modified nucleotide: the term "modified nucleotide" includes any chemical moiety that differs in structure from a natural nucleotide but is capable of performing at least one function of the natural nucleotide. In some embodiments, the modified nucleotides comprise modifications at sugar, base, and/or internucleotide linkages. In some embodiments, the modified nucleotides comprise modified sugars, modified nucleobases, and/or modified internucleotide linkages. In some embodiments, the modified nucleotide is capable of having at least one function of the nucleotide, e.g., forming a subunit in a polymer capable of base pairing with a nucleic acid comprising at least a complementary base sequence.
The analogues: the term "analog" includes any chemical moiety that is structurally different from the class of reference chemical moieties or moieties but is capable of performing at least one function of the class of such reference chemical moieties or moieties. By way of non-limiting example, a nucleotide analog differs in structure from a nucleotide, but is capable of performing at least one function of the nucleotide; nucleobase analogs are structurally different from nucleobases, but capable of performing at least one function of a nucleobase; and the like.
A nucleoside: the term "nucleoside" refers to a moiety in which a nucleobase or modified nucleobase is covalently bound to a sugar or modified sugar.
Modified nucleosides: the term "modified nucleoside" refers to a moiety that is derived from or is chemically similar to a natural nucleoside, but contains chemical modifications that distinguish it from the natural nucleoside. Non-limiting examples of modified nucleosides include those comprising modifications at the base and/or sugar. Non-limiting examples of modified nucleosides include those having a 2' modification at the sugar. Non-limiting examples of modified nucleosides also include abasic nucleosides (which lack nucleobases). In some embodiments, the modified nucleoside can have at least one function of the nucleoside, e.g., forming a moiety in a polymer that can base pair with a nucleic acid comprising at least a complementary base sequence.
Nucleoside analogs: the term "nucleoside analog" refers to a chemical moiety that is chemically different from a natural nucleoside but capable of performing at least one function of the nucleoside. In some embodiments, the nucleoside analogs comprise analogs of a sugar and/or analogs of a nucleobase. In some embodiments, the modified nucleoside can have at least one function of the nucleoside, e.g., forming a moiety in a polymer that can base pair with a nucleic acid comprising a complementary base sequence.
Sugar: the term "saccharide" refers to a monosaccharide or polysaccharide in a closed and/or open form. In some embodiments, the saccharide is a monosaccharide. In some embodiments, the saccharide is a polysaccharide. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties. As used herein, the term "saccharide" also encompasses structural analogs used in place of conventional saccharide molecules, such as diols, polymers forming the backbone of nucleic acid analogs, diol nucleic acids ("GNAs"), and the like. As used herein, the term "sugar" also encompasses structural analogs, such as modified sugars and nucleotide sugars, that are used in place of natural nucleotides or naturally occurring nucleotides.
Modified sugar: the term "modified sugar" refers to a moiety that can replace a sugar. The modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical properties of the sugar.
A nucleobase: the term "nucleobase" refers to a portion of a nucleic acid that participates in hydrogen bonding, which binds one nucleic acid strand to another complementary strand in a sequence-specific manner. The most common naturally occurring nucleobases are adenine (a), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the naturally occurring nucleobase is a modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the naturally occurring nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleobase is a "modified nucleobase", e.g., a nucleobase other than adenine (a), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the modified nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the modified nucleobases mimic the spatial arrangement, electronic properties, or some other physicochemical properties of the nucleobases, and retain the properties of hydrogen bonding, which binds one nucleic acid strand to another in a sequence-specific manner. In some embodiments, the modified nucleobases can pair with all five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting melting behavior, recognition by intracellular enzymes, or activity of the oligonucleotide duplex. As used herein, the term "nucleobase" also encompasses structural analogs, such as modified nucleobases and nucleobase analogs, that are used in place of natural nucleotides or naturally occurring nucleotides.
Modified nucleobases: the terms "modified nucleobase," "modified base," and the like refer to a chemical moiety that is chemically different from a nucleobase but capable of performing at least one function of the nucleobase. In some embodiments, the modified nucleobase is a nucleobase comprising a modification. In some embodiments, the modified nucleobase is capable of at least one function of a nucleobase, e.g., forming a moiety in a polymer capable of base pairing with a nucleobase comprising at least a complementary base sequence.
Blocking group: the term "blocking group" refers to a group that masks the reactivity of a functional group. The functional group can then be masked by removing the blocking group. In some embodiments, the blocking group is a protecting group.
The method comprises the following steps: the term "moiety" refers to a particular segment or functional group of a molecule. Chemical moieties are generally recognized chemical entities that are embedded in or attached to a molecule.
Solid support: the term "solid support" refers to any support capable of synthesizing nucleic acids. In some embodiments, the term refers to a glass or polymer that is insoluble in the medium used in the reaction step performed to synthesize the nucleic acid, and derivatized to include reactive groups. In some embodiments, the solid support is highly cross-linked polystyrene (HCP) or Controlled Pore Glass (CPG). In some embodiments, the solid support is Controlled Pore Glass (CPG). In some embodiments, the solid support is a hybrid support of Controlled Pore Glass (CPG) and highly cross-linked polystyrene (HCP).
Homology: "homology" or "identity" or "similarity" refers to sequence similarity between two nucleic acid molecules. Homology and identity can be determined individually by comparing the positions aligned for comparison purposes in each sequence. When equivalent positions in the compared sequences are occupied by the same base, then the molecules are identical at that position; when an equivalent site is occupied by the same or similar nucleic acid residues (e.g., similar in steric and/or electronic properties), then the molecules may be referred to as homologous (similar) at that position. The expression as percentage of homology/similarity or identity refers to a function of the number of identical or similar nucleic acids at positions shared by the compared sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, less than 35% identity, less than 30% identity, or less than 25% identity with a sequence described herein. The absence of residues (amino acids or nucleic acids) or the presence of additional residues also reduces identity and homology/similarity when comparing two sequences.
In some embodiments, the term "homology" describes a mathematically based comparison of sequence similarity that is used to identify genes with similar functions or motifs. The nucleic acid sequences described herein can be used as "query sequences" to search public databases, for example, to identify other family members, related sequences, or homologs. In some embodiments, Altschul et al, (1990) j.mol.biol. [ journal of molecular biology ] 215: the NBLAST and XBLAST programs (version 2.0) of 403-10 perform such searches. In some embodiments, a BLAST nucleotide search may be performed with NBLAST program (score 100, word length 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of the present disclosure. In some embodiments, to obtain gapped alignments for comparison purposes, a gap can be determined as described in Altschul et al, (1997) Nucleic Acids Res [ Nucleic Acids research ]25 (17): 3389 Using gapped BLAST as described in 3402-. When using BLAST and gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and BLAST) can be used (see www.ncbi.nlm.nih.gov).
Identity: as used herein, "identity" means the percentage of identical nucleotide residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching (i.e., to account for gaps and insertions). Identity can be readily calculated by known methods, including but not limited to those known in the art, including but not limited to those cited in WO 2017/192679.
Oligonucleotide: the term "oligonucleotide" refers to a polymer or oligomer of nucleotides and may comprise any combination of natural and non-natural nucleobases, sugars and internucleotide linkages.
The oligonucleotide may be single-stranded or double-stranded. A single-stranded oligonucleotide may have a double-stranded region (formed by two portions of a single-stranded oligonucleotide), and a double-stranded oligonucleotide comprising two oligonucleotide strands may have a single-stranded region, e.g., a region in which the two oligonucleotide strands are not complementary to each other. Exemplary oligonucleotides include, but are not limited to, structural genes, genes comprising control and termination regions, self-replicating systems (such as viral DNA or plasmid DNA), single and double stranded RNAi agents and other RNA interfering agents (RNAi or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, micrornas, microrna mimetics, supermir, aptamers, antimirs, antagomirs, Ul adapters, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.
Internucleotide linkage: as used herein, the phrase "internucleotide linkage" generally refers to a linkage that links the nucleoside units of an oligonucleotide or nucleic acid. In some embodiments, the internucleotide linkage is a phosphodiester linkage (natural phosphate linkage) as found in naturally occurring DNA and RNA molecules. In some embodiments, the internucleotide linkage comprises a modified internucleotide linkage. In some embodiments, the internucleotide linkage is a "modified internucleotide linkage," wherein each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, such organic or inorganic moieties are selected from, but not limited to, S, ═ Se, ═ NR ', -SR', -SeR′、-N(R′)2，、B(R′)3-S-, -Se-, and-N (R ') -, wherein each R' is independently as defined and described in the disclosure. In some embodiments, the internucleotide linkage is a phosphotriester linkage, a phosphorothioate diester linkage, or a combination thereofOr a modified phosphorothioate triester linkage. In some embodiments, the internucleotide linkage is one of a PNA (peptide nucleic acid) or PMO (phosphorodiamidate morpholino oligomer) linkage, for example. It is understood by one of ordinary skill in the art that internucleotide linkages may exist as either anions or cations at a given pH due to the presence of acid or base moieties in the linkage.
Non-limiting examples of modified internucleotide linkages are the modified internucleotide linkages designated s, s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17 and s18 as described in WO 2017/210647.
For example, (Rp, Sp) -ATsCs1GA has 1) phosphorothioate internucleotide linkage between T and CAnd 2) between C and G withStructural phosphorothioate triester internucleotide linkages. Unless otherwise indicated, the Rp/Sp identity preceding the oligonucleotide sequence describes the configuration of the chirally bonded phosphorus atom in the internucleotide linkage from 5 'to 3' of the oligonucleotide sequence. For example, in (Rp, Sp) -ATsCs1GA, the phosphorus in the "s" linkage between T and C has the Rp configuration, and the phosphorus in the "s 1" linkage between C and G has the Sp configuration. In some embodiments, "all- (Rp)" or "all- (Sp)" are used to indicate that all of the chirally bonded phosphorus atoms in the oligonucleotide have the same Rp or Sp configuration, respectively.
Oligonucleotide type: as used herein, the phrase "oligonucleotide type" is used to define a probe having a particular base sequenceColumns, backbone linkage patterns (i.e., patterns of internucleotide linkage types (e.g., phosphates, phosphorothioates, etc.), backbone chiral center patterns (i.e., bonded phosphorus stereochemistry pattern (Rp/Sp)), and backbone phosphorus modification patterns (e.g., "-XLR in formula I)1"pattern of groups"). In some embodiments, oligonucleotides having a commonly specified "type" are structurally identical to each other.
One skilled in the art will appreciate that the synthetic methods of the present disclosure provide a degree of control during synthesis of the oligonucleotide strand such that each nucleotide unit of the oligonucleotide strand can be designed and/or selected in advance to have a particular stereochemistry at and/or a particular modification at the linkage phosphorous, and/or to have a particular base, and/or to have a particular sugar. In some embodiments, the oligonucleotide strands are designed and/or selected in advance to have a particular combination of stereocenters at the point of linkage to the phosphate. In some embodiments, the oligonucleotide strands are designed and/or defined to have a particular combination of modifications at the point of linkage to the phosphate. In some embodiments, the oligonucleotide strands are designed and/or selected to have a particular combination of bases. In some embodiments, the oligonucleotide strands are designed and/or selected to have a particular combination of one or more of the above structural features. In some embodiments, the present disclosure provides compositions comprising or consisting of a plurality of oligonucleotide molecules (e.g., chirally controlled oligonucleotide compositions). In some embodiments, all such molecules are of the same type (i.e., structurally identical to one another). However, in many embodiments, provided compositions comprise a plurality of different types of oligonucleotides (typically in predetermined relative amounts).
Chiral control: as used herein, "chiral control" refers to controlling the stereochemical identity of a chirally bonded phosphorus in a chiral internucleotide linkage within an oligonucleotide. In some embodiments, control is achieved by chiral elements not present in the sugar and base portions of the oligonucleotide, for example, in some embodiments, control is achieved by using one or more chiral auxiliary agents during oligonucleotide preparation, which are typically part of the chiral phosphoramidite used during oligonucleotide preparation, as exemplified in the present disclosure. In contrast to chiral control, one of ordinary skill in the art recognizes that if conventional oligonucleotide synthesis is used to form a chiral internucleotide linkage, such conventional oligonucleotide synthesis without the use of a chiral auxiliary agent cannot control the stereochemistry at the chiral internucleotide linkage. In some embodiments, the stereochemistry of each chirally bonded phosphorus in the chiral internucleotide linkages within the oligonucleotide is controlled.
Chirally controlled oligonucleotide composition: as used herein, the terms "chirally controlled oligonucleotide composition," "chirally controlled nucleic acid composition," and the like, refer to a composition comprising a plurality of oligonucleotides (or nucleic acids) that share: 1) a common base sequence, 2) a common pattern of backbone linkages, and 3) a common pattern of backbone phosphorus modification, wherein the plurality of oligonucleotides (or nucleic acids) share the same stereochemistry at one or more chiral internucleotide linkages (chiral controlled internucleotide linkages whose chiral phosphorus linkages are either Rp or Sp in the composition, rather than a random mixture of Rp and Sp as with achiral controlled internucleotide linkages). The level of the plurality of oligonucleotides (or nucleic acids) in the chirally controlled oligonucleotide composition is predetermined/controlled (e.g., prepared by chirally controlled oligonucleotides to stereoselectively form one or more chiral internucleotide linkages). In some embodiments, about 1% -100% (e.g., about 5% -100%, 10% -100%, 20% -100%, 30% -100%, 40% -100%, 50% -100%, 60% -100%, 70% -100%, 80% -100%, 90% -100%, 95% -100%, 50% -90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%) of all oligonucleotides in the chirality-controlled oligonucleotide composition, 98% or 99%) are the plurality of oligonucleotides. In some embodiments, about 1% -100% (e.g., about 5% -100%, 10% -100%, 20% -100%, 30% -100%, 40% -100%, 50% -100%, 60% -100%, 70% -100%, 80% -100%, 90% -100%, 95% -100%, 50% -90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% of all oligonucleotides in a chirally controlled oligonucleotide composition that share a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone phosphorus modification, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) is the plurality of oligonucleotides. In some embodiments, the predetermined level is of all oligonucleotides in the composition; or all oligonucleotides in the composition that share a common base sequence (e.g., base sequences of multiple oligonucleotides or one oligonucleotide type); or all oligonucleotides in the composition that share a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone phosphorus modifications are the plurality of oligonucleotides; or about 1% -100% (e.g., about 5% -100%, 10% -100%, 20% -100%, 30% -100%, 40% -100%, 50% -100%, 60% -100%, 70% -100%, 80% -100%, 90% -100%, 95% -100%, 50% -90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, or all of the oligonucleotides in a composition that share a common base sequence, a common base modification pattern, a common sugar modification pattern, a common pattern of internucleotide linkage types, and/or a common pattern of internucleotide linkage modifications(s), 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%). In some embodiments, the plurality of oligonucleotides have the same stereochemistry at about 1-50 (e.g., about 1-10, 1-20, 5-10, 5-20, 10-15, 10-20, 10-25, 10-30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) chiral internucleotide linkages. In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 1% -100% (e.g., about 5% -100%, 10% -100%, 20% -100%, 30% -100%, 40% -100%, 50% -100%, 60% -100%, 70% -100%, 80% -100%, 90% -100%, 95% -100%, 50% -90%, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) of the chiral internucleotide linkages. In some embodiments, each chiral internucleotide linkage is a chirally controlled internucleotide linkage, and the composition is a fully chirally controlled oligonucleotide composition. In some embodiments, not all chiral internucleotide linkages are chirally controlled internucleotide linkages, and the composition is a partially chirally controlled oligonucleotide composition. In some embodiments, the chirally controlled oligonucleotide compositions comprise a non-random level or a controlled level of individual oligonucleotide types or nucleic acid types. For example, in some embodiments, a chirally controlled oligonucleotide composition comprises one oligonucleotide type. In some embodiments, the chirally controlled oligonucleotide composition comprises more than one oligonucleotide type. In some embodiments, the chirally controlled oligonucleotide composition comprises a plurality of oligonucleotide types. In some embodiments, a chirally controlled oligonucleotide composition is a composition of oligonucleotides of one oligonucleotide type, the composition comprising a non-random level or a controlled level of a plurality of oligonucleotides of the oligonucleotide type.
Chiral purity: as used herein, the phrase "chirally pure" is used to describe an oligonucleotide or composition thereof in which all or substantially all (the remainder being impurities) of the oligonucleotide molecules are present in a single diastereomeric form with respect to the bonded phosphorus atom.
Predetermining: predetermined means intentionally selected or not random or controlled, e.g., as opposed to occurring randomly, or without control. One of ordinary skill in the art reading the present specification will appreciate that the present disclosure provides techniques that allow for the selection of specific chemical and/or stereochemical characteristics to be incorporated into oligonucleotide compositions and further allow for the controlled preparation of oligonucleotide compositions having such chemical and/or stereochemical characteristics. Such provided compositions are "predetermined" as described herein. Compositions that may contain certain oligonucleotides are not "predetermined" compositions due to the accidental production of the oligonucleotides by a process that is uncontrolled to intentionally produce a particular chemical and/or stereochemical characteristic. In some embodiments, the predetermined composition is a composition that can be intentionally replicated (e.g., by repeating a controlled process). In some embodiments, the predetermined level of the plurality of oligonucleotides in the composition means that the absolute amount and/or the relative amount (ratio, percentage, etc.) of the plurality of oligonucleotides in the composition is controlled. In some embodiments, the predetermined level of the plurality of oligonucleotides in the composition is obtained by chiral controlled oligonucleotide preparation.
Bonding phosphorus: as defined herein, the phrase "bonded phosphorus" is used to indicate that the particular phosphorus atom referred to is a phosphorus atom present in an internucleotide linkage corresponding to a phosphodiester internucleotide linkage as is present in naturally occurring DNA and RNA. In some embodiments, the bonded phosphorus atom is located in a modified internucleotide linkage, wherein each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, the bonded phosphorus atom is P in formula I. In some embodiments, the bonded phosphorus atom is chiral. In some embodiments, the bonded phosphorus atom is achiral.
P-modification: as used herein, the term "P-modification" refers to any modification at the point of bonding to a phosphorus other than a stereochemical modification. In some embodiments, the P-modification comprises adding, substituting, or removing a pendant moiety covalently attached to a bonded phosphorus. In some embodimentsWherein the "P-modification" is-X-L-R1Wherein X, L and R1Each of which is independently as defined and described in the present disclosure.
Block (Blockmer): as used herein, the term "block" refers to an oligonucleotide chain that characterizes the structural feature pattern of each individual nucleotide unit by the presence of at least two consecutive nucleotide units sharing common structural features at the internucleotide phospho-link. Common structural features mean common stereochemistry at or common modification at the phosphorus linkage. In some embodiments, the at least two contiguous nucleotide units that share a common structural feature at the internucleotide phosphorus linkage are referred to as a "block. In some embodiments, provided oligonucleotides are block entities.
In some embodiments, the block is a "steric block," e.g., at least two consecutive nucleotide units have the same stereochemistry at the point of linkage to a phosphorus. Such at least two consecutive nucleotide units form a "stereo block".
In some embodiments, the block is a "P-modified block," e.g., at least two consecutive nucleotide units have the same modification at the point of linkage to the phosphorus. Such at least two consecutive nucleotide units form a "P-modified block". For example, (Rp, Sp) -atsscga is a P-modified block, since at least two consecutive nucleotide units Ts and Cs have the same P-modification (i.e., are both phosphorothioate diesters). In the same oligonucleotide of (Rp, Sp) -atsscga, TsCs form a block, and the block is a P-modified block.
In some embodiments, the block is a "linkage block," e.g., at least two consecutive nucleotide units have the same stereochemistry and the same modification at the linkage phosphorus. At least two consecutive nucleotide units form a "linkage block". For example, (Rp, Rp) -atsscga is a linker block because at least two consecutive nucleotide units Ts and Cs have the same stereochemistry (both Rp) and P-modification (both phosphorothioate). In the same oligonucleotide of (Rp, Rp) -atsscga, TsCs form a block, and the block is a linkage block.
In some embodiments, the block comprises one or more blocks independently selected from the group consisting of a steric block, a P-modified block, and a linked block. In some embodiments, a block is a stereoblock for one block, and/or a P-modifying block for another block, and/or a linking block for yet another block.
Alternate (altmer): as used herein, the term "alternating" refers to an oligonucleotide strand characterized by a pattern of structural features characterizing each individual nucleotide unit, wherein no two consecutive nucleotide units in the oligonucleotide strand share a particular structural feature at an internucleotide phosphate linkage. In some embodiments, the alternates are designed such that they contain a repeating pattern. In some embodiments, the alternates are designed such that they do not contain a repeating pattern. In some embodiments, the provided oligonucleotides are alternators.
In some embodiments, the alternans are "stereo-alternans," e.g., no two consecutive nucleotide units have the same stereochemistry at a bonded phosphorus.
In some embodiments, the alternans are "P-modified alternans," e.g., no two consecutive nucleotide units have the same modification at a phosphorus linkage. For example, all- (Sp) -CAs1GsT, where the P-modifications of each bonded phosphorus are different from each other.
In some embodiments, the alternans are "linked alternans," e.g., no two consecutive nucleotide units have the same stereochemistry or the same modification at the point of linkage to the phosphate.
Monomer (Unimer): as used herein, the term "unimer" refers to an oligonucleotide strand that characterizes the pattern of structural features of each individual nucleotide unit such that all nucleotide units within the strand share at least one common structural feature at the internucleotide phosphate linkage. Common structural features mean common stereochemistry at or common modification at the phosphorus linkage. In some embodiments, provided oligonucleotides are unimers.
In some embodiments, the homopolymer is a "stereounimer," e.g., all nucleotide units have the same stereochemistry at the point of linkage to the phosphorus.
In some embodiments, the homopolymer is a "P-modified homopolymer," e.g., all nucleotide units have the same modification at the point of phosphorus bonding.
In some embodiments, the monopolymer is a "linked monopolymer," e.g., all nucleotide units have the same stereochemistry and the same modification at the point of linkage to the phosphorus.
Notch body (gapmer): as used herein, the term "gapmer" refers to an oligonucleotide strand characterized in that at least one internucleotide phospholinkage of said oligonucleotide strand is a phosphodiester linkage, such as those found in naturally occurring DNA or RNA. In some embodiments, more than one internucleotide phospholinkage in the oligonucleotide strand is a phosphodiester linkage, such as those found in naturally occurring DNA or RNA. In some embodiments, the provided oligonucleotides are gapmers.
Skipper (skipmer): as used herein, the term "skipper" refers to a type of notch in which all other internucleotide phospholinkages in the oligonucleotide strand are phosphodiester linkages, such as those found in naturally occurring DNA or RNA, and all other internucleotide phospholinkages in the oligonucleotide strand are modified internucleotide linkages. In some embodiments, provided oligonucleotides are skips.
For the purposes of this disclosure, chemical Elements are identified according to the Periodic Table of the Elements (CAS version, Handbook of Chemistry and Physics, 67 th edition, 1986-87, inner cover).
The methods and structures described herein with respect to the compounds and compositions of the present disclosure are also applicable to the pharmaceutically acceptable acid or base addition salts and all stereoisomeric forms of these compounds and compositions.
Description of certain embodiments
Oligonucleotides provide molecular tools suitable for various applications. For example, oligonucleotides (e.g., oligonucleotides targeting C9orf 72) are useful in therapeutic, diagnostic, and research applications, including the treatment of various conditions, disorders, and diseases. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) is limited, for example, by their susceptibility to endonucleases and exonucleases. Therefore, various synthetic counterparts have been developed to circumvent these drawbacks. These synthetic counterparts include synthetic oligonucleotides containing chemical modifications, such as base modifications, sugar modifications, backbone modifications, etc., which, among other things, make these molecules less susceptible to degradation and improve other properties of the oligonucleotide. From a structural point of view, internucleotide linkage modifications may introduce chirality, and certain properties of oligonucleotides may be affected by the configuration of the phosphorus atoms forming the backbone of the oligonucleotide. For example, in vitro studies have shown that the properties of antisense oligonucleotides, such as binding affinity, sequence-specific binding to complementary RNA, stability against nucleases, are influenced inter alia by the chirality of the backbone phosphorus atoms. Various modifications were effective for the C9orf72 oligonucleotide.
Oligonucleotides and compositions
In some embodiments, the present disclosure provides an oligonucleotide comprising a contiguous region of nucleotide units:
(NuM)t[(NuO)n(NuM)m]y
wherein:
each NuMIndependently is a nucleotide unit comprising a modified internucleotide linkage;
each NuOIndependently is a nucleotide unit comprising a natural phosphate linkage;
t, n and m are each independently 1-20; and is
y is 1 to 10.
In some embodiments, such oligonucleotides provide improved properties, such as improved stability and/or activity, as demonstrated in the present disclosure.
In some embodiments, y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, y is 5. In some embodiments, y is 6. In some embodiments, y is 7. In some embodiments, y is 8. In some embodiments, y is 9. In some embodiments, y is 10.
As defined herein, each NuMIndependently comprise a modified internucleotide linkage. In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage. In some embodiments, the modified internucleotide linkage has formula I or a salt form thereof. In some embodiments, the modified internucleotide linkage is chiral and has formula I or a salt form thereof. In some embodiments, the modified internucleotide linkage is a phosphorothioate diester linkage. In some embodiments, the modified internucleotide linkages are chiral and chirally controlled. In some embodiments, each modified internucleotide linkage is chirally controlled. In some embodiments, NuMThe internucleotide linkage of (a) is a chirally controlled phosphorothioate diester linkage. In some embodiments, the Nu of the provided oligonucleotidesMComprising different types of modified internucleotide linkages. In some embodiments, the Nu of the provided oligonucleotidesMComprising chiral internucleotide linkages having bonded phosphorus atoms of different configurations. In some embodiments, the Nu of the provided oligonucleotidesMComprising different types of modified internucleotide linkages. In some embodiments, the Nu of the provided oligonucleotidesMComprising chiral internucleotide linkages having bonded phosphorus atoms of different configurations. In some embodiments, NuMIs Sp at its point of attachment to the phosphorus. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 NuMEach independently comprises a chiral internucleotide linkage that is Sp at its phosphorus linkage. In some embodiments, NuMEach chiral internucleotide linkage of (a) is Sp at its bonded phosphorus. In some embodiments, NuMIs Rp where it is bonded to the phosphorus. In some embodiments, NuMIs Rp at its bonding phosphorus, and NuMAt least one chirality ofThe internucleotide linkage is Sp where it is bonded to the phosphorus. Including for NuMAdditional nucleotide units of modified internucleotide linkages of (a) are known in the art and/or described in the present disclosure, and can be used in accordance with the present disclosure.
Each Nu, as defined hereinOIndependently a nucleotide unit comprising a natural phosphate linkage. In some embodiments, at least one NuOIs a nucleotide unit comprising a natural phosphate linkage, wherein the natural phosphate linkage is bound to a 5' -nucleotide unit and a carbon atom of a sugar unit of the nucleotide unit, wherein the carbon atom is bound to less than two hydrogen atoms. In some embodiments, each NuOIndependently is a nucleotide unit comprising a natural phosphate linkage, wherein the natural phosphate linkage is bonded to a carbon atom of the 5' -nucleotide unit and the sugar unit of the nucleotide unit, wherein the carbon atom is bonded to less than two hydrogen atoms. In some embodiments, at least one NuOcomprising-C (R)5s)2-, said structure being directly bound to NuOAnd NuOThe ring portion of the sugar unit of (a). In some embodiments, each NuOIndependently contain-C (R)5s)2-, said structure being directly bound to NuOAnd NuOThe ring portion of the sugar unit of (a).
In some embodiments, each NuOIndependently have the structure of formula N-I:
or a salt form thereof, wherein:
BA is an optionally substituted group selected from: c1-30Cycloaliphatic radical, C6-30Aryl, C having 1-10 heteroatoms5-30Heteroaryl, C having 1-10 heteroatoms3-30Heterocyclyl, natural nucleobase moiety and modified nucleobase moiety;
LOis a natural phosphate linkage;
Lsis-C (R)5s)2-or L;
each R5sAnd Rs is independently-F, -Cl, -Br, -I, -CN, -N3、-NO、-NO2、-L-R′、-L-OR′、-L-SR′、-L-N(R′)2-O-L-OR ', -O-L-SR ', OR-O-L-N (R ')2；
Each L is independently a covalent bond or is selected from C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron, and silicon1-30Aliphatic radical and C1-30A divalent optionally substituted straight or branched chain radical of a heteroaliphatic group, wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-; and one or more carbon atoms are optionally and independently substituted with CyLReplacement;
ring a is an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms;
s is 0 to 20;
each R' is independently-R, -C (O) OR OR-S (O)2R; and is
Each R is independently-H, or an optionally substituted group selected from: c1-30Aliphatic, C having 1-10 heteroatoms1-30Heteroaliphatic, C6-30Aryl radical, C6-30Arylaliphatic, C having 1-10 heteroatoms6-30Aryl heteroaliphatic, 5-30 membered heteroaryl having 1-10 heteroatoms, anda 3-to 30-membered heterocyclic group having 1 to 10 hetero atoms, or
Two R groups optionally and independently form a covalent bond together, or
Two or more R groups on the same atom optionally and independently form, with the atom, an optionally substituted 3-to 30-membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms other than the atom; or
Two or more R groups on two or more atoms optionally and independently form, with their intervening atoms, an optionally substituted 3-to 30-membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms in addition to the intervening atoms.
In some embodiments of the present invention, the,has a structureWherein R is1s、R2s、R3sAnd R4sEach of which is independently RsAnd as described in this disclosure. In some embodiments of the present invention, the,has a structureWherein R is1s、R2s、R3sAnd R4sEach independently as described in this disclosure. In some embodiments of the present invention, the,has a structureWherein R is1s、R2s、R3sAnd R4sEach independently as described in this disclosure.
In some embodiments, Lsis-C (R)5s)2-. In some implementationsIn the examples, one R5sis-H and Lsis-CHR5s-. In some embodiments, each R5sIndependently is R. In some embodiments, -C (R)5s)2-is-C (R)2-. In some embodiments, one R5sis-H and-C (R)5s)2-is-CHR-. In some embodiments, R is not hydrogen. In some embodiments, R is optionally substituted C1-6Aliphatic. In some embodiments, R is optionally substituted C1-6An alkyl group. In some embodiments, R is substituted. In some embodiments, R is unsubstituted. In some embodiments, R is methyl. Other exemplary R groups are broadly described in this disclosure. In some embodiments, -C (R)5s)2C of-is chiral and is R. In some embodiments, -C (R)5s)2C of-is chiral and is S. In some embodiments, -C (R)5s)2-is- (R) -CHMe-. In some embodiments, -C (R)5S)2-is- (S) -CHMe-.
In some embodiments, the contiguous region of nucleotide units comprises a backbone chiral center pattern (bonded phosphorous) (Np) t [ (Op) n (sp) m ] y, wherein each variable is independently as described in the present disclosure. In some embodiments, the contiguous nucleotide unit region comprises a backbone chiral center pattern (bonded phosphorous) (Sp) t [ (Op) n (Sp) m ] y, wherein each variable is independently as described in the present disclosure.
In some embodiments, the disclosure provides oligonucleotides comprising one or two wings and a core and comprising or having a wing-core-wing, or wing-core structure. In some embodiments, provided oligonucleotides comprise or have a wing-core-wing structure. In some embodiments, provided oligonucleotides comprise or have a core-wing structure. In some embodiments, provided oligonucleotides comprise or have a wing-core structure. In some embodiments, the core is a region of contiguous nucleotide units as described in the present disclosure. In some embodiments, each wing independently comprises one or more nucleobases as described in the disclosure.
In some embodiments, the wing-core-wing motif is described as "X-Y-Z," where "X" represents the length of the 5 'wing, "Y" represents the length of the core, and "Z" represents the length of the 3' wing. In some embodiments, the core is positioned proximate each of the 5 'wing and the 3' wing. In some embodiments, X and Z are the same or different lengths, and/or have the same or different modifications or modification patterns. In a preferred embodiment, Y is between 8 and 15 nucleotides. X, Y or Z can be any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides. In some embodiments, the oligonucleotides described herein have or comprise a wing-core-wing structure, e.g., 5-10-5, 5-10-4, 4-10-3, 3-10-3, 2-10-2, 5-9-5, 5-9-4, 4-9-5, 5-8-4, 4-8-5, 5-7-5, 4-7-5, 5-7-4, or 4-7-4. In some embodiments, the oligonucleotides described herein have or comprise a wing-core or core-wing structure, e.g., 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13, 5-8, or 6-8. In some embodiments, the wing or core is a block, and the wing-core, core-wing, or wing-core-wing structure is a block comprising two or three blocks.
In some embodiments, the oligonucleotide has a wing-core-wing structure, wherein the length (in bases) of the first wing is represented by X, the length of the core is represented by Y, and the length of the second wing is represented by Z, wherein X-Y-Z is any one of: 1-5-1, 1-6-1, 1-7-1, 1-8-1, 1-9-1, 1-10-1, 1-11-1, 1-12-1, 1-13-1, 1-14-1, 1-15-1, 1-16-1, 1-17-1, 1-18-1, 1-19-1, 1-20-1, 1-5-2, 1-6-2, 1-7-2, 1-8-2, 1-9-2, 1-10-2, 1-11-2, 1-12-2, 1-13-2, 1-14-2, 1-15-2, 1-16-2, 1-17-2, 1-18-2, 1-19-2, 1-20-2, 1-5-3, 1-6-3, 1-7-3, 1-8-3, 1-9-3, 1-10-3, 1-11-3, 1-12-3, 1-13-3, 1-14-3, 1-15-3, 1-16-3, 1-17-3, 1-18-3, 1-19-3, 1-20-3, 1-5-4, 1-6-4, 1-7-4, 1-8-4, 1-9-4, 1-10-4, 1-11-4, 1-12-4, 1-13-4, 1-14-4, 1-15-4, 1-16-4, 1-17-4, 1-18-4, 1-19-4, 1-20-4, 1-5-5, 1-6-5, 1-7-5, 1-8-5, 1-9-5, 1-10-5, 1-11-5, 1-12-5, 1-13-5, 1-14-5, 1-15-5, 1-16-5, 1-17-5, 1-18-5, 1-19-5, 1-20-5, 2-5-1, 2-6-1, 2-7-1, 2-8-1, 2-9-1, 2-10-1, 2-12-1, 2-13-1, 2-14-1, 2-15-1, 2-16-1, 2-17-1, 2-18-1, 2-19-1, 2-20-1, 2-5-2, 2-6-2, 2-7-2, 2-8-2, 2-9-2, 2-10-2, 2-12-2, 2-13-2, 2-14-2, 2-15-2, 2-16-2, 2-17-2, 2-18-2, 2-19-2, 2-20-2, 2-5-3, 2-6-3, 2-7-3, 2-8-3, 2-9-3, 2-10-3, 2-12-3, 2-13-3, 2-14-3, 2-15-3, 2-16-3, 2-17-3, 2-18-3, 2-19-3, 2-20-3, 2-5-4, 2-6-4, 2-7-4, 2-8-4, 2-9-4, 2-10-4, 2-12-4, 2-13-4, 2-14-4, 2-15-4, 2-16-4, 2-17-4, 2-18-4, 2-19-4, 2-20-4, 2-5-5, 2-6-5, 2-7-5, 2-8-5, 2-9-5, 2-10-5, 2-12-5, 2-13-5, 2-14-5, 2-15-5, 2-16-5, 2-17-5, 2-18-5, 2-19-5, 2-20-5, 3-5-1, 3-6-1, 3-7-1, 3-8-1, 3-9-1, 3-10-1, 3-13-1, 3-14-1, 3-15-1, 3-16-1, 3-17-1, 3-18-1, 3-19-1, 3-20-1, 3-5-2, 3-6-2, 3-7-2, 3-8-2, 3-9-2, 3-10-2, 3-13-2, 3-14-2, 3-15-2, 3-16-2, 3-17-2, 3-18-2, 3-19-2, 3-20-2, 3-5-3, 3-6-3, 3-7-3, 3-8-3, 3-9-3, 3-10-3, 3-13-3, 3-14-3, 3-15-3, 3-16-3, 3-17-3, 3-18-3, 3-19-3, 3-20-3, 3-5-4, 3-6-4, 3-7-4, 3-8-4, 3-9-4, 3-10-4, 3-13-4, 3-14-4, 3-15-4, 3-16-4, 3-17-4, 3-18-4, 3-19-4, 3-20-4, 3-5-5, 3-6-5, 3-7-5, 3-8-5, 3-9-5, 3-10-5, 3-13-5, 3-14-5, 3-15-5, 3-16-5, 3-17-5, 3-18-5, 3-19-5, 3-20-5, 4-5-1, 4-6-1, 4-7-1, 4-8-1, 4-9-1, 4-10-1, 4-14-1, 4-13-1, 4-14-1, 4-15-1, 4-16-1, 4-17-1, 4-18-1, 4-19-1, 4-20-1, 4-5-2, 4-6-2, 4-7-2, 4-8-2, 4-9-2, 4-10-2, 4-14-2, 4-13-2, 4-14-2, 4-15-2, 4-16-2, 4-17-2, 4-18-2, 4-19-2, 4-20-2, 4-5-3, 4-6-3, 4-7-3, 4-8-3, 4-9-3, 4-10-3, 4-14-3, 4-13-3, 4-14-3, 4-15-3, 4-16-3, 4-17-3, 4-18-3, 4-19-3, 4-20-3, 4-5-4, 4-6-4, 4-7-4, 4-8-4, 4-9-4, 4-10-4, 4-14-4, 4-13-4, 4-14-4, 4-15-4, 4-16-4, 4-17-4, 4-18-4, 4-19-4, 4-20-4, 4-5-5, 4-6-5, 4-7-5, 4-8-5, 4-9-5, 4-10-5, 4-14-5, 4-13-5, 4-14-5, 4-15-5, 4-16-5, 4-17-5, 4-18-5, 4-19-5, 4-20-5, 5-5-1, 5-6-1, 5-7-1, 5-8-1, 5-9-1, 5-10-1, 5-15-1, 5-12-1, 5-13-1, 5-14-1, 5-15-1, 5-16-1, 5-17-1, 5-18-1, 5-19-1, 5-20-1, 5-5-2, 5-6-2, 5-7-2, 5-8-2, 5-9-2, 5-10-2, 5-15-2, 5-12-2, 5-13-2, 5-14-2, 5-15-2, 5-16-2, 5-17-2, 5-18-2, 5-19-2, 5-20-2, 5-5-3, 5-6-3, 5-7-3, 5-8-3, 5-9-3, 5-10-3, 5-15-3, 5-12-3, 5-13-3, 5-14-3, 5-15-3, 5-16-3, 5-17-3, 5-18-3, 5-19-3, 5-20-3, 5-5-4, 5-6-4, 5-7-4, 5-8-4, 5-9-4, 5-10-4, 5-15-4, 5-12-4, 5-13-4, 5-14-4, 5-15-4, 5-16-4, 5-17-4, 5-18-4, 5-19-4, 5-20-4, 5-5-5, 5-6-5, 5-7-5, 5-8-5, 5-9-5, 5-10-5, 5-15-5, 5-12-5, 5-13-5, 5-14-5, 5-15-5, 5-16-5, 5-17-5, 5-18-5, 5-19-5, 5-20-5, 1-5-6, 1-6-6, 1-7-6, 1-8-6, 1-9-6, 1-10-6, 1-11-6, 1-12-6, 1-13-6, 1-14-6, 1-15-6, 1-16-6, 1-17-6, 1-18-6, 1-19-6, 1-20-6, 2-5-6, 2-6-6, 2-7-6, 2-8-6, 2-9-6, 2-10-6, 2-11-6, 2-12-6, 2-13-6, 2-14-6, 2-15-6, 2-16-6, 2-17-6, 2-18-6, 2-19-6, 2-20-6, 3-5-6, 3-6-6, 3-7-6, 3-8-6, 3-9-6, 3-10-6, 3-11-6, 3-12-6, 3-13-6, 3-14-6, 3-15-6, 3-16-6, 3-17-6, 3-18-6, 3-19-6, 3-20-6, 4-5-6, 4-6-6, 4-7-6, 4-8-6, 4-9-6, 4-10-6, 4-11-6, 4-12-6, 4-13-6, 4-14-6, 4-15-6, 4-16-6, 4-17-6, 4-18-6, 4-19-6, 4-20-6, 5-5-6, 5-6-6, 5-7-6, 5-8-6, 5-9-6, 5-10-6, 5-11-6, 5-12-6, 5-13-6, 5-14-6, 5-15-6, 5-16-6, 5-17-6, 5-18-6, 5-19-6, 5-20-6, 6-5-6, 6-6-6, 6-7-6, 6-8-6, 6-9-6, 6-10-6, 6-11-6, 6-12-6, 6-13-6, 6-14-6, 6-15-6, 6-16-6, 6-17-6, 6-18-6, 6-19-6, 6-20-6, 7-5-6, 7-6-6, 7-7-6, 7-8-6, 7-9-6, 7-10-6, 7-11-6, 7-12-6, 7-13-6, 7-14-6, 7-15-6, 7-16-6, 7-17-6, 7-18-6, 7-19-6, 7-20-6, 1-5-7, 1-6-7, 1-7-7, 1-8-7, 1-9-7, 1-10-7, 1-11-7, 1-12-7, 1-13-7, 1-14-7, 1-15-7, 1-16-7, 1-17-7, 1-18-7, 1-19-7, 1-20-7, 2-5-7, 2-6-7, 2-7-7, 2-8-7, 2-9-7, 2-10-7, 2-11-7, 2-12-7, 2-13-7, 2-14-7, 2-15-7, 2-16-7, 2-17-7, 2-18-7, 2-19-7, 2-20-7, 3-5-7, 3-6-7, 3-7-7, 3-8-7, 3-9-7, 3-10-7, 3-11-7, 3-12-7, 3-13-7, 3-14-7, 3-15-7, 3-16-7, 3-17-7, 3-18-7, 3-19-7, 3-20-7, 4-5-7, 4-6-7, 4-7-7, 4-8-7, 4-9-7, 4-10-7, 4-11-7, 4-12-7, 4-13-7, 4-14-7, 4-15-7, 4-16-7, 4-17-7, 4-18-7, 4-19-7, 4-20-7, 5-5-7, 5-6-7, 5-7-7, 5-8-7, 5-9-7, 5-10-7, 5-11-7, 5-12-7, 5-13-7, 5-14-7, 5-15-7, 5-16-7, 5-17-7, 5-18-7, 5-19-7, 5-20-7, 6-5-7, 6-6-7, 6-7-7, 6-8-7, 6-9-7, 6-10-7, 6-11-7, 6-12-7, 6-13-7, 6-14-7, 6-15-7, 6-16-7, 6-17-7, 6-18-7, 6-19-7, 6-20-7, 7-5-7, 7-6-7, 7-7-7, 7-8-7, 7-9-7, 7-10-7, 7-11-7, 7-12-7, 7-13-7, 7-14-7, 7-15-7, 7-16-7, 7-17-7, 7-18-7, 7-19-7, or 7-20-7.
In some embodiments, the disclosure provides an oligonucleotide comprising or having a wing-core-wing, or wing-core structure, wherein:
the core contains a pattern of backbone chiral centers (bonded phosphorus):
(Np)t[(Op/Rp)n(Sp)m]y，
wherein:
np is Rp or Sp;
sp represents the S configuration of a chirally bonded phosphorus of a chirally modified internucleotide linkage;
op represents a natural phosphate-linked achiral-linked phosphorus; and is
Rp represents the S configuration of the chirally bonded phosphorus of the chirally modified internucleotide linkage; and is
Each wing independently comprises one or more nucleobases.
In some embodiments, the disclosure provides an oligonucleotide comprising or having a wing-core-wing, or wing-core structure, wherein:
the core is or comprises a region of contiguous nucleotide units (Nu)M)t[(NuO)n(NuM)m]y, the contiguous region of nucleotide units has a pattern of backbone chiral centers (bonded phosphorus) (Np) t [ (Op) n (Sp) m]y，
Wherein each variable is independently as described in the present disclosure.
In some embodiments, (Np) t [ (Op/Rp) n (sp) m ] y comprises at least one Op. In some embodiments, (Np) t [ (Op/Rp) n (sp) m ] y comprises at least one Rp. In some embodiments, (Np) t [ (Op/Rp) n (Sp) m ] y is (Np) t [ (Op) n (Sp) m ] y. In some embodiments, (Np) t [ (Op/Rp) n (Sp) m ] y is (Np) t [ (Rp) n (Sp) m ] y.
In some embodiments, the wings comprise one or more sugar modifications. In some embodiments, the two wings of the wing-core-wing structure comprise different sugar modifications. In some embodiments, the sugar modification provides improved stability compared to the absence of the sugar modification.
In some embodiments, certain sugar modifications (e.g., 2 '-MOE) provide greater stability compared to 2' -OMe under otherwise identical conditions. In some embodiments, the wings comprise a 2' -MOE modification. In some embodiments, each nucleoside unit comprising a pyrimidine base (e.g., C, U, T, etc.) that is flanking comprises a 2' -MOE modification. In some embodiments, each sugar unit of the wings comprises a 2' -MOE modification. In some embodiments, each nucleoside unit of the wings that includes a purine base (e.g., A, G, etc.) does not include a 2 ' -MOE modification (e.g., 2 ' -OMe, no 2 ' -modification, etc.). In some embodiments, each nucleoside unit of the wings that comprises a purine base comprises a 2' -OMe modification. In some embodiments, each internucleotide linkage at the 3 '-position of the sugar unit comprising the 2' -MOE modification is a native phosphate linkage. In some embodiments, each internucleotide linkage at the 3 ' -position of the sugar unit comprising the 2 ' -MOE modification is a natural phosphate linkage, except that if the wing is the 5 ' wing of the core, the first internucleotide linkage of the wing is a modified internucleotide linkage (e.g., a phosphorothioate diester linkage), and the internucleotide linkage connecting the 3 ' terminal nucleoside unit of the wing with the 5 ' terminal nucleoside unit of the core is a modified internucleotide linkage (e.g., a phosphorothioate diester linkage); and where the wing is 3 ' wing to core, the last internucleotide linkage of the wing is a modified internucleotide linkage (e.g., phosphorothioate diester linkage), and the internucleotide linkage connecting the 3 ' terminal nucleoside unit of the core with the 5 ' terminal nucleoside unit of the wing is a modified internucleotide linkage (e.g., phosphorothioate diester linkage) (see, e.g., WV-7127, WV-7128, etc.). In some embodiments, such wings are 5' wings. In some embodiments, such wings are 3' wings.
In some embodiments, the wings do not comprise a 2' -MOE modification. In some embodiments, the wings comprise a 2' -OMe modification. In some embodiments, each nucleoside unit of the wing independently comprises a 2' -OMe modification. The present disclosure encompasses, among other things, the recognition that under certain conditions, an oligonucleotide having a 2 '-OMe modification has poorer stability than a comparable oligonucleotide having a 2' -MOE modification. In some embodiments, modified non-natural internucleotide linkages (such as phosphorothioate diester linkages, in some cases particularly Sp phosphorothioate diester linkages) may be used to improve the properties (e.g., stability) of the oligonucleotide. In some embodiments, the wings do not comprise a 2' -MOE modification, and each internucleotide linkage between the nucleoside units of the wings is a modified internucleotide linkage. In some embodiments, the wings do not comprise a 2 '-MOE modification, each nucleoside unit of the wings comprises a 2' -OMe modification, and each internucleotide linkage between the nucleoside units of the wings is a modified internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate diester linkage. In some embodiments, the modified internucleotide linkage is a chirally controlled internucleotide linkage. In some embodiments, the modified internucleotide linkage is a chirally controlled internucleotide linkage, wherein the linked phosphorus has an Sp configuration. In some embodiments, the modified internucleotide linkage is a chirally controlled internucleotide linkage, wherein the linkage phosphorus has an Rp configuration. In some embodiments, the modified internucleotide linkage is an Sp phosphorothioate diester linkage. In some embodiments, the modified internucleotide linkage is an Rp phosphorothioate diester linkage. In some embodiments, such wings are 5' wings. In some embodiments, such wings are 3' wings.
The present disclosure specifically contemplates that 2' -modifications and/or modified internucleotide linkages can be used, alone or in combination, to fine tune the properties (e.g., stability) and/or activity of an oligonucleotide.
In some embodiments, the wings comprise one or more natural phosphate linkages. In some embodiments, the wings comprise one or more consecutive natural phosphate linkages. In some embodiments, the wings comprise one or more natural phosphate linkages and one or more modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is a phosphorothioate diester linkage. In some embodiments, the modified internucleotide linkage is an Sp phosphorothioate diester linkage.
In some embodiments, the flap does not comprise a natural phosphate linkage, and each internucleotide linkage of the flap is independently a modified internucleotide linkage. In some embodiments, the modified internucleotide linkages are chiral and chirally controlled. In some embodiments, the modified internucleotide linkage is a phosphorothioate diester linkage. In some embodiments, the modified internucleotide linkage is an Sp phosphorothioate diester linkage.
In some embodiments, for oligonucleotides comprising or being in a wing-core-wing structure, the two wings differ in that they contain different levels and/or types of chemical modifications, backbone chiral center stereochemistry, and/or patterns thereof. In some embodiments, the two wings differ in that they contain different levels and/or types of sugar modifications, and/or internucleotide linkages, and/or internucleotide linkage stereochemistry, and/or patterns thereof. For example, in some embodiments, one wing comprises a 2' -OR modification, wherein R is optionally substituted C1-6An alkyl group (e.g., 2-MOE), and the other wing does not comprise such modifications, or comprises (e.g., by number and/or percentage) a lower level of such modifications; additionally and alternatively, one wing comprises a natural phosphate linkage while the other wing does not comprise a natural phosphate linkage or comprises a lower level (e.g., by number and/or percentage) of a natural phosphate linkage; additionally and alternatively, one wing may comprise a particular type of modified internucleotide linkage (e.g., phosphorothioate diester internucleotide linkage), while the other wing does not comprise a native phosphate linkage or comprises a lower level (e.g., by number and/or percentage) of that type of modified internucleotide linkage; additionally and alternatively, one flap may comprise a chirally modified internucleotide linkage containing a specifically configured (e.g., Rp or Sp) bonded phosphorus atom, while additionallyA wing that contains no or a lower level of chirally modified internucleotide linkages containing said configurationally bound phosphorus atom; additionally or alternatively, each wing may comprise a different pattern of sugar modifications, internucleotide linkages, and/or backbone chiral centers. In some embodiments, one wing comprises one OR more native phosphate linkages and one OR more 2 '-OR modifications, wherein R is not-H OR-Me, and the other wing does not comprise a native phosphate linkage and does not comprise a 2' -OR modification, wherein R is not-H OR-Me. In some embodiments, one wing comprises one or more native phosphate linkages and one or more 2 '-MOE modifications, and each internucleotide linkage in the other wing is a phosphorothioate linkage, and each sugar unit of the other wing comprises a 2' -OMe modification. In some embodiments, one wing comprises one or more native phosphate linkages and one or more 2 '-MOE modifications, and each internucleotide linkage in the other wing is an Sp phosphorothioate linkage, and each sugar unit of the other wing comprises a 2' -OMe modification.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein one wing comprises the 2' -OMe and the other wing comprises a bicyclic sugar. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein one wing comprises a 2 '-OMe and the other wing comprises a bicyclic sugar, and the majority of the sugar in the core comprises a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein a majority of the sugars in one wing comprise the 2' -OMe and a majority of the sugars in the other wing are bicyclic sugars. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugar in one wing comprises a 2 '-OMe, and the majority of the sugar in the other wing is a bicyclic sugar, and the majority of the sugar in the core comprises a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein a majority of the sugars in one wing comprise 2 '-OMe, and in the other wing, at least one sugar is a bicyclic sugar and at least one sugar comprises 2' -OMe. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise 2 ' -OMe, and in the other wing, at least one sugar is a bicyclic sugar and at least one sugar comprises a 2 ' -OMe, and the majority of the sugars in the core comprise a 2 ' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein a majority of the sugars in one wing are bicyclic sugars, and in the other wing, at least one sugar is a bicyclic sugar and at least one sugar comprises a 2' -OMe. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing are bicyclic sugars, and in the other wing, at least one sugar is a bicyclic sugar and at least one sugar comprises a 2 '-OMe, and the majority of the sugars in the core comprise a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise 2 '-OMe, and in the other wing, at least two of the sugars are bicyclic sugars and at least two of the sugars comprise 2' -OMe. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise 2 ' -OMe, and in the other wing, at least two of the sugars are bicyclic sugars and at least two of the sugars comprise 2 ' -OMe, and the majority of the sugars in the core comprise a 2 ' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein a majority of the sugars in one wing are bicyclic sugars, and in the other wing, at least two of the sugars are bicyclic sugars and at least two of the sugars comprise a 2' -OMe. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing are bicyclic sugars, and in the other wing, at least two of the sugars are bicyclic sugars and at least two of the sugars comprise 2 '-OMe, and the majority of the sugars in the core comprise a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein each sugar in one wing comprises a 2' -OMe and each sugar in the other wing comprises a bicyclic sugar. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein each sugar in one wing comprises a 2 '-OMe and each sugar in the other wing comprises a bicyclic sugar, and the majority of the sugars in the core comprise a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein each sugar in one wing comprises a bicyclic sugar, each sugar in the other wing comprises a 2 '-OMe, and each sugar in the core comprises a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein one wing comprises a bicyclic sugar and the other wing comprises a 2' -MOE. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein one wing comprises a bicyclic sugar and the other wing comprises a 2 '-MOE, and the majority of the sugar in the core comprises a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein a majority of the sugars in one wing comprise bicyclic sugars and a majority of the sugars in the other wing comprise a 2' -MOE. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise bicyclic sugars, and the majority of the sugars in the other wing comprise 2 '-MOE, and the majority of the sugars in the core comprise 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein a majority of the sugars in one wing comprise bicyclic sugars, and in the other wing, at least one sugar comprises a 2' -MOE and at least one sugar is a bicyclic sugar. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein a majority of the sugars in one wing comprise bicyclic sugars, and in the other wing, at least one sugar comprises a 2 '-MOE and at least one sugar is a bicyclic sugar, and a majority of the sugars in the core comprise a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise a 2 '-MOE, and in the other wing at least one sugar comprises a 2' -MOE and at least one sugar is a bicyclic sugar. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise a 2 ' -MOE, and in the other wing, at least one sugar comprises a 2 ' -MOE and at least one sugar is a bicyclic sugar, and the majority of the sugars in the core comprise a 2 ' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein a majority of the sugars in one wing comprise bicyclic sugars, and in the other wing, at least two of the sugars comprise a 2' -MOE and at least two of the sugars are bicyclic sugars. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise bicyclic sugars, and in the other wing, at least two of the sugars comprise a 2 '-MOE and at least two of the sugars are bicyclic sugars, and the majority of the sugars in the core comprise a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise a 2 '-MOE, and in the other wing, at least two of the sugars comprise a 2' -MOE and at least two of the sugars are bicyclic sugars. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise a 2 ' -MOE, and in the other wing, at least two of the sugars comprise a 2 ' -MOE and at least two of the sugars are bicyclic sugars, and the majority of the sugars in the core comprise a 2 ' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein each sugar in one wing is a bicyclic sugar and each sugar in the other wing comprises a 2' -MOE. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein each sugar in one wing is a bicyclic sugar and each sugar in the other wing comprises a 2 '-MOE, and the majority of the sugars in the core comprise a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein each sugar in one wing comprises a 2 '-MOE, each sugar in the other wing is a bicyclic sugar, and each sugar in the core comprises a 2' -deoxy.
In some embodiments, the bicyclic sugar is LNA, cEt, or BNA.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein one wing comprises 2 '-OMe and the other wing comprises 2' -F. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein one wing comprises 2 ' -OMe and the other wing comprises 2 ' -F, and the majority of the sugars in the core comprise a 2 ' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugar in one wing comprises 2 '-OMe and the majority of the sugar in the other wing is 2' -F. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugar in one wing comprises 2 ' -OMe, and the majority of the sugar in the other wing is 2 ' -F, and the majority of the sugar in the core comprises a 2 ' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise 2 ' -OMe, and in the other wing, at least one sugar is 2 ' -F and at least one sugar comprises 2 ' -OMe. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise 2 '-OMe, and in the other wing, at least one sugar is 2' -F and at least one sugar comprises 2 '-OMe, and the majority of the sugars in the core comprise a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing are 2 ' -F, and in the other wing, at least one sugar is 2 ' -F and at least one sugar comprises 2 ' -OMe. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing are 2 '-F, and in the other wing, at least one sugar is 2' -F and at least one sugar comprises 2 '-OMe, and the majority of the sugars in the core comprise a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise 2 ' -OMe, and in the other wing, at least two of the sugars are 2 ' -F and at least two of the sugars comprise 2 ' -OMe. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise 2 '-OMe, and in the other wing, at least two of the sugars are 2' -F and at least two of the sugars comprise 2 '-OMe, and the majority of the sugars in the core comprise a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing are 2 ' -F, and in the other wing, at least two of the sugars are 2 ' -F and at least two of the sugars comprise 2 ' -OMe. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing are 2 '-F, and in the other wing, at least two of the sugars are 2' -F and at least two of the sugars comprise 2 '-OMe, and the majority of the sugars in the core comprise a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein each sugar in one wing comprises 2 '-OMe and each sugar in the other wing comprises 2' -F. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein each sugar in one wing comprises 2 ' -OMe and each sugar in the other wing comprises 2 ' -F, and the majority of the sugars in the core comprise 2 ' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein each sugar in one wing comprises 2 ' -F, each sugar in the other wing comprises 2 ' -OMe, and each sugar in the core comprises a 2 ' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein one wing comprises 2 '-F and the other wing comprises 2' -MOE. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein one wing comprises 2 ' -F and the other wing comprises 2 ' -MOE, and the majority of the sugars in the core comprise 2 ' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise 2 '-F and the majority of the sugars in the other wing comprise 2' -MOE. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise 2 ' -F, and the majority of the sugars in the other wing comprise 2 ' -MOE, and the majority of the sugars in the core comprise 2 ' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise 2 ' -F, and in the other wing, at least one sugar comprises 2 ' -MOE and at least one sugar is 2 ' -F. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing are 2 '-F, and in the other wing, at least one sugar comprises a 2' -MOE and at least one sugar is 2 '-F, and the majority of the sugars in the core comprise a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise a 2 ' -MOE, and in the other wing, at least one sugar comprises a 2 ' -MOE and at least one sugar is a 2 ' -F. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise a 2 '-MOE, and in the other wing, at least one sugar comprises a 2' -MOE and at least one sugar is a 2 '-F, and the majority of the sugars in the core comprise a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise 2 ' -F, and in the other wing, at least two of the sugars comprise 2 ' -MOE and at least two of the sugars are 2 ' -F. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise 2 '-F, and in the other wing, at least two of the sugars comprise 2' -MOE and at least two of the sugars are 2 '-F, and the majority of the sugars in the core comprise 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise a 2 ' -MOE, and in the other wing, at least two of the sugars comprise a 2 ' -MOE and at least two of the sugars are 2 ' -F. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein the majority of the sugars in one wing comprise a 2 '-MOE, and in the other wing, at least two of the sugars comprise a 2' -MOE and at least two of the sugars are 2 '-F, and the majority of the sugars in the core comprise a 2' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein each sugar in one wing is 2 '-F and each sugar in the other wing comprises a 2' -MOE. In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein each sugar in one wing is 2 ' -F and each sugar in the other wing comprises a 2 ' -MOE, and the majority of the sugars in the core comprise a 2 ' -deoxy.
In some embodiments, the oligonucleotide comprises a wing-core-wing structure, wherein each sugar in one wing comprises a 2 ' -MOE, each sugar in the other wing is a 2 ' -F, and each sugar in the core comprises a 2 ' -deoxy.
In some embodiments, the C9orf72 oligonucleotide has a wing-core-wing structure and has an asymmetric form. In some embodiments of the C9orf72 oligonucleotide having an asymmetric form, one wing is different from the other wing. In some embodiments of C9orf72 oligonucleotides having an asymmetric form, one wing differs from the other in sugar modifications or pattern thereof, or backbone internucleotide linkages or pattern thereof, or backbone chiral centers or pattern thereof. In some embodiments of oligonucleotides having an asymmetric form, the core comprises 1 or more 2' -deoxy sugars. In some embodiments of oligonucleotides having an asymmetric form, the core comprises 5 or more consecutive 2' -deoxy sugars. In some embodiments of oligonucleotides having an asymmetric form, the core comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more consecutive 2' -deoxy sugars. Some non-limiting examples of C9orf72 oligonucleotides having asymmetric forms are shown herein. In some embodiments of the C9orf72 oligonucleotide having an asymmetric form, the first wing and the second wing independently have a 2' -modification pattern of sugars that is or comprises: F. FF, FFF, FFFF, fffffff, FMMMF, LMMMm, M, MM, mMm, mMMm, mmmmmmmmm, mmmmmmm, MMMMM, or any wing of any oligonucleotide described herein, wherein the 2 '-modification pattern of the first wing and the second wing is different, and wherein M ═ 2' -OMe; m ═ 2' -MOE; f is 2' -F; and L ═ LNA. In some embodiments of oligonucleotides having an asymmetric form, the first wing and the second wing independently have an internucleotide linkage pattern that is or comprises PS, PO, PS-PS, PS-PO, PO-PS, PO-PO, PO-PS, PS-PO-PS, PS-PO-PS, PS-PS, PS-Xn-PS, or any wing of any of the oligonucleotides described herein, wherein the internucleotide linkage pattern of the first wing and the second wing is different, and wherein PS is a phosphorothioate; PO ═ phosphodiester; xn is any neutral internucleotide linkage. In some embodiments of oligonucleotides having an asymmetric form, the first wing and the second wing independently have a stereochemical pattern of internucleotide linkages which is or comprises a PO, SR, Sp, Rp, Sp-PO, Rp-PO, PO-Sp, PO-Rp, PO-PO-PO, Sp-PO-PO, Rp-PO-PO-Rp, Rp-PO-Rp-PO-Rp, Rp-Rp-PO-PO-Rp, Sp-PO-PO-Sp, Sp-Sp-Sp-Sp, Sp-Sp-Sp, A stereochemical pattern of any internucleotide linkage of Sp-Xn-Sp, SR-PO-SR, SR-SR, SR-Xn-SR, or any wing of any of the oligonucleotides described herein, wherein the stereochemical pattern of the internucleotide linkage of the first wing and the second wing is different, and wherein SR is a stereorandom internucleotide linkage (e.g., achiral controlled); PO ═ phosphodiester (which lacks a chiral center); sp ═ internucleotide linkages in the Sp configuration; an internucleotide linkage in the Rp configuration; xn ═ neutral internucleotide linkages, which may independently be stereocontrolled (in either the Rp or Sp configuration) or stereorandom. In some embodiments of oligonucleotides having an asymmetric form, the first wing is a 5 'wing (the wing closer to the 5' end of the oligonucleotide) and the second wing is a 3 'wing (the wing closer to the 3' end of the oligonucleotide). In some embodiments of oligonucleotides having an asymmetric form, the first wing is a 3 'wing (the wing closer to the 3' end of the oligonucleotide) and the second wing is a 5 'wing (the wing closer to the 5' end of the oligonucleotide). In some embodiments, the first wing and the second wing have the same or different lengths.
In some embodiments, an oligonucleotide having an asymmetric structure (e.g., one wing that is chemically different from the other wing) has improved biological activity compared to an oligonucleotide having the same base sequence but a different structure (e.g., a symmetric structure in which the two wings have the same pattern of chemical modification; or another asymmetric structure). In some embodiments, the improved biological activity comprises an improved reduction in expression, activity, and/or level of a gene or gene product. In some embodiments, the improved biological activity is improved delivery to the nucleus. In some embodiments, the improved biological activity is improved delivery into the nucleus of a cell, and one wing in the oligonucleotide having an asymmetric structure comprises 2 '-F or two or more 2' -fs. In some embodiments, the improved biological activity is improved delivery into the nucleus of a cell, and one wing in the oligonucleotide having an asymmetric structure comprises a 2 '-MOE or two or more 2' -MOEs. In some embodiments, the improved biological activity is improved delivery into the nucleus of a cell, and one wing in the oligonucleotide having an asymmetric structure comprises a 2 '-OMe or two or more 2' -OMe. In some embodiments, the improved biological activity is improved delivery into the nucleus of a cell, and one wing in the oligonucleotide having an asymmetric structure comprises a bicyclic sugar or two or more bicyclic sugars.
In some embodiments, the core does not comprise a 2' -substitution, and each sugar unit is a native sugar unit present in the unmodified native DNA. In some embodiments, the core comprises one or more 2' -halo modifications. In some embodiments, the core comprises one or more 2' -F modifications. In some embodiments, no less than 70%, 80%, 90%, or 100% of the internucleotide linkages in the core are modified internucleotide linkages. In some embodiments, no less than 70%, 80%, or 90% of the internucleotide linkages in the core are independently modified internucleotide linkages having the Sp configuration, and the core further contains 1, 2, 3, 4, or 5 internucleotide linkages selected from the group consisting of modified internucleotide linkages having the Rp configuration and natural phosphate linkages. In some embodiments, the core further contains 1 or 2 internucleotide linkages selected from the group consisting of modified internucleotide linkages having the Rp configuration and natural phosphate linkages. In some embodiments, the core also contains 1 and no more than 1 internucleotide linkage selected from the group consisting of modified internucleotide linkages having the Rp configuration and natural phosphate linkages, and the remaining internucleotide linkages are independently modified internucleotide linkages having the Sp configuration. In some embodiments, the core also contains 2 and no more than 2 internucleotide linkages each independently selected from modified internucleotide linkages having the Rp configuration and natural phosphate linkages, and the remaining internucleotide linkages are independently modified internucleotide linkages having the Sp configuration. In some embodiments, the core also contains 1 and no more than 1 native phosphate linkage, and the remaining internucleotide linkages are independently modified internucleotide linkages having the Sp configuration. In some embodiments, the core also contains 2 and no more than 2 native phosphate linkages, and the remaining internucleotide linkages are independently modified internucleotide linkages having the Sp configuration. In some embodiments, the core also contains 1 and no more than 1 modified internucleotide linkage having the Rp configuration, and the remaining internucleotide linkages are independently modified internucleotide linkages having the Sp configuration. In some embodiments, the core also contains 2 and no more than 2 modified internucleotide linkages having the Rp configuration, and the remaining internucleotide linkages are independently modified internucleotide linkages having the Sp configuration. In some embodiments, two native phosphate linkages or two modified internucleotide linkages having an Rp configuration are spaced apart by two or more modified internucleotide linkages having an Sp configuration. In some embodiments, the modified internucleotide linkage is of formula I. In some embodiments, the modified internucleotide linkage is a phosphorothioate diester linkage.
The core and wings may have various lengths. In some embodiments, the core comprises no less than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases. In some embodiments, the flap comprises no less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobases. In some embodiments, the flap comprises no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobases. In some embodiments, for a wing-core-wing structure, the two wings are of the same length, e.g., have 5 nucleobases. In some embodiments, the two wings are of different lengths. In some embodiments, the core is no less than 40%, 45%, 50%, 60%, 70%, 80%, or 90% of the total oligonucleotide length as measured by the percentage of nucleoside units within the core. In some embodiments, the core is not less than 50% of the total oligonucleotide length.
In some embodiments, the present disclosure provides oligonucleotides comprising additional chemical moieties attached to the oligonucleotide moiety, optionally via a linker. In some embodiments, the present disclosure provides compositions comprising (RD) b-LM1-LM2-LM3The oligonucleotide of (a), wherein:
each RDIndependently a chemical moiety;
LM1、LM2and LM3Each of which is independently a covalent bond or an optionally substituted linear or branched divalent or multivalent radical selected from C1-30Aliphatic radical and C having 1 to 10 heteroatoms1-30A heteroaliphatic group wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-; and one or more carbon atoms are optionally and independently substituted with CyLReplacement;
each CyLIndependently is an optionally substituted tetravalent group selected from: c3-20Cycloaliphatic radical, C6-20An aromatic ring, a 5-to 20-membered heteroaromatic ring having 1-10 heteroatoms, and a 3-to 20-membered heterocyclic ring; and is
b is 1 to 1000.
LM1、LM2And LM3Each independently is a covalent bond, or is selected from C having 1-5 heteroatoms1-10Aliphatic radical and C1-10Divalent or polyvalent optionally substituted straight or branched chain groups of heteroaliphatic groups, wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-; and one or more carbon atoms are optionally and independently replaced by CyL.
In some embodiments, LM1Comprising one or more of-N (R') -and one or more of-C (O) -. In some embodiments, linker or LM1Is or compriseWherein n isLIs 1 to 8. In some embodiments, the linker is-LM1-LM2-LM3-isOr a salt form thereof, wherein nLIs 1 to 8. In some embodiments, the linker is-LM1-LM2-LM3-is
Or a salt form thereof, wherein:
nLis 1 to 8.
Each amino group is independently attached to a moiety; and is
The P atom is attached to the 5' -OH of the oligonucleotide.
In some embodiments, the moiety and linker, or (R)D)b-LM1-LM2-LMB-, is or compriseIn some embodiments, the moiety and linker, or (R)D)b-LM1-LM2-LMB-, is or compriseIn some embodiments, the moiety and linker, or (R)D)b-LM1-LM2-LMB-, is or compriseIn some embodiments, the moiety and linker, or (R)D)b-LM1-LM2-LMB-, is or compriseIn some embodiments, the moiety and linker, or (R)D)b-LM1-LM2-LMB-, is or compriseIn some embodiments, the moiety and linker, or (R)D)b-LM1-LM2-LMB-, is or compriseIn some embodiments, the moiety and linker, or (R)D)b-LM1-LM2-LM3-, is or compriseIn some embodiments, linker or LM1Is or compriseIn some embodiments, the moiety and linker, or (R)D)b-LM1-LM2-LM3-, is or comprises:in some embodiments, the moiety and linker, or (R)D)b-LM1-LM2-LMB-, is or comprises:
in some embodiments, nLIs 1 to 8. In some embodiments, nLIs 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, nLIs 1. In some embodiments, nLIs 2. In some embodiments, nLIs 3. In some embodiments, nLIs 4. In some embodiments, nLIs 5. In some embodiments, nLIs 6. In some embodiments, nLIs 7. In some embodiments, nLIs 8.
In some embodiments, LM2Is a covalent bond or an optionally substituted linear or branched divalent radical selected from C1-10Aliphatic radical and C having 1 to 5 heteroatoms1-10A heteroaliphatic group wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-; and one or more carbon atoms are optionally and independently replaced by CyL. In some embodiments, LM2Is a covalent bond or an optionally substituted linear or branched divalent radical selected from C1-10Aliphatic radical and C having 1 to 5 heteroatoms1-10A heteroaliphatic group wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N (R ') -, -C (O) S-, -C (O) O-, -P (O) (OR') -, -P (O) (SR ') -, OR-P (O) (R') -. In some embodiments, LM2Is a covalent bond or an optionally substituted linear or branched divalent C1-10An aliphatic group wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-, -O-, -S-, -N (R') -, or-C (O) -. In some embodiments, LM2is-NH- (CH)2)6-, wherein-NH-is bound to LM1。
In some embodiments, LM3is-P (O ') (OR') -, -P (O ') - (SR') -, -P (O ') - (R') -, -P (O ') - (NR') -, -P (S ') - (OR') -, -P (S ') - (SR') -, -P (S ') - (R') -, -P (S ') - (NR') -, -P (R ') -, -P (OR') -, -P (SR ') -, -P (NR') -, -P (OR ') - [ B (R')3]-, -OP (OR ') -, -OP (O)) - (SR') -, -OP (O)) - (R ') -, -OP (O)) - (NR') -, -OP (S)) - (OR ') -, -OP (S)) - (SR') -, -OP (S)) - (R ') -, -OP (S)) - (NR') -, -OP (R ') -, -OP (OR') -, -OP (SR ') -, -OP (NR') -, OR-OP (OR ') [ B (R') -)3]-. In some embodiments, LM3is-OP (O) (OR ') -OR-OP (O) (SR') -, wherein-O-is bound to LM2. In some embodiments, the P atom is attached to a sugar unit, a nucleobase unit or an internucleotide linkage. In some embodiments, the P atom is attached to the-OH group via the formation of a P-O bond. In some embodiments, the P atom is attached to the 5' -OH group via the formation of a P — O bond.
In some embodiments, LM1Is a covalent bond. In some embodiments, LM2Is a covalent bond. In some embodiments, LM3Is a covalent bond. In some embodiments, LM1Is L as described in this disclosureM2. In some embodiments, LM1Is L as described in this disclosureM3. In some embodiments, LM2Is L as described in this disclosureM1. In some embodiments, LM2Is L as described in this disclosureM3. In some embodiments, LM3Is L as described in this disclosureM1. In some embodiments, LM3Is L as described in this disclosureM2. In some embodiments, LMIs L as described in this disclosureM1. In some embodiments, LMIs L as described in this disclosureM2. In some embodiments, LMIs L as described in this disclosureM3. In some embodiments, LMIs LM1-LM2Wherein L isM1And LM2Each independently as described in the present disclosure. In some embodiments, LMIs LM1-LM3Wherein L isM1And LM3Each independently as described in the present disclosure. In some embodiments, LMIs LM2-LM3Wherein L isM2And LM3Each independently as described in the present disclosure. In some embodiments, LMIs LM1-LM2-LM3Wherein L isM1、LM2And LM3Each independently as described in the present disclosure.
In some embodiments, each RDIndependently a chemical moiety as described in this disclosure. In some embodiments, RDIs a targeting moiety. In some embodiments, RDIs or comprises a carbohydrate moiety. In some embodiments, RDIs or comprises a lipid moiety. In some embodiments, RDIs or comprises, for example, a ligand moiety for a cellular receptor such as a delta receptor, asialoglycoprotein receptor, etc. In some embodiments, the ligand moiety is or comprises an anisamide moiety, which may be a ligand moiety of a delta receptor. In some embodiments, the ligand moiety is or comprises a GalNAc moiety, which may be a ligand moiety of an asialoglycoprotein receptor. In some embodiments, RDSelected from optionally substituted phenyl,
Wherein n' is 0 or 1 and each of the other variables is independently as described in the disclosure. In some embodiments, RsIs F. In some embodiments, RsIs OMe. In some embodiments, RsIs OH. In some embodiments, RsIs NHAc. In some embodiments, RsIs NHCOCF3. In some embodiments, R' is H. In some embodiments, R is H. In some embodiments, R2sIs NHAc, and R5sIs OH. In some embodiments, R2sIs p-anisoyl, and R5sIs OH. In some embodiments, R2sIs NHAc, and R5sIs p-anisoyl. In some embodiments, R2sIs OH, and R5sIs p-anisoyl. In some embodiments, RDIs selected fromRDOther embodiments of (a) include other chemical moiety embodiments such as those described in example, example 2, etc.
In some embodiments, n' is 1. In some embodiments, n' is 0.
In some embodiments, n "is 1. In some embodiments, n "is 2.
In some embodiments, the disclosure provides a provided compound, e.g., an oligonucleotide of a provided composition, having a structure of formula O-I:
or a salt thereof, wherein:
REis a 5' terminal group;
each BA is independently an optionally substituted group selected from: c3-30Cycloaliphatic, C6-30Aryl, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon5-30Heteroaryl, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron, and silicon3-30Heterocyclyl, natural nucleobase moieties, and modified nucleobase moieties;
each Rsindependently-F, -Cl, -Br, -I, -CN, -N3、-NO、-NO2、-L-R′、-L-OR′、-L-SR′、-L-N(R′)2-O-L-OR ', -O-L-SR ' OR-O-L-N (R ')2；
s is 0 to 20;
Lsis-C (R)5s)2-or L;
each L is independently a covalent bond or is selected from C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron, and silicon1-30Aliphatic radical and C1-30A divalent optionally substituted straight or branched chain radical of a heteroaliphatic group, wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-; and one or more carbon atoms are optionally and independently substituted with CyLReplacement;
each CyLIndependent of each otherIs an optionally substituted tetravalent group selected from: c3-20Cycloaliphatic ring, C6-20An aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron, and silicon;
each ring a is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
each LPIndependently an internucleotide linkage;
z is 1 to 1000;
L3Eis L or-L-L-;
R3Eis-R ', -L-R ', -OR ' OR a solid support;
each R' is independently-R, -C (O) OR, OR-S (O)2R；
Each R is independently-H, or an optionally substituted group selected from: c1-30Aliphatic, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon1-30Heteroaliphatic group, C6-30Aryl radical, C6-30Arylaliphatic, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon6-30Aryl heteroaliphatics, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, or
Two R groups optionally and independently form a covalent bond together, or
Two or more R groups on the same atom optionally and independently form, with the atom, an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, other than the atom; or
Two or more R groups on two or more atoms optionally and independently form, with the intervening atoms, an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, excluding the intervening atoms.
In some embodiments, each LPIndependently have the structure of formula I:
or a salt form thereof, wherein:
PLis P (═ W), P or P → B (R')3；
W is O, S or Se;
R1is-L-R, halogen, -CN, -NO2、-Si(R′)3-OR ', -SR ', OR-N (R ')2；
X, Y and Z are each independently-O-, -S-, -N (-L-R)1) -, or L;
each R' is independently-R, -C (O) OR, OR-S (O)2R；
Each L is independently a covalent bond or is selected from C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron, and silicon1-30Aliphatic radical and C1-30A divalent optionally substituted straight or branched chain radical of a heteroaliphatic group, wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-; and one or more carbon atoms are optionally and independently substituted with CyLReplacement;
each R is independently-H, or an optionally substituted group selected from: c1-30Aliphatic, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon1-30Heteroaliphatic group, C6-30Aryl radical, C6-30Arylaliphatic, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon6-30Aryl heteroaliphatics, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, or
Two R groups optionally and independently form a covalent bond together, or
Two or more R groups on the same atom optionally and independently form, with the atom, an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, other than the atom; or
Two or more R groups on two or more atoms optionally and independently form, with the intervening atoms, an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, excluding the intervening atoms.
In some embodiments, each LPIndependently have the structure of formula I, and REis-C (R)5s)3、-L-pDB、-C(R5s)2OH、-L-R5sor-L-p5s-L-R5sOr a salt form thereof, wherein each variable is independently as described in the disclosure.
In some embodiments, each LPIndependently have the structure of formula I, and REis-C (R)5s)3、-L-pDB、-C(R5s)2OH、-L-R5sor-L-P5s-L-R5sOr a salt form thereof, wherein each variable is independently as described in the disclosure.
In some embodiments, REis-C (R)5s)3、-C(R5s)2OH or-L-R5s；
Each BA is independently an optionally substituted group selected from: c having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon5-30Heteroaryl, and C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron, and silicon3-30A heterocyclic group;
each ring a is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and is
Each LPIndependently have the structure of formula I, wherein each variable is independently as described in the present disclosure.
In some embodiments, REis-C (R)5s)3、-C(R5s)2OH or-L-R5s
Each BA is independently an optionally substituted C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon5-30Heteroaryl, wherein the heteroaryl comprises one or more heteroatoms selected from oxygen and nitrogen;
each ring a is independently an optionally substituted 5-to 10-membered monocyclic or bicyclic saturated ring having 0-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, wherein the ring comprises at least one oxygen atom; and is
Each LPIndependently have the structure of formula I, wherein each variable is independently as described in the present disclosure.
In some embodiments, REis-C (R)5s)3、-C(R5s)2OH or-L-R5s；
Each BA is independently an optionally substituted or protected nucleobase selected from adenine, cytosine, guanosine, thymine and uracil;
each ring a is independently an optionally substituted 5-to 7-membered monocyclic or bicyclic saturated ring having one or more oxygen atoms; and is
Each LPIndependently have the structure of formula I, wherein each variable is independently as described in the present disclosure.
In some embodiments, REIs a 5' terminal group as described herein. In some embodiments, REIs a 5' end group as described herein. In some embodiments, REis-C (R)5s)3、-L-PDB、-C(R5s)2OH、-L-R5sor-L-P5s-L-R5sOr a salt form thereof, wherein each variable is independently as described in the disclosure. In some embodiments, REis-CH2And (5) OH. In some embodiments, REis-CH2OP(O)(OR)2Or a salt form thereof, wherein each R is independently as described in the disclosure. In some embodiments, REis-CH2OP(O)(OH)2Or a salt form thereof. In some embodiments, REis-CH2Op (o) (or) (sr) or a salt form thereof, wherein each R is independently as described in the disclosure. In some embodiments, REis-CH2OP (O) (SH) (OH) or a salt form thereof. In some embodiments, REIs (E) -CH ═ CHP (O) (OR)2Or a salt form thereof, wherein each R is independently as described in the disclosure. In some embodiments, REIs (E) -CH ═ CHP (O) (OH)2。
In some embodiments, REis-CH2And (5) OH. In some embodiments, REis-CH2OP(O)(R)2Or a salt form thereof, wherein each R is independently as described in the disclosure. In some embodiments, REis-CH2OP(O)(OR)2Or a salt form thereof, wherein each R is independently as described in the disclosure. In some embodiments, REis-CH2OP(O)(OH)2Or a salt form thereof. In some embodiments, REis-CH2Op (o) (or) (sr) or a salt form thereof, wherein each R is independently as described in the disclosure. In some embodiments, REis-CH2OP (O) (SH) (OH) or a salt form thereof. In some embodiments, REIs (E) -CH ═ CHP (O) (OR)2Or a salt form thereof, wherein each R is independently as described in the disclosure. In some embodiments, REIs (E) -CH ═ CHP (O) (OH)2。
In some embodiments, REis-CH (R)5s) -OH, wherein R5sAs described in this disclosure. In some embodiments, REis-CH (R)5s)-OP(O)(R)2Or a salt form thereof, wherein each R5sAnd R is independently as described in the disclosure. In some embodiments, REis-CH (R)5s)-OP(O)(OR)2Or a salt form thereof, wherein each R5sAnd R is independently as described in the disclosure. In some embodiments, REis-CH (R)5s)-OP(O)(OH)2Or a salt form thereof. In some embodiments, REis-CH (R)5s) -op (o), (or) (sr) or a salt form thereof. In some embodiments, REis-CH (R)5s) -op (o) (oh) (sh) or a salt form thereof. In some embodiments, REIs- (R) -CH (R)5s) -OH, wherein R5sAs described in this disclosure. In some embodiments, REIs- (R) -CH (R)5s)-OP(O)(R)2Or a salt form thereof, wherein each R5s and R is independently as described in the disclosure. In some embodiments, REIs- (R) -CH (R)5s)-OP(O)(OR)2Or a salt form thereof, wherein each R5sAnd R is independently as described in the disclosure. In some embodiments, REIs- (R) -CH (R)5s)-OP(O)(OH)2Or a salt form thereof. In some embodiments, REIs- (R) -CH (R)5s) -op (o), (or) (sr) or a salt form thereof. In some embodiments, REIs- (R) -CH (R)5s) -op (o) (oh) (sh) or a salt form thereof. In some embodiments, REIs- (S) -CH (R)5s) -OH, wherein R5sAs described in this disclosure. In some embodiments, REIs- (S) -CH (R)5s)-OP(O)(R)2Or a salt form thereof, wherein each R5sAnd R is independently as described in the disclosure. In some embodiments, REIs- (S) -CH (R)5s)-OP(O)(OR)2Or a salt form thereof, wherein each R5sAnd R is independently as described in the disclosure. In some embodiments, REIs- (S) -CH (R)5s)-OP(O)(OH)2Or a salt form thereof. In some embodiments, REIs- (S) -CH (R)5s) -op (o), (or) (sr) or a salt form thereof. In some embodiments, REIs- (S) -CH (R)5s) -op (o) (oh) (sh) or a salt form thereof. In some embodiments, R5sIs optionally substituted C1、C2、C3Or C4An aliphatic group. In some embodiments, R5sIs C1、C2、C3Or C4Aliphatic or haloaliphatic. In some embodiments, R5sIs optionally substituted-CH3. In some embodiments, R5sis-CH3。
In some embodiments, BA is an optionally substituted group selected from: c3-30Cycloaliphatic radical, C6-30Aryl, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon5-30Heteroaryl, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon3-30Heterocyclyl, natural nucleobase moiety and modified nucleobase moiety. In some embodiments, BA is an optionally substituted group selected from: c having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon5-30Heteroaryl, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon3-30Heterocyclyl, natural nucleobase moieties, and modified nucleobase moieties. In some embodiments, BA is an optionally substituted group selected from: c having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon5-30Heteroaryl, natural nucleobase moieties and modified nucleobase moieties. In some embodiments, BA is optionally substituted C having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur5-30A heteroaryl group. In some embodiments, BA is an optionally substituted natural nucleobase and tautomers thereof. In some embodiments, BA is a protected natural nucleobase and tautomers thereof. Various nucleobase protecting groups for oligonucleotide synthesis are known and can be used in accordance with the present disclosure. In some embodiments, BA is an optional draw selected from adenine, cytosine, guanosine, thymine and uracilAnd (b) nucleobases of generations and tautomers thereof. In some embodiments, BA is an optionally protected nucleobase selected from adenine, cytosine, guanosine, thymine and uracil and tautomers thereof.
In some embodiments, BA is optionally substituted C3-30A cycloaliphatic radical. In some embodiments, BA is optionally substituted C6-30And (4) an aryl group. In some embodiments, BA is optionally substituted C3-30A heterocyclic group. In some embodiments, BA is optionally substituted C5-30A heteroaryl group. In some embodiments, BA is an optionally substituted natural base moiety. In some embodiments, BA is an optionally substituted modified base moiety. BA is an optionally substituted group selected from: c3-30Cycloaliphatic radical, C6-30Aryl radical, C3-30Heterocyclic radical and C5-30A heteroaryl group. In some embodiments, BA is an optionally substituted group selected from: c3-30Cycloaliphatic radical, C6-30Aryl radical, C3-30Heterocyclic group, C5-30Heteroaryl and natural nucleobase moieties.
In some embodiments, BA is attached via an aromatic ring. In some embodiments, BA is attached via a heteroatom. In some embodiments, BA is attached via a ring heteroatom of the aromatic ring. In some embodiments, BA is attached via a ring nitrogen atom of the aromatic ring.
In some embodiments, BA is a natural nucleobase moiety. In some embodiments, BA is an optionally substituted native nucleobase moiety. In some embodiments, BA is a substituted native nucleobase moiety. In some embodiments, BA is the native nucleobase A, T, C, U or G. In some embodiments, BA is an optionally substituted group selected from the natural nucleobases A, T, C, U and G.
In some embodiments, BA is an optionally substituted group prepared by reacting a compound selected from the group consisting ofOr a tautomer thereof by removal of-H. In some embodiments, BA is an optionally substituted group,said radicals being derived fromremoving-H to form. In some embodiments, BA is an optionally substituted group selected fromAnd tautomeric forms thereof. In some embodiments, BA is an optionally substituted group selected fromIn some embodiments, BA is an optionally substituted group prepared by reacting a compound selected from the group consisting ofAnd tautomers thereof are formed by removing-H. In some embodiments, BA is an optionally substituted group prepared by reacting a compound selected from the group consisting ofremoving-H to form. In some embodiments, BA is an optionally substituted group selected fromAnd tautomeric forms thereof. In some embodiments, BA is an optionally substituted group selected fromIn some embodiments, BA is optionally substitutedOr a tautomeric form thereof. In some casesIn the examples, BA is optionally substitutedIn some embodiments, BA is optionally substitutedOr a tautomeric form thereof. In some embodiments, BA is optionally substitutedIn some embodiments, BA is optionally substitutedOr a tautomeric form thereof. In some embodiments, BA is optionally substitutedIn some embodiments, BA is optionally substitutedOr a tautomeric form thereof. In some embodiments, BA is optionally substitutedIn some embodiments, BA is optionally substitutedOr a tautomeric form thereof. In some embodiments, BA is optionally substitutedIn some embodiments, BA isIn some embodiments, BA isIn some embodiments, BA isIn some embodiments, BA isIn some embodiments, BA isIn some embodiments, the BA of the 5' terminal nucleoside unit of a provided oligonucleotide (e.g., an oligonucleotide of formula VIII) is an optionally substituted group, by reaction with a ligand selected from the group consisting ofremoving-H to form. In some embodiments, the BA of the 5' terminal nucleoside unit is an optionally substituted group selected fromIn some embodiments, the BA of the 5' terminal nucleoside unit is an optionally substituted group obtained by reacting a compound selected from the group consisting ofremoving-H to form. In some embodiments, the BA of the 5' terminal nucleoside unit is an optionally substituted group selected fromIn some embodiments, the BA of the 5' terminal nucleoside unit is optionally substitutedIn some embodiments, the BA of the 5' terminal nucleoside unit is optionally substitutedIn some embodiments, the BA of the 5' terminal nucleoside unit is optionally substitutedIn some embodiments, the BA of the 5' terminal nucleoside unit is optionally substitutedIn some embodiments, the BA of the 5' terminal nucleoside unit is optionally substitutedIn some embodiments, the BA of the 5' terminal nucleoside unit isIn some embodiments, the BA of the 5' terminal nucleoside unit isIn some embodiments, the BA of the 5' terminal nucleoside unit isIn some embodiments, the BA of the 5' terminal nucleoside unit isIn some embodiments, the BA of the 5' terminal nucleoside unit is
In some embodiments, BA isIn some embodiments, BA isIn some embodiments, BA isIn some embodiments, BA isIn some embodiments, BA isIn some embodiments, BA isIn some embodiments, BA isIn some embodiments, BA isIn some embodiments, BA isIn some embodiments, BA isIn some embodiments, the protecting group is-Ac. In some embodiments, the protecting group is-Bz. In some embodiments, the protecting group is-iBu for the nucleobase.
In some embodiments, BA is an optionally substituted purine base residue. In some embodiments, BA is a protected purine base residue. In some embodiments, BA is an optionally substituted adenine residue. In some embodiments, BA is a protected adenine residue. In some embodiments, BA is an optionally substituted guanine residue. In some embodiments, BA is a protected guanine residue. In some embodiments, BA is an optionally substituted cytosine residue. In some embodiments, BA is a protected cytosine residue. In some embodiments, BA is an optionally substituted thymine residue. In some embodiments, BA is a protected thymine residue. In some embodiments, BA is an optionally substituted uracil residue. In some embodiments, BA is a protected uracil residue. In some embodiments, BA is an optionally substituted 5-methylcytosine residue. In some embodiments, BA is a protected 5-methylcytosine residue.
In some embodiments, BA is a protected base residue as used in oligonucleotide preparation. In some embodiments, the BAs are the base residues shown in US 201I/0294124, US 2015/0211006, US 2015/0197540, and WO2015/107425 (each of which is incorporated herein by reference).
In some embodiments, BA is the modified nucleobase shown in WO 2017/192679.
In some embodiments, each Rsindependently-H, halogen, -CN, -N as described in the disclosure3、-NO、-NO2、-Ls-R′、-Ls-Si(R)3、-Ls-OR′、-Ls-SR′、-Ls-N(R′)2、-O-Ls-R′、-O-Ls-Si(R)3、-O-Ls-OR′、-O-Ls-SR', or-O-Ls-N(R′)2. In some embodiments, Rsis-H. In some embodiments, RsIs not-H.
In some embodiments, RsIs R', wherein R is as described in the disclosure. In some embodiments, RsIs R, wherein R is as described in the disclosure. In some embodiments, RsIs optionally substituted C1-30A heteroaliphatic group. In some embodiments, RsContaining one or more silicon atoms. In some embodiments, Rsis-CH2Si(Ph)2CH3。
In some embodiments, Rsis-Ls-R'. In some embodiments, Rsis-Ls-R', wherein-LsIs an optionally substituted divalent C1-30A heteroaliphatic group. In some embodiments, Rsis-CH2Si(Ph)2CH3。
In some embodiments, Rsis-F. In thatIn some embodiments, Rsis-Cl. In some embodiments, Rsis-Br. In some embodiments, Rsis-I. In some embodiments, Rsis-CN. In some embodiments, Rsis-N3. In some embodiments, Rsis-NO. In some embodiments, Rsis-NO2. In some embodiments, Rsis-Ls-Si(R)3. In some embodiments, Rsis-Si (R)3. In some embodiments, Rsis-Ls-R'. In some embodiments, Rsis-R'. In some embodiments, Rsis-Ls-OR'. In some embodiments, Rsis-OR'. In some embodiments, Rsis-Ls-SR'. In some embodiments, Rsis-SR'. In some embodiments, Rsis-Ls-N(R′)2. In some embodiments, Rsis-N (R')2. In some embodiments, Rsis-O-Ls-R'. In some embodiments, Rsis-O-Ls-Si(R)3. In some embodiments, Rsis-O-Ls-OR'. In some embodiments, Rsis-O-Ls-SR'. In some embodiments, Rsis-O-Ls-N(R′)2. In some embodiments, RsIs a 2' -modification as described in the present disclosure. In some embodiments, Rsis-OR, wherein R is as described in the disclosure. In some embodiments, Rsis-OR, wherein R is optionally substituted C1-6An aliphatic group. In some embodiments, Rsis-OMe. In some embodiments, Rsis-OCH2CH2OMe。
In some embodiments, s is 0-20. In some embodiments, s is 1-20. In some embodiments, s is 1-5. In some embodiments, s is 1. In some embodiments, s is 2. In some embodiments, s is 3. In some embodiments, s is 4. In some embodiments, s is 5. In some embodiments, s is 6. In some embodiments, s is 7. In some embodiments, s is 8. In some embodiments, s is 9. In some embodiments, s is 10. In some embodiments, s is 11. In some embodiments, s is 12. In some embodiments, s is 13. In some embodiments, s is 14. In some embodiments, s is 15. In some embodiments, s is 16. In some embodiments, s is 17. In some embodiments, s is 18. In some embodiments, s is 19. In some embodiments, s is 20.
In some embodiments, LsIs L, wherein L is as described in the present disclosure. In some embodiments, L is an optionally substituted divalent methylene group.
As described herein, each L is independently a covalent bond, or is selected from the group consisting of C having 1-10 heteroatoms independently selected from the group consisting of oxygen, nitrogen, sulfur, phosphorus, boron, and silicon1-30Aliphatic radical and C1-30A divalent optionally substituted straight or branched chain radical of a heteroaliphatic group, wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-; and one or more carbon atoms are optionally and independently substituted with CyLAnd (6) replacing.
In some embodiments, L is a covalent bond or an optionally substituted linear or branched divalent group selected from C1-30Aliphatic radical and C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon1-30Heteroaliphatic radicals in which one or more methylene units are presentOptionally and independently replaced by an optionally substituted group selected from: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-, and one or more carbon atoms optionally and independently through CyLAnd (6) replacing. In some embodiments, L is a covalent bond or an optionally substituted linear or branched divalent C1-30An aliphatic group wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-, and one or more carbon atoms optionally and independently through CyLAnd (6) replacing. In some embodiments, L is a covalent bond, or is a divalent optionally substituted straight or branched C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon1-30An aliphatic group wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-, and one or more carbon atoms are optionally and independently CyLAnd (6) replacing. In some embodiments, L is a covalent bond or an optionally substituted linear or branched divalent group selected from C1-30Aliphatic radical and C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon1-30A heteroaliphatic group wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N (R') -, -C (O) S-or-C (O) O-, and one or more carbon atoms optionally and independently via CyLAnd (6) replacing. In some embodiments, L is a covalent bond or an optionally substituted linear or branched divalent group selected from C1-10Aliphatic radical and C having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon1-10A heteroaliphatic group wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from: c1-6Alkylene radical, C1-6Alkenylene, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′) -, -C (O) S-and-C (O) O-, and one or more carbon atoms optionally and independently passing through CyLAnd (6) replacing. In some embodiments, L is a covalent bond or an optionally substituted linear or branched divalent group selected from C1-10Aliphatic radical and C having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon1-10A heteroaliphatic group wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from: -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N (R') -, -C (O) S-and-C (O) O-.
In some embodiments, L is a covalent bond. In some embodiments, L is optionally substituted divalent C1-30An aliphatic group. In some embodiments, L is an optionally substituted divalent C having 1-10 heteroatoms independently selected from boron, oxygen, nitrogen, sulfur, phosphorus, and silicon1-30A heteroaliphatic group.
In some embodiments, the aliphatic moiety (e.g., aliphatic moiety of L, R, etc.) is monovalent or divalent or multivalent, and (prior to any optional substitution) may contain any number of carbon atoms within its range, e.g., C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11、C12、C13、C14、C15、C16、C17、C18、C19、C20、C21、C22、C23、C24、C25、C26、C27、C28、C29、C30And the like. In some embodiments, the heteroaliphatic moiety (e.g., L, R, etc.) is monovalent or divalent or polyvalent, and can contain any number of carbon atoms within its range (prior to any optional substitution), e.g., C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11、C12、C13、C14、C15、C16、C17、C18、C19、C20、C21、C22、C23、C24、C25、C26、C27、C28、C29、C30And the like.
In some embodiments, one or more methylene units are optionally and independently substituted by-O-, -S-, -N (R') -, -C (O) -, -S (O) -)2-, -P (O) (OR ') -, -P (O) (SR') -, -P (S) (OR ') -, OR-P (S) (OR') -. In some embodiments, the methylene unit is replaced with-O-. In some embodiments, the methylene unit is replaced with-S-. In some embodiments, the methylene unit is replaced with-N (R') -. In some embodiments, the methylene unit is replaced with-c (o) -. In some embodiments, the methylene unit is replaced with-s (o) -. In some embodiments, the methylene unit is substituted with-S (O)2-replacing. In some embodiments, the methylene unit is replaced by-p (o) (OR') -o. In some embodiments, the methylene unit is replaced by-p (o) (SR') -o. In some embodiments, the methylene unit is replaced by-p (o) (R') -o. In some embodiments, the methylene unit is replaced by-p (o) (NR') -. In some embodiments, the methylene unit is replaced by-p(s) (OR') -l. In some embodiments, the methylene unit is replaced by-p-(s), (SR') -l. In some embodiments, the methylene unit is replaced by-p(s) (R') -l. In some embodiments, the methylene unit is replaced by-p(s) (NR') -. In some embodiments, the methylene unit is replaced with-P (R') -. In some embodiments, the methylene unit is replaced with-P (OR') -. In some embodiments, the methylene unit is replaced with-P (SR') -. In some embodiments, the methylene unit is replaced by-P (NR') -. In some embodiments, the methylene unit is substituted with-P (OR ') [ B (R')3]-replacing. In some embodiments, one or more methylene units are optionally and independently substituted by-O-, -S-, -N (R') -, -C (O) -, -S (O) -)2-, -P (O) (OR ') -, -P (O) (SR') -, -P (S) (OR ') -, OR-P (S) (OR') -. In some casesIn the examples, the methylene units are substituted with-OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-substitutions, each of which may independently be an internucleotide linkage.
In some embodiments, L is-CH, e.g., when linked to R2-. In some embodiments, L-C (R)2-, wherein at least one R is not hydrogen. In some embodiments, L is-CHR-. In some embodiments, R is hydrogen. In some embodiments, L is-CHR-, wherein R is not hydrogen. In some embodiments, the C of-CHR-is chiral. In some embodiments, L is- (R) -CHR-, wherein the C of-CHR-is chiral. In some embodiments, L is- (S) -CHR-, wherein the C of-CHR-is chiral. In some embodiments, R is optionally substituted C1-6Aliphatic. In some embodiments, R is optionally substituted C1-6An alkyl group. In some embodiments, R is optionally substituted C1-5Aliphatic. In some embodiments, R is optionally substituted C1-5An alkyl group. In some embodiments, R is optionally substituted C1-4Aliphatic. In some embodiments, R is optionally substituted C1-4An alkyl group. In some embodiments, R is optionally substituted C1-3Aliphatic. In some embodiments, R is optionally substituted C1-3An alkyl group. In some embodiments, R is optionally substituted C2An aliphatic group. In some embodiments, R is optionally substituted methyl. In some embodiments, R is C1-6An aliphatic group. In some embodiments, R is C1-6An alkyl group. In some embodiments, R is C1-5An aliphatic group. In some embodiments, R is C1-5An alkyl group. In some embodiments, R is C1-4An aliphatic group. In some embodiments, R is C1-4An alkyl group. In some embodiments, R is C1-3An aliphatic group. In some embodiments, R is C1-3An alkyl group. In some embodiments, R is C2An aliphatic group. In some embodiments, R is methyl. In some embodiments, R is C1-6Marinated fatsA group. In some embodiments, R is C1-6A haloalkyl group. In some embodiments, R is C1-5A haloaliphatic group. In some embodiments, R is C1-5A haloalkyl group. In some embodiments, R is C1-4A haloaliphatic group. In some embodiments, R is C1-4A haloalkyl group. In some embodiments, R is C1-3A haloaliphatic group. In some embodiments, R is C1-3A haloalkyl group. In some embodiments, R is C2A haloaliphatic group. In some embodiments, R is methyl substituted with one or more halogens. In some embodiments, R is-CF3. In some embodiments, L is optionally substituted-CH ═ CH-. In some embodiments, L is optionally substituted (E) -CH ═ CH-. In some embodiments, L is optionally substituted (Z) -CH ═ CH-. In some embodiments, L is-C ≡ C-.
In some embodiments, L comprises at least one phosphorus atom. In some embodiments, at least one methylene unit of L is replaced by: -P (O ') - (OR') -, -P (O ') - (SR') -, -P (O ') - (R') -, -P (O ') - (NR') -, -P (S ') - (OR') -, -P (S ') - (SR') -, -P (S ') - (R') -, -P (S ') - (NR') -, -P (R ') -, -P (OR') -, -P (SR ') -, -P (NR') -, -P (OR ') - [ B (R')3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-。
In some embodiments, CyLIs an optionally substituted tetravalent group selected from: c3-20Cycloaliphatic radical, C6-20An aromatic ring, a 5-to 20-membered heteroaromatic ring having 1-10 heteroatoms independently selected from the group consisting of oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-to 20-membered heterocyclic ring having 1-10 heteroatoms independently selected from the group consisting of oxygen, nitrogen, sulfur, phosphorus, boron, and silicon.
In some embodiments, CyLIs monocyclic. In some embodiments, CyLIs bicyclic. In some embodiments, CyLIs polycyclic.
In some embodiments, CyLIs saturated. In some embodiments, CyLIs partially unsaturated. In some embodiments, CyLIs aromatic. In some embodiments, CyLIs or comprises a saturated cyclic moiety. In some embodiments, CyLIs or contains a partially unsaturated cyclic moiety. In some embodiments, CyLIs or contains an aromatic ring moiety.
In some embodiments, CyLIs optionally substituted C as described in the disclosure3-20Cycloaliphatic rings (e.g., those described for R but tetravalent). In some embodiments, the ring is optionally substituted saturated C3-20A cycloaliphatic ring. In some embodiments, the ring is an optionally substituted partially unsaturated C3-20A cycloaliphatic ring. The cycloaliphatic ring can have various sizes as described in this disclosure. In some embodiments, the loop is 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered. In some embodiments, the ring is 3-membered. In some embodiments, the ring is 4-membered. In some embodiments, the ring is 5-membered. In some embodiments, the ring is 6-membered. In some embodiments, the ring is 7-membered. In some embodiments, the ring is 8-membered. In some embodiments, the ring is 9-membered. In some embodiments, the ring is 10-membered. In some embodiments, the ring is an optionally substituted cyclopropyl moiety. In some embodiments, the ring is an optionally substituted cyclobutyl moiety. In some embodiments, the ring is an optionally substituted cyclopentyl moiety. In some embodiments, the ring is an optionally substituted cyclohexyl moiety. In some embodiments, the ring is an optionally substituted cycloheptyl moiety. In some embodiments, the ring is an optionally substituted cyclooctyl moiety. In some embodiments, the cycloaliphatic ring is a cycloalkyl ring. In some embodiments, the cycloaliphatic ring is monocyclic. In some embodiments, the cycloaliphatic ring is bicyclic. In some embodiments, the cycloaliphatic ring is polycyclic. In some embodiments, the ring is a cycloaliphatic moiety having a higher valence as described for R in this disclosure.
In some embodiments, CyLIs an optionally substituted 6-to 20-membered aromatic ring. In some embodiments, the ringIs an optionally substituted tetravalent phenyl moiety. In some embodiments, the ring is a tetravalent phenyl moiety. In some embodiments, the ring is an optionally substituted naphthalene moiety. The rings may have different sizes as described in this disclosure. In some embodiments, the aryl ring is 6 membered. In some embodiments, the aryl ring is 10 membered. In some embodiments, the aryl ring is 14 membered. In some embodiments, the aryl ring is monocyclic. In some embodiments, the aryl ring is bicyclic. In some embodiments, the aryl ring is polycyclic. In some embodiments, the ring is an aryl moiety with a higher valence as described for R in this disclosure.
In some embodiments, CyLIs an optionally substituted 5-to 20-membered heteroaromatic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, CyLIs an optionally substituted 5-to 20-membered heteroaromatic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroaryl rings, as described in this disclosure, may be of various sizes and contain various numbers and/or types of heteroatoms. In some embodiments, the heteroaryl ring contains no more than one heteroatom. In some embodiments, the heteroaryl ring contains more than one heteroatom. In some embodiments, the heteroaryl ring contains no more than one type of heteroatom. In some embodiments, the heteroaryl ring contains more than one type of heteroatom. In some embodiments, the heteroaryl ring is 5-membered. In some embodiments, the heteroaryl ring is 6-membered. In some embodiments, the heteroaryl ring is 8-membered. In some embodiments, the heteroaryl ring is 9-membered. In some embodiments, the heteroaryl ring is 10-membered. In some embodiments, the heteroaryl ring is monocyclic. In some embodiments, the heteroaryl ring is bicyclic. In some embodiments, the heteroaryl ring is polycyclic. In some embodiments, the heteroaryl ring is a nucleobase moiety, e.g., A, T, C, G, U, and the like. In some embodiments, the ring is a heteroaryl moiety with a higher valence as described for R in the disclosure.
In some embodiments, CyLIs a compound having 1-10 substituents independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon3-to 20-membered heterocyclic ring of the heteroatom(s). In some embodiments, CyLIs a 3-to 20-membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, the heterocyclyl ring is saturated. In some embodiments, the heterocyclyl ring is partially unsaturated. The heterocyclyl ring can have various sizes as described in this disclosure. In some embodiments, the loop is 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered. In some embodiments, the ring is 3-membered. In some embodiments, the ring is 4-membered. In some embodiments, the ring is 5-membered. In some embodiments, the ring is 6-membered. In some embodiments, the ring is 7-membered. In some embodiments, the ring is 8-membered. In some embodiments, the ring is 9-membered. In some embodiments, the ring is 10-membered. Heterocyclyl rings may contain various numbers and/or types of heteroatoms. In some embodiments, the heterocyclyl ring contains no more than one heteroatom. In some embodiments, the heterocyclyl ring contains more than one heteroatom. In some embodiments, the heterocyclyl ring contains no more than one type of heteroatom. In some embodiments, the heterocyclyl ring contains more than one type of heteroatom. In some embodiments, the heterocyclyl ring is monocyclic. In some embodiments, the heterocyclyl ring is bicyclic. In some embodiments, the heterocyclyl ring is polycyclic. In some embodiments, the ring is a heterocyclyl moiety with a higher valence as described for R in this disclosure.
As one of ordinary skill in the art will readily appreciate, many suitable ring moieties are broadly described in the present disclosure and can be used in accordance with the present disclosure, such as those described for R (which can have a higher Cy)L)。
In some embodiments, CyLIs a sugar moiety in nucleic acids. In some embodiments, CyLIs an optionally substituted furanose moiety. In some embodiments, CyLIs a pyranose moiety. In some embodiments, CyLIs an optionally substituted furanose moiety present in the DNA. In some embodiments, CyLIs an optionally substituted furanose moiety present in the RNA. In some embodiments of the present invention, the,CyLis an optionally substituted 2' -deoxyribofuranose moiety. In some embodiments, CyLIs an optionally substituted ribofuranose moiety. In some embodiments, the substitution provides a sugar modification as described in the present disclosure. In some embodiments, the optionally substituted 2 '-deoxyribofuranose moiety and/or the optionally substituted ribofuranose moiety comprises a substitution at the 2' position. In some embodiments, the 2 'position is a 2' -modification as described in the present disclosure. In some embodiments, the 2' -modification is-F. In some embodiments, the 2' -modification is-OR, wherein R is as described in the disclosure. In some embodiments, R is not hydrogen. In some embodiments, CyLIs a modified sugar moiety, such as in LNA. In some embodiments, CyLIs a modified sugar moiety, such as in ENA. In some embodiments, CyLIs the terminal sugar portion of the oligonucleotide that links the internucleotide linkage to the nucleobase. In some embodiments, CyLIs the terminal sugar moiety of the oligonucleotide, e.g., when the terminal is attached to a solid support, optionally via a linker. In some embodiments, CyLIs a sugar moiety linking two internucleotide linkages to a nucleobase. Exemplary sugars and sugar moieties are broadly described in this disclosure.
In some embodiments, CyLIs a nucleobase moiety. In some embodiments, the nucleobase is a natural nucleobase, such as A, T, C, G, U and the like. In some embodiments, the nucleobase is a modified nucleobase. In some embodiments, CyLIs an optionally substituted nucleobase moiety selected from A, T, C, G, U and 5 mC. Exemplary nucleobases and nucleobase moieties are broadly described in this disclosure.
In some embodiments, two CyLThe moieties being bound to each other, one of CyLIs a sugar moiety and the other is a nucleobase moiety. In some embodiments, such sugar moieties and nucleobase moieties form a nucleoside moiety. In some embodiments, the nucleoside moiety is native. In some embodiments, the nucleoside moiety is modified. In some embodiments, CyLIs an optionally substituted natural nucleoside moiety selected from: adenosine (I)5-methyluridine, cytidine, guanosine, uridine, 5-methylcytidine, 2 ' -deoxyadenosine, thymidine, 2 ' -deoxycytidine, 2 ' -deoxyguanosine, 2 ' -deoxyuridine and 5-methyl-2 ' -deoxycytidine. Exemplary nucleosides and nucleoside moieties are broadly described in the present disclosure.
In some embodiments, for example at LsIn (1), CyLIs an optionally substituted nucleoside moiety bonded to an internucleotide linkage, such as-OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, -OP (OR ') [ B (R')3]O-, etc., which may form an optionally substituted nucleotide unit. Exemplary nucleotide and nucleoside moieties are broadly described in this disclosure.
In some embodiments, each ring a is independently an optionally substituted 3-20 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, ring a is an optionally substituted ring, as described in the present disclosure. In some embodiments, the ring isIn some embodiments, the ring isIn some embodiments, ring a is or comprises a ring of a sugar moiety. In some embodiments, ring a is or comprises a ring of a modified sugar moiety.
In some embodiments, the saccharide unit has the structureWherein each variable is independently as described in the present disclosure. In some embodiments, the nucleoside unit has the structureWherein each variable is independently as described in the present disclosure. In some embodiments, a nucleotide unit (e.g., Nu)M、NuOEtc.) have a structureWherein each variable is independently as described in the present disclosure. In some embodiments, for NuO，LPIs a natural phosphate linkage, and Lsis-C (R) as described in this disclosure5s)2-。
In some embodiments, R1s、R2s、R3s、R4sAnd R5sEach independently is RsWherein R issAs described in this disclosure.
In some embodiments, R1sIs RsWherein R issAs described in this disclosure. In some embodiments, R1sAt the 1 'position (BA at the 1' position). In some embodiments, R1sis-H. In some embodiments, R1sis-F. In some embodiments, R1sis-Cl. In some embodiments, R1sis-Br. In some embodiments, R1sis-I. In some embodiments, R1sis-CN. In some embodiments, R1sis-N3. In some embodiments, R1sis-NO. In some embodiments, R1sis-NO2. In some embodiments, R1sis-L-R'. In some embodiments, R1sis-R'. In some embodiments, R1sis-L-OR'. In some embodiments, R1sis-OR'. In some embodiments, R1sis-L-SR'. In some embodiments, R1sis-SR'. In some embodiments, R1sIs L-L-N (R')2. In some embodiments, R1sis-N (R')2. In some embodiments, R1sis-OR ', wherein R' is optionally substituted C1-6An aliphatic group. In some embodiments, R1sis-OR ', wherein R' is optionally substituted C1-6An alkyl group. In some embodiments, R1sis-OMe. In some embodiments, R1sis-MOE. In some embodiments, R1sIs hydrogen. In some embodiments, R at one 1' positionsIs hydrogen and R in the other 1' positionsIs not hydrogen, as described herein. In some embodiments, R at two 1' positionssIs hydrogen. In some embodiments, R at one 1' positionsIs hydrogen and the other 1' position is linked to an internucleotide linkage. In some embodiments, R1sis-F. In some embodiments, R1sis-Cl. In some embodiments, R1sis-Br. In some embodiments, R1sis-I. In some embodiments, R1sis-CN. In some embodiments, R1sis-N3. In some embodiments, R1sis-NO. In some embodiments, R1sis-NO2. In some embodiments, R1sis-L-R'. In some embodiments, R1sis-R'. In some embodiments, R1sis-L-OR'. In some embodiments, R1sis-OR'. In some embodiments, R1sis-L-SR'. In some embodiments, R1sis-SR'. In some embodiments, R1sis-L-N (R')2. In some embodiments, R1sis-N (R')2. In some embodiments, R1sis-OR ', wherein R' is optionally substituted C1-6An aliphatic group. In some embodiments, R1sis-OR ', wherein R' is optionally substituted C1-6An alkyl group. In some embodiments, R1sis-OH. In some embodiments, R1sis-OMe. In some embodiments, R1sis-MOE. In some embodiments, R1sIs hydrogen. In some embodiments, one R1sAt the 1' position is hydrogen, and the other R1sAt the other 1' position is other than hydrogen, as described herein. In some embodiments, R at two 1' positions1sIs hydrogen. In some embodiments, R1sis-O-Ls-OR'. In some embodiments, R1sis-O-Ls-OR', wherein LsIs optionally substituted C1-6Alkylene, and R' is optionally substituted C1-6An aliphatic group. In some embodiments, R1sis-O- (optionally substituted C)1-6Alkylene) -OR'. In some embodiments, R1sis-O- (optionally substituted C)1-6Alkylene) -OR ', wherein R' is optionally substituted C1-6An alkyl group. In some embodiments, R1sis-OCH2CH2OMe。
In some embodiments, R2sIs RsWherein R issAs described in this disclosure. In some embodiments, if there are two R at the 2' position2sThen an R2sis-H and the other is not-H. In some embodiments, R2sAt the 2 'position (BA at the 1' position). In some embodiments, R2sis-H. In some embodiments, R2sis-F. In some embodiments, R2sis-Cl. In some embodiments, R2sis-Br. In some embodiments, R2sis-I. In some embodiments, R2sis-CN. In some embodiments, R2sis-N3. In some embodiments, R2sis-NO. In some embodiments, R2sis-NO2. In some embodiments, R2sis-L-R'. In some embodiments, R2sis-R'. In some embodiments, R2sis-L-OR'. In some embodiments, R2sis-OR'. In some embodiments, R2sis-L-SR'. In some embodiments, R2sis-SR'. In some embodiments, R2sIs L-L-N (R')2. In some embodiments, R2sis-N (R')2. In some embodiments, R2sis-OR ', wherein R' is optionally substituted C1-6An aliphatic group. In some embodiments, R2sis-OR ', wherein R' is optionally substituted C1-6An alkyl group. In some embodiments, R2sis-OMe. In some embodiments, R2sis-MOE. In some embodiments, R2sIs hydrogen. In some embodiments, at one 2' positionRsIs hydrogen and R in the other 2' positionsIs not hydrogen, as described herein. In some embodiments, R at two 2' positionssIs hydrogen. In some embodiments, R at one 2' positionsIs hydrogen and the other 2' position is linked to an internucleotide linkage. In some embodiments, R2sis-F. In some embodiments, R2sis-Cl. In some embodiments, R2sis-Br. In some embodiments, R2sis-I. In some embodiments, R2sis-CN. In some embodiments, R2sis-N3. In some embodiments, R2sis-NO. In some embodiments, R2sis-NO2. In some embodiments, R2sis-L-R'. In some embodiments, R2sis-R'. In some embodiments, R2sis-L-OR'. In some embodiments, R2sis-OR'. In some embodiments, R2sis-L-SR'. In some embodiments, R2sis-SR'. In some embodiments, R2sis-L-N (R')2. In some embodiments, R2sis-N (R')2. In some embodiments, R2sis-OR ', wherein R' is optionally substituted C1-6An aliphatic group. In some embodiments, R2sis-OR ', wherein R' is optionally substituted C1-6An alkyl group. In some embodiments, R2sis-OH. In some embodiments, R2sis-OMe. In some embodiments, R2sis-MOE. In some embodiments, R2sIs hydrogen. In some embodiments, one R2sAt the 2' position is hydrogen and the other R2sAt the other 2' position is other than hydrogen, as described herein. In some embodiments, R at two 2' positions2sIs hydrogen. In some embodiments, R2sis-O-Ls-OR'. In some embodiments, R2sis-O-Ls-OR', wherein LsIs optionally substituted C1-6Alkylene, and R' is optionally substituted C1-6An aliphatic group. In some embodiments, R2sis-O- (optionally substituted C)1-6Alkylene) -OR'. In some embodiments, R2sis-O- (optionally substituted C)1-6Alkylene) -OR ', wherein R' is optionally substituted C1-6An alkyl group. In some embodiments, R2sis-OCH2CH2OMe。
In some embodiments, R3sIs RsWherein R issAs described in this disclosure. In some embodiments, R3sAt the 3 'position (BA at the 1' position). In some embodiments, R3sis-H. In some embodiments, R3sis-F. In some embodiments, R3sis-Cl. In some embodiments, R3sis-Br. In some embodiments, R3sis-I. In some embodiments, R3sis-CN. In some embodiments, R3sis-N3. In some embodiments, R3sis-NO. In some embodiments, R3sis-NO2. In some embodiments, R3sis-L-R'. In some embodiments, R3sis-R'. In some embodiments, R3sis-L-OR'. In some embodiments, R3sis-OR'. In some embodiments, R3sis-L-SR'. In some embodiments, R3sis-SR'. In some embodiments, R3sis-L-N (R')2. In some embodiments, R3sis-N (R')2. In some embodiments, R3sis-OR ', wherein R' is optionally substituted C1-6An aliphatic group. In some embodiments, R3sis-OR ', wherein R' is optionally substituted C1-6An alkyl group. In some embodiments, R3sis-OMe. In some embodiments, R3sis-MOE. In some embodiments, R3sIs hydrogen. In some embodiments, R at one 3' positionsIs hydrogen and R in the other 3' positionsIs not hydrogen, as described herein. In some embodiments, R at two 3' positionssIs hydrogen. In some embodiments, R at one 3' positionsIs hydrogen and the other 3' position is linked to an internucleotide linkage. In some casesIn the examples, R3sis-F. In some embodiments, R3sis-Cl. In some embodiments, R3sis-Br. In some embodiments, R3sis-I. In some embodiments, R3sis-CN. In some embodiments, R3sis-N3. In some embodiments, R3sis-NO. In some embodiments, R3sis-NO2. In some embodiments, R3sis-L-R'. In some embodiments, R3sis-R'. In some embodiments, R3sis-L-OR'. In some embodiments, R3sis-OR'. In some embodiments, R3sis-L-SR'. In some embodiments, R3sis-SR'. In some embodiments, R3sIs L-L-N (R')2. In some embodiments, R3sis-N (R')2. In some embodiments, R3sis-OR ', wherein R' is optionally substituted C1-6An aliphatic group. In some embodiments, R3sis-OR ', wherein R' is optionally substituted C1-6An alkyl group. In some embodiments, R3sis-OH. In some embodiments, R3sis-OMe. In some embodiments, R3sis-MOE. In some embodiments, R3sIs hydrogen.
In some embodiments, R4sIs RsWherein R issAs described in this disclosure. In some embodiments, R4sAt the 4 'position (BA at the 1' position). In some embodiments, R4sis-H. In some embodiments, R4sis-F. In some embodiments, R4sis-Cl. In some embodiments, R4sis-Br. In some embodiments, R4sis-I. In some embodiments, R4sis-CN. In some embodiments, R4sis-N3. In some embodiments, R4sis-NO. In some embodiments, R4sis-NO2. In some embodiments, R4sis-L-R'. In some embodiments, R4sis-R'. In some embodiments, R4sis-L-OR'. In some embodimentsIn, R4sis-OR'. In some embodiments, R4sis-L-SR'. In some embodiments, R4sis-SR'. In some embodiments, R4sis-L-N (R')2. In some embodiments, R4sis-N (R')2. In some embodiments, R4sis-OR ', wherein R' is optionally substituted C1-6An aliphatic group. In some embodiments, R4sis-OR ', wherein R' is optionally substituted C1-6An alkyl group. In some embodiments, R4sis-OMe. In some embodiments, R4sis-MOE. In some embodiments, R4sIs hydrogen. In some embodiments, R at one 4' positionsIs hydrogen and R in the other 4' positionsIs not hydrogen, as described herein. In some embodiments, R at two 4' positionssIs hydrogen. In some embodiments, R at one 4' positionsIs hydrogen and the other 4' position is linked to an internucleotide linkage. In some embodiments, R4sis-F. In some embodiments, R4sis-Cl. In some embodiments, R4sis-Br. In some embodiments, R4sis-I. In some embodiments, R4sis-CN. In some embodiments, R4sis-N3. In some embodiments, R4sis-NO. In some embodiments, R4sis-NO2. In some embodiments, R4sis-L-R'. In some embodiments, R4sis-R'. In some embodiments, R4sis-L-OR'. In some embodiments, R4sis-OR'. In some embodiments, R4sis-L-SR'. In some embodiments, R4sis-SR'. In some embodiments, R4sIs L-L-N (R')2. In some embodiments, R4sis-N (R')2. In some embodiments, R4sis-OR ', wherein R' is optionally substituted C1-6An aliphatic group. In some embodiments, R4sis-OR ', wherein R' is optionally substituted C1-6An alkyl group. In some embodiments, R4sis-OH. In thatIn some embodiments, R4sis-OMe. In some embodiments, R4sis-MOE. In some embodiments, R4sIs hydrogen.
In some embodiments, R5sIs RsWherein R issAs described in this disclosure. In some embodiments, R5sIs R', wherein R is as described in the disclosure. In some embodiments, R5sis-H. In some embodiments, two or more R5sAre attached to the same carbon atom and at least one is not-H. In some embodiments, R5sIs not-H. In some embodiments, R5sis-F. In some embodiments, R5sis-Cl. In some embodiments, R5sis-Br. In some embodiments, R5sis-I. In some embodiments, R5sis-CN. In some embodiments, R5sis-N3. In some embodiments, R5sis-NO. In some embodiments, R5sis-NO2. In some embodiments, R5sis-L-R'. In some embodiments, R5sis-R'. In some embodiments, R5sis-L-OR'. In some embodiments, R5sis-OR'. In some embodiments, R5sis-L-SR'. In some embodiments, R5sis-SR'. In some embodiments, R5sIs L-L-N (R')2. In some embodiments, R5sis-N (R')2. In some embodiments, R5sis-OR ', wherein R' is optionally substituted C1-6Aliphatic. In some embodiments, R5sis-OR ', wherein R' is optionally substituted C1-6An alkyl group. In some embodiments, R5sis-OH. In some embodiments, R5sis-OMe. In some embodiments, R5sis-MOE. In some embodiments, R5sIs hydrogen.
In some embodiments, R5sIs optionally substituted C as described in the disclosure1-6Aliphatic radicals, e.g. C, as described for R or other variables1-6Aliphatic radical examples. In some implementationsIn the examples, R5sIs optionally substituted C1-6An alkyl group. In some embodiments, R5sIs methyl. In some embodiments, R5sIs ethyl.
In some embodiments, R5sAre protected hydroxyl groups suitable for oligonucleotide synthesis. In some embodiments, R5sis-OR ', wherein R' is optionally substituted C1-6Aliphatic. In some embodiments, R5sIs DMTrO-. Exemplary protecting groups for use in accordance with the present disclosure are widely known. For other examples, see Greene, t.w.; wuts, P.G.M.protective Groups in Organic Synthesis [ protecting Groups in Organic Synthesis]2 nd edition; wiley publication]: new york, 1991; and WO/2011/005761, WO/2013/012758, WO/2014/012081, WO/2015/107425, WO/2010/064146, WO/2014/010250, WO/2011/108682, WO/2012/039448 and WO/2012/073857, the protecting groups of each being hereby incorporated by reference.
In some embodiments, R1s、R2s、R3s、R4sAnd R5sAre R and may form a ring with one or more intervening atoms as described in this disclosure. In some embodiments, R2sAnd R4sAre R that together form a ring, and the sugar moiety may be a bicyclic sugar moiety, such as a LNA sugar moiety.
In some embodiments, Lsis-C (R)5s)2-, wherein each R5sIndependently as described in this disclosure. In some embodiments, R5sOne is H and the other is not H. In some embodiments, R5sNone of which is H. In some embodiments, Lsis-CHR5sWherein each R5sIndependently as described in this disclosure. In some embodiments, -C (R)5s)2-is an optionally substituted 5' -C of a sugar moiety. In some embodiments, -C (R)5s)2C of-is linked to the phosphorane and sugar wing moiety. In some embodiments, -C (R)5s)2C of (A) has the R configuration. In some embodiments, -C (R)5s)2C of-has the S configuration. As described in this disclosure, in some embodiments, R5sIs optionally substituted C1-6An aliphatic group; in some embodiments, R5sIs methyl.
In some embodiments, provided compounds comprise one or more optionally substituted divalent or multivalent rings, such as ring A, CyLA ring formed by two or more R groups (R and (combinations of) variables that may be R) taken together, and the like. In some embodiments, the ring is a cycloaliphatic, aryl, heteroaryl, or heterocyclic group as described for R, but divalent or polyvalent. As will be appreciated by those skilled in the art, the ring portion described for one variable (e.g., ring a) may also be applicable to other variables (e.g., Cy), if the requirements for the other variables (e.g., number of heteroatoms, valency, etc.) are metL). Example rings are broadly described in this disclosure.
In some embodiments, a ring (e.g., optionally substituted ring A, R, etc.) is a 3-20 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon.
In some embodiments, a ring may have any size within its range, such as 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-membered.
In some embodiments, the ring is monocyclic. In some embodiments, the ring is saturated and monocyclic. In some embodiments, the ring is monocyclic and partially saturated. In some embodiments, the ring is monocyclic and aromatic.
In some embodiments, the ring is bicyclic. In some embodiments, the ring is polycyclic. In some embodiments, the bicyclic or polycyclic ring comprises two or more monocyclic moieties, each of which can be saturated, partially saturated, or aromatic, and each of which can contain no heteroatoms or 1-10 heteroatoms. In some embodiments, the bicyclic or polycyclic ring comprises a saturated monocyclic ring. In some embodiments, the bicyclic or polycyclic ring comprises a saturated monocyclic ring that is free of heteroatoms. In some embodiments, bicyclic or polycyclic rings comprise a saturated monocyclic ring containing one or more heteroatoms. In some embodiments, the bicyclic or polycyclic ring comprises a partially saturated monocyclic ring. In some embodiments, the bicyclic or polycyclic ring comprises a partially saturated monocyclic ring that is free of heteroatoms. In some embodiments, bicyclic or polycyclic rings comprise a partially saturated monocyclic ring containing one or more heteroatoms. In some embodiments, the bicyclic or polycyclic ring comprises an aromatic monocyclic ring. In some embodiments, the bicyclic or polycyclic ring comprises an aromatic monocyclic ring that is free of heteroatoms. In some embodiments, bicyclic or polycyclic rings comprise aromatic monocyclic rings containing one or more heteroatoms. In some embodiments, bicyclic or polycyclic rings comprise saturated and partially saturated rings, each of which independently contains one or more heteroatoms. In some embodiments, bicyclic rings comprise a saturated ring and a partially saturated ring, each independently comprising no heteroatoms or one or more heteroatoms. In some embodiments, bicyclic rings comprise an aromatic ring and a partially saturated ring, each independently containing no heteroatoms or one or more heteroatoms. In some embodiments, polycyclic includes saturated and partially saturated rings, each independently containing no heteroatoms or one or more heteroatoms. In some embodiments, polycyclic rings include aromatic rings and partially saturated rings, each independently containing no heteroatoms or one or more heteroatoms. In some embodiments, polycyclic contains aromatic and saturated rings, each independently containing no heteroatoms or one or more heteroatoms. In some embodiments, polycyclic includes aromatic, saturated, and partially saturated rings, each independently containing no heteroatoms or one or more heteroatoms. In some embodiments, the ring comprises at least one heteroatom. In some embodiments, the ring comprises at least one nitrogen atom. In some embodiments, the ring comprises at least one oxygen atom. In some embodiments, the ring comprises at least one sulfur atom.
As understood by those skilled in the art in light of this disclosure, the rings are typically optionally substituted. In some embodiments, the ring is unsubstituted. In some embodiments, the ring is substituted. In some embodiments, the ring is substituted on one or more of its carbon atoms. In some embodiments, the ring is at a heteroatom thereofAre substituted on one or more of them. In some embodiments, the ring is substituted on one or more of its carbon atoms and one or more of its heteroatoms. In some embodiments, two or more substituents may be located on the same ring atom. In some embodiments, all available ring atoms are substituted. In some embodiments, not all available ring atoms are substituted. In some embodiments, in the structures provided, where the ring is indicated as being connected to other structures (e.g., in the structures providedRing a) of (a), optionally substituted means that the remaining substitutable ring positions (if any) are also optionally substituted in addition to those structures already attached.
In some embodiments, the ring is divalent or polyvalent C3-30A cycloaliphatic ring. In some embodiments, the ring is divalent or polyvalent C3-20A cycloaliphatic ring. In some embodiments, the ring is divalent or polyvalent C3-10A cycloaliphatic ring. In some embodiments, the ring is a divalent or polyvalent 3-30 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, the ring is a divalent or polyvalent 3-7 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, the ring is a divalent or polyvalent 3-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, the ring is a divalent or polyvalent 4-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, the ring is a divalent or polyvalent 5-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, the ring is a divalent or polyvalent 6-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, the ring is a divalent or polyvalent 7-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, the ring is a divalent or polyvalent cyclohexyl ring. In some embodiments, the ring is a divalent or polyvalent cyclopentyl ring. In some embodiments, the ring is a divalent or polyvalent cyclobutyl ring. In some embodiments, the ring is a divalent or polyvalent cyclopropyl ring.
In some embodiments, the ring is divalent or polyvalent C6-30An aryl ring. In some embodiments, the ring isDivalent or polyvalent benzene rings.
In some embodiments, the ring is a divalent or polyvalent 8-10 membered bicyclic saturated ring, partially unsaturated ring, or aryl ring. In some embodiments, the ring is a bivalent or multivalent 8-10 membered bicyclic saturated ring. In some embodiments, the ring is a divalent or polyvalent 8-10 membered bicyclic partially unsaturated ring. In some embodiments, the ring is a divalent or polyvalent 8-10 membered bicyclic aryl ring. In some embodiments, the ring is a divalent or polyvalent naphthyl ring.
In some embodiments, the ring is a divalent or polyvalent 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, the ring is a divalent or polyvalent 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, the ring is a divalent or polyvalent 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, the ring is a divalent or polyvalent 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.
In some embodiments, the ring is a divalent or polyvalent 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the ring is a divalent or polyvalent 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, and oxygen.
In some embodiments, the ring is a divalent or polyvalent 5-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, the ring is a divalent or polyvalent 6-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In certain embodiments, the ring is a divalent or polyvalent 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the ring is a divalent or multivalent 5, 6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the ring is a divalent or polyvalent 5, 6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, the ring is a divalent or polyvalent 6, 6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, the ring is a divalent or polyvalent 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, the ring is a divalent or polyvalent 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, the ring is a divalent or polyvalent 5-7 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, the ring is a divalent or polyvalent 5-6 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, the ring is a divalent or polyvalent 5-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, the ring is a divalent or polyvalent 6-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, the ring is a divalent or polyvalent 7-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the ring is a divalent or polyvalent 3-membered heterocyclic ring having one heteroatom selected from nitrogen, oxygen, or sulfur. In some embodiments, the ring is a divalent or polyvalent 4-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the ring is a divalent or polyvalent 5-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the ring is a divalent or polyvalent 6-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the ring is a divalent or polyvalent 7-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, the ring is a divalent or polyvalent 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the ring is a divalent or polyvalent 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, the ring is a divalent or polyvalent 5, 6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, the ring is a divalent or polyvalent 6, 6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, the ring formed by two or more groups together (typically optionally substituted) is a monocyclic saturated 5-7 membered ring having no heteroatoms other than intervening heteroatoms (if present). In some embodiments, the ring formed by two or more groups together is a monocyclic saturated 5-membered ring having no heteroatoms other than intervening heteroatoms (if present). In some embodiments, the ring formed by two or more groups together is a monocyclic saturated 6-membered ring having no heteroatoms other than intervening heteroatoms (if present). In some embodiments, the ring formed by two or more groups together is a monocyclic saturated 7-membered ring having no heteroatoms other than intervening heteroatoms (if present).
In some embodiments, the ring formed by two or more groups together is a bicyclic, saturated, partially unsaturated, or aryl 5-30 membered ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, except for intervening heteroatoms (if present). In some embodiments, the ring formed by two or more groups together is a bicyclic, saturated, partially unsaturated, or aryl 5-30 membered ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur, except for intervening heteroatoms (if present). In some embodiments, the rings formed by two or more groups together are bicyclic and saturated 8-10 membered bicyclic rings having no heteroatom other than an intervening heteroatom (if present). In some embodiments, the rings formed by two or more groups together are bicyclic and saturated 8-membered bicyclic rings having no heteroatom other than an intervening heteroatom (if present). In some embodiments, the rings formed by two or more groups together are bicyclic and saturated 9-membered bicyclic rings having no heteroatom other than an intervening heteroatom (if present). In some embodiments, the ring formed by two or more groups together is free of intervening heteroatoms (ifIf present) bicyclic and saturated 10-membered bicyclic rings having no other heteroatoms than nitrogen. In some embodiments, the ring formed by two or more groups together is bicyclic and comprises a 5-membered ring fused to a 5-membered ring. In some embodiments, the ring formed by two or more groups together is bicyclic and comprises a 5-membered ring fused to a 6-membered ring. In some embodiments, the 5-membered ring comprises one or more intervening nitrogen atoms, phosphorus atoms, and oxygen atoms as ring atoms. In some embodiments, the ring formed by two or more groups together comprises a ring system having the following backbone structure:
in some embodiments, the ring formed by two or more groups together is a polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, excluding intervening heteroatoms (if present). In some embodiments, the ring formed by two or more groups together is a polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur, except intervening heteroatoms (if present).
In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises a 5-10 membered monocyclic ring, the ring atoms of which comprise one or more intervening nitrogen, phosphorus, and/or oxygen atoms. In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises a 5-9 membered monocyclic ring, the ring atoms of which comprise one or more intervening nitrogen, phosphorus, and/or oxygen atoms. In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises a 5-8 membered monocyclic ring, the ring atoms of which comprise one or more intervening nitrogen, phosphorus, and/or oxygen atoms. In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises a 5-7 membered monocyclic ring, the ring atoms of which comprise one or more intervening nitrogen, phosphorus, and/or oxygen atoms. In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises a 5-6 membered monocyclic ring, the ring atoms of which comprise one or more intervening nitrogen, phosphorus, and/or oxygen atoms.
In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises a 5-membered monocyclic ring, the ring atoms of which comprise one or more intervening nitrogen, phosphorus, and/or oxygen atoms. In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises a 6-membered monocyclic ring, the ring atoms of which comprise one or more intervening nitrogen, phosphorus, and/or oxygen atoms. In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises a 7-membered monocyclic ring, the ring atoms of which comprise one or more intervening nitrogen, phosphorus, and/or oxygen atoms. In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises an 8-membered monocyclic ring, the ring atoms of which comprise one or more intervening nitrogen, phosphorus, and/or oxygen atoms. In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises a 9-membered monocyclic ring, the ring atoms of which comprise one or more intervening nitrogen, phosphorus, and/or oxygen atoms. In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises a 10 membered monocyclic ring, the ring atoms of which comprise one or more intervening nitrogen, phosphorus, and/or oxygen atoms.
In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises 5-membered rings whose ring atoms consist of carbon atoms with intervening nitrogen, phosphorus, and oxygen atoms. In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises 6 membered rings, the ring atoms of which consist of carbon atoms with intervening nitrogen, phosphorus, and oxygen atoms. In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises 7 membered rings whose ring atoms consist of carbon atoms with intervening nitrogen, phosphorus, and oxygen atoms. In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises 8 membered rings whose ring atoms are composed of carbon atoms with intervening nitrogen, phosphorus, and oxygen atoms. In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises 9 membered rings whose ring atoms consist of carbon atoms with intervening nitrogen, phosphorus, and oxygen atoms. In some embodiments, the ring formed by two or more groups together is monocyclic, bicyclic, or polycyclic and comprises 10 membered rings whose ring atoms are composed of carbon atoms with intervening nitrogen, phosphorus, and oxygen atoms.
In some embodiments, the rings described herein are unsubstituted. In some embodiments, the rings described herein are substituted. In some embodiments, the substituents are selected from those described in the example compounds provided in this disclosure.
As described herein, each LPIndependently are internucleotide linkages as described in the present disclosure, e.g., natural phosphate linkages, phosphorothioate diester linkages, modified internucleotide linkages, chiral internucleotide linkages, and the like. In some embodiments, each LPIndependently a linkage having the structure of formula I. In some embodiments, L3Eis-Ls-or-Ls-Ls-. In some embodiments, L3Eis-Ls-. In some embodiments, L3Eis-Ls-Ls-. In some embodiments, L3EIs a covalent bond. In some embodiments, L3EAre linkers for oligonucleotide synthesis. In some embodiments, L3EIs a linker for solid phase oligonucleotide synthesis. Various types of linkers are known and may be used in accordance with the present disclosure. In some embodiments, the linker is a succinate linker (-O-C (O) -CH)2-CH2-C (O) -. In some embodiments, the linker is an oxalyl linker (-O-C (O) -). In some embodiments, L3EIs a succinyl-piperidine linker (SP). In some embodiments, L3EIs a succinyl linker. In some embodiments, L3EIs a Q-linker.
In some embodiments, R3Eis-R', -Ls-R ', -OR' OR a solid support. In some embodiments, R3Eis-R'. In some embodiments, R3Eis-Ls-R'. In some embodiments, R3Eis-OR'. In some embodiments, R3EIs a solid support. In some embodiments, R3Eis-H. In some embodiments, -L3-R3Eis-H. In some embodiments, R3Eis-OH. In some embodiments, -L3-R3Eis-OH. In some embodiments, R3EIs optionally substituted C1-6An aliphatic group. In some embodiments, R3EIs optionally substituted C1-6An alkyl group. In some embodiments, R3Eis-OR'. In some embodiments, R3Eis-OH. In some embodiments, R3Eis-OR ', wherein R' is not hydrogen. In some embodiments, R3Eis-OR ', wherein R' is optionally substituted C1-6An alkyl group.
In some embodiments, R3EAre 3' -end caps (e.g., those used in RNAi technology).
In some embodiments, R3EIs a solid support. In some embodiments, R3EIs a solid support for oligonucleotide synthesis. Various types of solid supports are known and may be used in accordance with the present disclosure. In some embodiments, the solid support is an HCP. In some embodiments, the solid support is CPG.
In some embodiments, R' is-R, -C (O) OR, OR-S (O)2R, wherein R is as described in the disclosure. In some embodiments, R' is R, wherein R is as described in the disclosure. In some embodiments, R' is-c (o) R, wherein R is as described in the disclosure. In some embodiments, R' is-c (o) OR, wherein R is as described in the disclosure. In some embodiments, R' is-S (O)2R, wherein R is asDescribed in this disclosure. In some embodiments, R' is hydrogen. In some embodiments, R' is not hydrogen. In some embodiments, R' is R, wherein R is optionally substituted C as described in the disclosure1-20An aliphatic group. In some embodiments, R' is R, wherein R is optionally substituted C as described in the disclosure1-20A heteroaliphatic group. In some embodiments, R' is R, wherein R is optionally substituted C as described in the disclosure6-20And (4) an aryl group. In some embodiments, R' is R, wherein R is optionally substituted C as described in the disclosure6-20An arylaliphatic group. In some embodiments, R' is R, wherein R is optionally substituted C as described in the disclosure6-20An aryl heteroaliphatic group. In some embodiments, R' is R, wherein R is an optionally substituted 5-20 membered heteroaryl as described in the present disclosure. In some embodiments, R' is R, wherein R is an optionally substituted 3-20 membered heterocyclyl as described in the present disclosure. In some embodiments, two or more R' are R, and optionally and independently together form an optionally substituted ring as described in the present disclosure.
In some embodiments, each R is independently-H, or an optionally substituted group selected from: c1-30Aliphatic radical, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon1-30Heteroaliphatic radical, C6-30Aryl radical, C6-30Arylaliphatic group, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon6-30An aryl heteroaliphatic, a 5-to 30-membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-to 30-membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon.
Two R groups optionally and independently form a covalent bond together, or
Two or more R groups on the same atom optionally and independently form, with the atom, an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, in addition to the atom; or
Two or more R groups on two or more atoms optionally and independently form, with the intervening atoms, an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, excluding the intervening atoms.
In some embodiments, each R is independently-H, or an optionally substituted group selected from: c1-30Aliphatic, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon1-30Heteroaliphatic group, C6-30Aryl radical, C6-30Arylaliphatic, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon6-30Aryl heteroaliphatics, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, or
Two R groups optionally and independently form a covalent bond together, or
Two or more R groups on the same atom optionally and independently form, with the atom, an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, in addition to the atom.
Two or more R groups on two or more atoms optionally and independently form, with the intervening atoms, an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, excluding the intervening atoms.
In some embodiments, each R is independently-H, or an optionally substituted group selected from: c1-20Aliphatic, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon1-20Heteroaliphatic group, C6-20Aryl radical, C6-20Arylaliphatic, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon6-20Aryl heteroaliphatic having 1-10 substituents independently selected from oxygen, nitrogen, sulfur, phosphorusAnd a 5-20 membered heteroaryl group of heteroatoms of silicon, and a 3-20 membered heterocyclic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or
Two R groups optionally and independently form a covalent bond together, or
Two or more R groups on the same atom optionally and independently form, with the atom, an optionally substituted 3-20 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, in addition to the atom;
two or more R groups on two or more atoms optionally and independently form, with the intervening atoms, an optionally substituted 3-20 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, excluding the intervening atoms.
In some embodiments, each R is independently-H, or an optionally substituted group selected from: c1-30Aliphatic, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon1-30Heteroaliphatic group, C6-30Aryl radical, C6-30Arylaliphatic, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon6-30An aryl heteroaliphatic, a 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon.
In some embodiments, each R is independently-H, or an optionally substituted group selected from: c1-20Aliphatic, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon1-20Heteroaliphatic group, C6-20Aryl radical, C6-20Arylaliphatic, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon6-20An aryl heteroaliphatic, a 5-20 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-20 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon.
In some embodiments, R is hydrogen. In some embodiments, R is not hydrogen. In some embodiments, R is an optionally substituted group selected from: c1-30Aliphatic radical, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon1-30Heteroaliphatic radical, C6-30An aryl group, a 5-to 30-membered heteroaromatic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-to 30-membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon.
In some embodiments, R is hydrogen or an optionally substituted group selected from: c1-20An aliphatic group; a phenyl group; a 3-to 7-membered saturated or partially unsaturated carbocyclic ring; an 8-to 10-membered bicyclic saturated, partially unsaturated, or aromatic ring; a 5-to 6-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 4-to 7-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 7-to 10-membered bicyclic saturated or partially unsaturated heterocycle having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or an 8-to 10-membered bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, R is optionally substituted C1-30An aliphatic group. In some embodiments, R is optionally substituted C1-20An aliphatic group. In some embodiments, R is optionally substituted C1-15An aliphatic group. In some embodiments, R is optionally substituted C1-10An aliphatic group. In some embodiments, R is optionally substituted C1-6An aliphatic group. In some embodiments, R is optionally substituted C1-6An alkyl group. In some embodiments, R is optionally substituted hexyl, pentyl, butyl, propyl, ethyl, or methyl. In some embodiments, R is an optionally substituted hexyl. In some embodiments, R is optionally substituted pentyl. In some embodiments, R is optionally substituted butyl. In some embodiments, R is optionally substituted propyl. In some embodiments, R is an optionally substituted ethyl. In some embodiments, R isOptionally substituted methyl. In some embodiments, R is hexyl. In some embodiments, R is pentyl. In some embodiments, R is butyl. In some embodiments, R is propyl. In some embodiments, R is ethyl. In some embodiments, R is methyl. In some embodiments, R is isopropyl. In some embodiments, R is n-propyl. In some embodiments, R is tert-butyl. In some embodiments, R is sec-butyl. In some embodiments, R is n-butyl. In some embodiments, R is- (CH)2)2CN。
In some embodiments, R is optionally substituted C3-30A cycloaliphatic radical. In some embodiments, R is optionally substituted C3-20A cycloaliphatic radical. In some embodiments, R is optionally substituted C3-10A cycloaliphatic radical. In some embodiments, R is optionally substituted cyclohexyl. In some embodiments, R is cyclohexyl. In some embodiments, R is an optionally substituted cyclopentyl. In some embodiments, R is cyclopentyl. In some embodiments, R is an optionally substituted cyclobutyl. In some embodiments, R is cyclobutyl. In some embodiments, R is optionally substituted cyclopropyl. In some embodiments, R is cyclopropyl.
In some embodiments, R is an optionally substituted 3-30 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 3-7 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 3-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 4-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 5-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 6-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 7-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is optionally substituted cycloheptyl. In some embodiments, R is cycloheptyl. In some embodiments, R is optionally substituted cyclohexyl. In some embodiments, R is cyclohexyl. In some embodiments, R is an optionally substituted cyclopentyl. In some embodiments, R is cyclopentyl. In some embodiments, R is an optionally substituted cyclobutyl. In some embodiments, R is cyclobutyl. In some embodiments, R is optionally substituted cyclopropyl. In some embodiments, R is cyclopropyl.
In some embodiments, when R is or comprises a ring structure (e.g., cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, etc.), the ring structure may be monocyclic, bicyclic, or polycyclic. In some embodiments, R is or comprises a monocyclic structure. In some embodiments, R is or comprises a bicyclic structure. In some embodiments, R is or comprises a polycyclic structure.
In some embodiments, R is an optionally substituted C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon1-30A heteroaliphatic group. In some embodiments, R is optionally substituted C having 1-10 heteroatoms1-20A heteroaliphatic group. In some embodiments, R is an optionally substituted C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, or silicon1-20A heteroaliphatic group, optionally including one or more oxidized forms of nitrogen, sulfur, phosphorus, or selenium. In some embodiments, R is optionally substituted C1-30A heteroaliphatic comprising 1-10 groups independently selected from:-N＝、≡N、-S-、-S(O)-、-S(O)2-、-O-、＝O、
in some embodiments, R is optionally substituted C6-30And (4) an aryl group. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is substituted phenyl.
In some embodiments, R is an optionally substituted 8-10 membered bicyclic saturated ring, partially unsaturated ring, or aryl ring. In some embodiments, R is an optionally substituted 8-10 membered bicyclic saturated ring. In some embodiments, R is an optionally substituted 8-10 membered bicyclic partially unsaturated ring. In some embodiments, R is an optionally substituted 8-10 membered bicyclic aryl ring. In some embodiments, R is optionally substituted naphthyl.
In some embodiments, R is an optionally substituted 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, R is an optionally substituted 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, R is an optionally substituted 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, R is an optionally substituted 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.
In some embodiments, R is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is a substituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an unsubstituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, and oxygen. In some embodiments, R is a substituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an unsubstituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, and oxygen.
In some embodiments, R is an optionally substituted 5-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted 6-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, R is an optionally substituted 5-membered monocyclic heteroaryl ring having one heteroatom selected from nitrogen, oxygen, and sulfur. In some embodiments, R is selected from optionally substituted pyrrolyl, furanyl, or thienyl.
In some embodiments, R is an optionally substituted 5-membered heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-membered heteroaryl ring having one nitrogen atom and another heteroatom selected from sulfur or oxygen. Exemplary R groups include, but are not limited to, optionally substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, or isoxazolyl.
In some embodiments, R is an optionally substituted 5-membered heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. Exemplary R groups include, but are not limited to, optionally substituted triazolyl, oxadiazolyl, or thiadiazolyl.
In some embodiments, R is an optionally substituted 5-membered heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. Exemplary R groups include, but are not limited to, optionally substituted tetrazolyl, oxatriazolyl, and thiatriazolyl.
In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1-4 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1-3 nitrogen atoms. In other embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1-2 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having four nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having three nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having two nitrogen atoms. In certain embodiments, R is an optionally substituted 6-membered heteroaryl ring having one nitrogen atom. Exemplary R groups include, but are not limited to, optionally substituted pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl.
In certain embodiments, R is an optionally substituted 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 1 heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted indolyl. In some embodiments, R is an optionally substituted azabicyclo [3.2.1] octyl. In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted azaindolyl. In some embodiments, R is optionally substituted benzimidazolyl. In some embodiments, R is optionally substituted benzothiazolyl. In some embodiments, R is optionally substituted benzoxazolyl. In some embodiments, R is optionally substituted indazolyl. In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having one heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted indolyl. In some embodiments, R is an optionally substituted benzofuranyl. In some embodiments, R is optionally substituted benzo [ b ] thienyl. In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted azaindolyl. In some embodiments, R is optionally substituted benzimidazolyl. In some embodiments, R is optionally substituted benzothiazolyl. In some embodiments, R is optionally substituted benzoxazolyl. In some embodiments, R is optionally substituted indazolyl. In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted oxazolopyridinyl, thiazolopyridinyl, or imidazopyridinyl. In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted purinyl, oxazolopyrimidinyl, thiazolopyrimidinyl, oxazolopyrazinyl, thiazolopyrazinyl, imidazopyrazinyl, oxazolopyridazinyl, thiazolopyridazinyl, or imidazopyridazinyl. In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, R is optionally substituted 1, 4-dihydropyrrolo [3, 2-b ] pyrrolyl, 4H-furo [3, 2-b ] pyrrolyl, 4H-thieno [3, 2-b ] pyrrolyl, furo [3, 2-b ] furyl, thieno [3, 2-b ] thienyl, 1H-pyrrolo [1, 2-a ] imidazolyl, pyrrolo [2, 1-b ] oxazolyl, or pyrrolo [2, 1-b ] thiazolyl. In some embodiments, R is optionally substituted dihydropyrroloimidazolyl, 1H-furoimidazolyl, 1H-thienoimidazolyl, furooxazolyl, furoisoxazolyl, 4H-pyrrolooxazolyl, 4H-pyrroloisoxazolyl, thienooxazolyl, thienoisoxazolyl, 4H-pyrrolothiazolyl, furothiazolyl, thienothiazolyl, 1H-imidazoimidazolyl, imidazooxazolyl, or imidazo [5, 1-b ] thiazolyl.
In certain embodiments, R is an optionally substituted 6, 6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6, 6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 6, 6-fused heteroaryl ring having 1 heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted quinolinyl. In some embodiments, R is optionally substituted isoquinolinyl. In some embodiments, R is an optionally substituted 6, 6-fused heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted quinazoline or quinoxaline.
In some embodiments, R is a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, R is a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, R is a 3-30 membered heterocyclic ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, R is a 3-30 membered heterocyclic ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.
In some embodiments, R is an optionally substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is a substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an unsubstituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-7 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 6-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 7-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 3-membered heterocyclic ring having one heteroatom selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted 4-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 7-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, R is an optionally substituted 3-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 4-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 7-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, R is an optionally substituted 4-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is nitrogen. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is oxygen. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 oxygen atoms. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 nitrogen atoms. In some embodiments, R is an optionally substituted 4-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is nitrogen. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is oxygen. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 oxygen atoms. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 nitrogen atoms.
In some embodiments, R is an optionally substituted 5-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is nitrogen. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is oxygen. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is sulfur. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having 2 oxygen atoms. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having 2 nitrogen atoms.
In some embodiments, R is an optionally substituted 6-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is nitrogen. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is oxygen. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is sulfur. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having 2 oxygen atoms. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having 2 nitrogen atoms.
In certain embodiments, R is a 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is optionally substituted oxiranyl, oxetanyl, tetrahydrofuryl, tetrahydropyranyl, oxepanyl, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl, thiazolidinyl, dithiolane, dioxanyl, morpholinyl, oxathietanyl, piperazinyl, thiomorpholinyl, dithianyl, dioxacycloheptyl, oxazepanyl, oxathiepinyl, dithepinyl, diazepanyl, dihydrofuranonyl, tetrahydropyranonyl, oxepinyl, pyrrolidinonyl, piperidinonyl, azepinyl, oxacycloheptanonyl, tetrahydropyranonyl, oxocycloheptanonyl, pyrrolidinonyl, piperidinonyl, azacycloheptanonyl, etc, Dihydrothienylone, tetrahydrothiopyranonyl, thiepinyl, oxazolidinonyl, oxaazacyclohexonyl, oxazepinyl, dioxapentonyl, dioxanone, dioxepinyl, oxathiepinyl, oxathiapyranonyl, oxathiepinyl, thiazolidinonyl, thiazinonenyl, thiazepinyl, imidazolidinonyl, tetrahydropyrimidinyl, diazepinyl, imidazolidinedionyl, oxazolidinedione, thiazolidinedioneyl, dioxolanedionyl, oxathiolanedionyl, piperazinedionyl, morpholine dione, thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, tetrahydrothiophenyl or tetrahydrothiopyranyl.
In certain embodiments, R is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted tetrahydropyridinyl, dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.
In some embodiments, R is an optionally substituted 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted indolinyl. In some embodiments, R is optionally substituted isoindolinyl. In some embodiments, R is an optionally substituted 1, 2, 3, 4-tetrahydroquinolinyl. In some embodiments, R is optionally substituted 1, 2, 3, 4-tetrahydroisoquinolinyl. In some embodiments, R is an optionally substituted azabicyclo [3.2.1] octyl.
In some embodiments, R is an optionally substituted 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted 1, 4-dihydropyrrolo [3, 2-b ] pyrrolyl, 4H-furo [3, 2-b ] pyrrolyl, 4H-thieno [3, 2-b ] pyrrolyl, furo [3, 2-b ] furyl, thieno [3, 2-b ] thienyl, 1H-pyrrolo [1, 2-a ] imidazolyl, pyrrolo [2, 1-b ] oxazolyl, or pyrrolo [2, 1-b ] thiazolyl. In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted dihydropyrroloimidazolyl, IH-furoimidazolyl, 1H-thienoimidazolyl, furooxazolyl, furoisoxazolyl, 4H-pyrrolooxazolyl, 4H-pyrroloisoxazolyl, thienooxazolyl, thienoisoxazolyl, 4H-pyrrolothiazolyl, furothiazolyl, thienothiazolyl, 1H-imidazoimidazolyl, imidazooxazolyl, or imidazo [5, 1-b ] thiazolyl. In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having one heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted indolyl. In some embodiments, R is an optionally substituted benzofuranyl. In some embodiments, R is optionally substituted benzo [ b ] thienyl. In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted azaindolyl. In some embodiments, R is optionally substituted benzimidazolyl. In some embodiments, R is optionally substituted benzothiazolyl. In some embodiments, R is optionally substituted benzoxazolyl. In some embodiments, R is optionally substituted indazolyl. In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted oxazolopyridinyl, thiazolopyridinyl, or imidazopyridinyl. In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted purinyl, oxazolopyrimidinyl, thiazolopyrimidinyl, oxazolopyrazinyl, thiazolopyrazinyl, imidazopyrazinyl, oxazolopyridazinyl, thiazolopyridazinyl, or imidazopyridazinyl. In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In certain embodiments, R is an optionally substituted 6, 6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6, 6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 6, 6-fused heteroaryl ring having one heteroatom selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted quinolinyl. In some embodiments, R is optionally substituted isoquinolinyl. In some embodiments, R is an optionally substituted 6, 6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted quinazolinyl, phthalazinyl, quinoxalinyl, or naphthyridinyl group. In some embodiments, R is an optionally substituted 6, 6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted pyridopyrimidinyl, pyridopyridazinyl, pyridopyrazinyl, or benzotriazinyl. In some embodiments, R is an optionally substituted 6, 6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted pyridotriazinyl, pteridinyl, pyrazinopyrazinyl, pyrazinopyridazinyl, pyridazinopyridazinyl, pyrimidopyridazinyl or pyrimidopyrimidinyl. In some embodiments, R is an optionally substituted 6, 6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, R is optionally substituted C6-30An arylaliphatic group. In some embodiments, R is optionally substituted C6-20An arylaliphatic group. In some embodiments, R is optionally substituted C6-10ArylaliphaticA group. In some embodiments, the aryl moiety of the arylaliphatic has 6, 10, or 14 aryl carbon atoms. In some embodiments, the aryl portion of the arylaliphatic has 6 aryl carbon atoms. In some embodiments, the aryl portion of the arylaliphatic has 10 aryl carbon atoms. In some embodiments, the aryl portion of the arylaliphatic has 14 aryl carbon atoms. In some embodiments, the aryl moiety is an optionally substituted phenyl.
In some embodiments, R is an optionally substituted C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon6-30An aryl heteroaliphatic group. In some embodiments, R is an optionally substituted C having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur6-30An aryl heteroaliphatic group. In some embodiments, R is an optionally substituted C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon6-20An aryl heteroaliphatic group. In some embodiments, R is an optionally substituted C having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur6-20An aryl heteroaliphatic group. In some embodiments, R is an optionally substituted C having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon6-10An aryl heteroaliphatic group. In some embodiments, R is an optionally substituted C having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur6-10An aryl heteroaliphatic group.
In some embodiments, two R groups optionally and independently form a covalent bond together. In some embodiments, -C ═ O is formed. In some embodiments, -C ═ C-is formed. In some embodiments, -C ≡ C-is formed.
In some embodiments, two or more R groups on the same atom optionally and independently form, with the atom, an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, in addition to the atom. In some embodiments, two or more R groups on the same atom optionally and independently form, with the atom, an optionally substituted 3-20 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, in addition to the atom. In some embodiments, two or more R groups on the same atom optionally and independently form, with the atom, an optionally substituted 3-10 membered monocyclic, bicyclic, or polycyclic ring having 0-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, in addition to the atom. In some embodiments, two or more R groups on the same atom optionally and independently form, with the atom, an optionally substituted 3-6 membered monocyclic, bicyclic, or polycyclic ring having 0-3 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, in addition to the atom. In some embodiments, two or more R groups on the same atom optionally and independently form, with the atom, an optionally substituted 3-5 membered monocyclic, bicyclic, or polycyclic ring having 0-3 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, in addition to the atom.
In some embodiments, two or more R groups on two or more atoms optionally and independently form, with the intervening atoms, an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, excluding the intervening atoms. In some embodiments, two or more R groups on two or more atoms optionally and independently form, with the intervening atoms, an optionally substituted 3-20 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, excluding the intervening atoms. In some embodiments, two or more R groups on two or more atoms optionally and independently form, with the intervening atoms, an optionally substituted 3-10 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, excluding the intervening atoms. In some embodiments, two or more R groups on two or more atoms optionally and independently form, with the intervening atoms, an optionally substituted 3-10 membered monocyclic, bicyclic, or polycyclic ring having 0-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, excluding the intervening atoms. In some embodiments, two or more R groups on two or more atoms optionally and independently form, with the intervening atoms, an optionally substituted 3-6 membered monocyclic, bicyclic, or polycyclic ring having 0-3 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, excluding the intervening atoms. In some embodiments, two or more R groups on two or more atoms optionally and independently form, with the intervening atoms, an optionally substituted 3-5 membered monocyclic, bicyclic, or polycyclic ring having 0-3 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, excluding the intervening atoms.
In some embodiments, the heteroatoms in the R groups or in the structures formed by two or more R groups together are selected from oxygen, nitrogen, and sulfur. In some embodiments, the formed ring is 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-or 20-membered. In some embodiments, the formed ring is saturated. In some embodiments, the formed ring is partially saturated. In some embodiments, the ring formed is aromatic. In some embodiments, the formed ring comprises a saturated ring portion, a partially saturated ring portion, or an aromatic ring portion. In some embodiments, the ring formed comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aromatic ring atoms. In some embodiments, the ring formed contains no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aromatic ring atoms. In some embodiments, the aromatic ring atoms are selected from carbon, nitrogen, oxygen, and sulfur.
In some embodiments, the ring formed by two or more R groups (or two or more groups selected from R and variables which may be R) taken together is C3-30Cycloaliphatic radical, C6-30Aryl, 5-to 30-membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, or 3-to 30-membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, the rings as described for R but divalent or polyvalent.
In some embodiments, PLIs P (═ W). In some embodiments, PLIs P. In some embodiments, PLIs P → B (R')3. In some embodiments, PLP of (a) is chiral. In some embodiments, PLP is Rp in some embodiments, PLP is Sp in some embodiments, the linkage of formula I is a phosphate linkage or salt form thereof. In some embodiments, the linkage of formula I is a phosphorothioate linkage or a salt form thereof. In some embodiments, PLIs P (═ W), where P is a chirally bonded phosphorus. In some embodiments, PLIs P (═ O), where P is a chirally bonded phosphorus.
In some embodiments, W is O. In some embodiments, W is S. In some embodiments, W is Se.
X, Y, Z and R1Each independently as described in the present disclosure, e.g., as described, in some embodiments, R1Is H. In some embodiments, -X-L-R1is-X-R1In some embodiments, -X-L-R1is-X-H. In some embodiments, Y and Z are O, and X is S. In some embodiments, Y and Z are O and X is O. Other embodiments of the variables are described independently in this disclosure.
In some embodiments, provided oligonucleotides have a structure of formula O-I. In some embodiments, the oligonucleotide of formula O-I comprises chemical modifications as described in the present disclosure (e.g., sugar modifications, base modifications, modified internucleotide linkages, and the like, and patterns thereof), stereochemistry (stereochemistry and patterns thereof of 5' -C, chiral phosphorus, and the like), base sequences, and the like. In some embodiments, provided oligonucleotides of formula O-I are oligonucleotides selected from table 1A, table 17, and the like.
In some embodiments, the present disclosure provides multimers of oligonucleotides. In some embodiments, at least one of the monomers is a C9orf72 oligonucleotide. In some embodiments, the multimer is a multimer of the same oligonucleotide. In some embodiments, the multimer is a multimer of structurally different oligonucleotides. In some embodiments, each oligonucleotide of the multimer independently performs its function via its own pathway, e.g., RNA interference (RNAi), RNaseH dependence, and the like. In some embodiments, providedOligonucleotides exist in oligomeric or polymeric form, in which one or more oligonucleotide moieties are linked by a linker (e.g., L, L)MEtc.) are linked together via nucleobases, sugars and/or internucleotide linkages of said oligonucleotide moieties. For example, in some embodiments, the multimeric compound provided has the structure (a)c)a-LM-(Ac)bWherein each variable is independently as described in the present disclosure.
In some embodiments, a provided compound, e.g., an oligonucleotide of a provided composition, has the following structure:
Ac-[-LM-(RD)a]b、[(Ac)a-LM]b-RD、(Ac)a-LM-(Ac)bor (A)c)a-LM-(RD)b，
Or a salt thereof, wherein:
Ac-[-LM-(RD)a]b、[(Ac)a-LM]b-RD、(Ac)a-LM-(Ac)bor (A)c)a-LM-(RD)b，
Or a salt thereof, wherein:
each A iscIndependently an oligonucleotide moiety (e.g., [ H ]]a-AcOr [ H ]]b-AcIs an oligonucleotide);
a is 1 to 1000;
b is 1 to 1000;
LMis a multivalent linker; and is
Each RDIndependently a chemical moiety.
In some embodiments, a provided compound, e.g., an oligonucleotide of a provided composition, has the following structure:
Ac-[-LM-(RD)a]b、[(Ac)a-LM]b-RD、(Ac)a-LM-(Ac)bor (A)c)a-LM-(RD)b，
Or a salt thereof, wherein:
each A iscIndependently an oligonucleotide moiety (e.g., [ H ]]a-AcOr [ H ]]b-AcIs an oligonucleotide);
a is 1 to 1000;
b is 1 to 1000;
each RDIndependently is RLD、RCDOr RTD；
RCDIs an optionally substituted linear or branched radical selected from C1-100Aliphatic radical and C having 1-30 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon1-100A heteroaliphatic group wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-; and one or more carbon atoms are optionally and independently substituted with CyLReplacement;
RLDis selected from C1-100An optionally substituted linear or branched group of aliphatic groups wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-; and one or more carbon atoms are optionally and independently substituted with CyLReplacement;
RTDis a targeting moiety;
each LMIndependently a covalent bond or an optionally substituted linear or branched divalent or polyvalent group selected from C1-100Aliphatic radical and C having 1-30 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon1-100A heteroaliphatic group wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-; and one or more carbon atoms are optionally and independently substituted with CyLReplacement;
each CyLIndependently is an optionally substituted tetravalent group selected from: c3-20Cycloaliphatic ring, C6-20Aryl rings, 5-20 membered heteroaryl rings having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, anda 3-20 membered heterocyclyl ring of 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon;
each R' is independently-R, -C (O) OR OR-S (O)2R; and is
Each R is independently-H, or an optionally substituted group selected from: c1-30Aliphatic, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon1-30Heteroaliphatic group, C6-30Aryl radical, C6-30Arylaliphatic, C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon6-30Aryl heteroaliphatics, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, or
Two R groups optionally and independently form a covalent bond together, or
Two or more R groups on the same atom optionally and independently form, with the atom, an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, other than the atom; or
Two or more R groups on two or more atoms optionally and independently form, with the intervening atoms, an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, excluding the intervening atoms.
In some embodiments, Ac-[-LM-(RD)a]b、[(Ac)a-LM]b-RDOr (A)c)a-LM-(RD)bAre conjugates of the provided oligonucleotides with one or more chemical moieties (e.g., targeting moieties, carbohydrate moieties, lipid moieties, etc.).
In some embodiments, (R)D)b-LMIs (R) as described in the present disclosureD)b-LM1-LM2。
In some embodiments, [ H ]]a-AcOr [ H ]]b-AcAre oligonucleotides as described in the present disclosure. In some embodiments, [ H ]]a-AcOr [ H ]]b-AcHas the formula O-I.
In some embodiments, RDAre additional chemical moieties as described in the present disclosure. In some embodiments, RDIs a targeting moiety as described in the present disclosure. In some embodiments, RDIs RTDWhich is a targeting moiety as described in the present disclosure (e.g., R is described as a targeting moiety)DThe targeting moiety of embodiments (a). In some embodiments, RDIs RCDWherein R isCDAs described in this disclosure. In some embodiments, RCDComprising one or more carbohydrate moieties. In some embodiments, RDIs RLD. In some embodiments, RLDIs a lipid moiety as described in the present disclosure.
In some embodiments, a is 1-100. In some embodiments, a is 1-50. In some embodiments, a is 1-40. In some embodiments, a is 1-30. In some embodiments, a is 1-20. In some embodiments, a is 1-15. In some embodiments, a is 1-10. In some embodiments, a is 1-9. In some embodiments, a is 1-8. In some embodiments, a is 1-7. In some embodiments, a is 1-6. In some embodiments, a is 1-5. In some embodiments, a is 1-4. In some embodiments, a is 1-3. In some embodiments, a is 1-2. In some embodiments, a is 1. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, a is 4. In some embodiments, a is 5. In some embodiments, a is 6. In some embodiments, a is 7. In some embodiments, a is 8. In some embodiments, a is 9. In some embodiments, a is 10. In some embodiments, a is greater than 10.
In some embodiments, b is 1-100. In some embodiments, b is 1-50. In some embodiments, b is 1-40. In some embodiments, b is 1-30. In some embodiments, b is 1-20. In some embodiments, b is 1-15. In some embodiments, b is 1-10. In some embodiments, b is 1-9. In some embodiments, b is 1-8. In some embodiments, b is 1-7. In some embodiments, b is 1-6. In some embodiments, b is 1-5. In some embodiments, b is 1-4. In some embodiments, b is 1-3. In some embodiments, b is 1-2. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3. In some embodiments, b is 4. In some embodiments, b is 5. In some embodiments, b is 6. In some embodiments, b is 7. In some embodiments, b is 8. In some embodiments, b is 9. In some embodiments, b is 10. In some embodiments, b is 1. In some embodiments, b is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or greater.
In some embodiments, z is 1-1000. In some embodiments, z +1 is the oligonucleotide length as described in the present disclosure. In some embodiments, z is not less than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In some embodiments, z is not less than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, z is no greater than 50, 60, 70, 80, 90, 100, 150, or 200. In some embodiments, z is 5-50, 10-50, 14-45, 14-40, 14-35, 14-30, 14-25, 14-100, 14-150, 14-200, 14-250, 14-300, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-100, 15-150, 15-200, 15-250, 15-300, 16-50, 16-45, 16-40, 16-35, 16-30, 16-25, 16-100, 16-150, 16-200, 16-250, 16-300, 17-50, 17-45, 17-40, 17-35, 17-30, 17-25, 17-100, 17-150, 17-200, 17-250, 17-300, 18-50, 18-45, 18-40, 18-35, 18-30, 18-25, 18-100, 18-150, 18-200, 18-250, 18-300, 19-50, 19-45, 19-40, 19-35, 19-30, 19-25, 19-100, 19-150, 19-200, 19-250, or 19-300. In some embodiments, z is 10. In some embodiments, z is 11. In some embodiments, z is 12. In some embodiments, z is 13. In some embodiments, z is 14. In some embodiments, z is 15. In some embodiments, z is 16. In some embodiments, z is 17. In some embodiments, z is 18. In some embodiments, z is 19. In some embodiments, z is 20. In some embodiments, z is 21. In some embodiments, z is 22. In some embodiments, z is 23. In some embodiments, z is 24. In some embodiments, z is 25. In some embodiments, z is 26. In some embodiments, z is 27. In some embodiments, z is 28. In some embodiments, z is 29. In some embodiments, z is 30. In some embodiments, z is 31. In some embodiments, z is 32. In some embodiments, z is 33. In some embodiments, z is 34.
In some embodiments, LMIs as described in this disclosureM1-LM2-LM3-. In some embodiments, LMIs L as described in this disclosureM1. In some embodiments, LMIs L as described in this disclosureM2. In some embodiments, LMIs L as described in this disclosureM3。
In some embodiments, at least one LMIs a sugar unit that binds directly to the provided oligonucleotide. In some embodiments, LMDirect binding to the sugar unit incorporates a lipid moiety into the oligonucleotide. In some embodiments, LMThe carbohydrate moiety is incorporated into the oligonucleotide directly bound to the sugar unit. In some embodiments, LMDirect binding to sugar unitsLDGroups are incorporated into the oligonucleotide. In some embodiments, LMDirect binding to sugar unitsCDGroups are incorporated into the oligonucleotide. In some embodiments, LMIs directly bound via the 5' -OH of the oligonucleotide chain. In some embodiments, LMIs directly bound via the 3' -OH of the oligonucleotide chain.
In some embodiments, at least one LMAre internucleotide linkage units that bind directly to the provided oligonucleotides. In some embodiments, LMDirect binding to internucleotide linkage units incorporates lipid moieties into oligonucleotides. In some embodiments, LMDirect binding to internucleotide linkage units incorporates carbohydrate moieties into the oligonucleotide. In some embodiments, LMDirect binding of an internucleotide linkage unit to RLDGroups are incorporated into the oligonucleotide. In some embodiments, LMDirect binding of an internucleotide linkage unit to RCDGroups are incorporated into the oligonucleotide.
In some embodiments, at least one LMIs a nucleobase unit that directly binds to the provided oligonucleotides. In some embodiments, LMDirect binding to nucleobase units incorporates lipid moieties into oligonucleotides. In some embodiments, LMDirect binding to nucleobase units incorporates carbohydrate moieties into oligonucleotides. In some embodiments, LMDirect binding of R to nucleobase unitLDGroups are incorporated into the oligonucleotide. In some embodiments, LMDirect binding of R to nucleobase unitCDGroups are incorporated into the oligonucleotide.
In some embodiments, LMIs divalent. In some embodiments, LMIs multivalent. In some embodiments, LMIs thatWherein L isMBinding directly to a nucleobase, for example as in:
in some embodiments, LMIs that
In some embodiments, LMIs that
In some embodiments, LMIs thatIn some embodiments, LMIs that
In some embodiments, RLDIs optionally substituted C10、C15、C16、C17、C18、C19、C20、C21、C22、C23、C24Or C25To C20、C21、C22、C23、C24、C25、C26、C27、C28、C29、C30、C35、C40、C45、C50、C60、C70Or C80An aliphatic group. In some embodiments, RLDIs optionally substituted C10-80An aliphatic group. In some embodiments, RLDIs optionally substituted C20-80An aliphatic group. In some embodiments, RLDIs optionally substituted C10-70An aliphatic group. In some embodiments, RLDIs optionally substituted C20-70An aliphatic group. In some embodiments, RLDIs optionally substituted C10-60An aliphatic group. In some embodiments, RLDIs optionally substituted C20-60An aliphatic group. In some embodiments, RLDIs optionally substituted C10-50An aliphatic group. In some embodiments, RLDIs optionally substituted C20-50An aliphatic group. In some embodiments, RLDIs optionally substituted C10-40An aliphatic group. In some embodiments, RLDIs optionally substituted C20-40An aliphatic group. In some embodiments, RLDIs optionally substituted C10-30An aliphatic group. In some embodiments, RLDIs optionally substituted C20-30An aliphatic group. In some embodiments, RLDIs unsubstituted C10、C15、C16、C17、C18、C19、C20、C21、C22、C23、C24Or C25To C20、C21、C22、C23、C24、C25、C26、C27、C28、C29、C30、C35、C40、C45、C50、C60、C70Or C80An aliphatic group. In some embodiments, RLDIs unsubstituted C10-80An aliphatic group. In some embodiments, RLDIs unsubstituted C20-80An aliphatic group. In some embodiments, RLDIs unsubstituted C10-70An aliphatic group. In some embodiments, RLDIs unsubstituted C20-70An aliphatic group. In some embodiments, RLDIs unsubstituted C10-60An aliphatic group. In some embodiments, RLDIs unsubstituted C20-60An aliphatic group. In some embodiments, RLDIs unsubstituted C10-50An aliphatic group. In some embodiments, RLDIs unsubstituted C20-50An aliphatic group. In some embodiments, RLDIs unsubstituted C10-40An aliphatic group. In some embodiments, RLDIs unsubstituted C20-40An aliphatic group. In some embodiments, RLDIs unsubstituted C10-30An aliphatic group. In some embodiments, RLDIs unsubstituted C20-30An aliphatic group.
In some embodiments, RLDIs not hydrogen. In some embodiments, RLDIs a lipid moiety. In some embodiments, RLDIs a targeting moiety. In some embodiments, RLDIs a targeting moiety comprising a carbohydrate moiety. In some embodiments, RLDIs a GalNAc moiety.
In some embodiments, RTDIs RLDWherein R isLDIndependently as described in this disclosure. In some embodiments, RTDIs RCDWherein R isCDIndependently as described in this disclosure.
In some embodiments, RCDIs an optionally substituted linear or branched radical selected from C1-30Aliphatic radical and C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon1-30A heteroaliphatic group wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-; and one or more carbon atoms optionally and independently passing through CyLAnd (6) replacing. In some embodiments, RCDIs an optionally substituted linear or branched radical selected from C1-30Aliphatic radical and C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon1-30A heteroaliphatic group wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-、-OP(O) (OR ') O-, -OP (O) (SR') O-, -OP (O)) (R ') O-, -OP (O)) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-; and one or more carbon atoms are independently replaced by a monosaccharide, disaccharide or polysaccharide moiety. In some embodiments, RCDIs an optionally substituted linear or branched radical selected from C1-30Aliphatic radical and C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon1-30A heteroaliphatic group wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, -C (R')2-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-; and one or more carbon atoms are independently replaced with a GalNac moiety.
In some embodiments, the present disclosure provides salts of oligonucleotides and pharmaceutical compositions thereof. In some embodiments, the salt is a pharmaceutically acceptable salt. In some embodiments, each hydrogen ion that can donate to the base (e.g., under conditions of aqueous solution, pharmaceutical composition, etc.) is not H+And (4) cation replacement. For example, in some embodiments, the pharmaceutically acceptable salt of the oligonucleotide is a full metal ion salt, wherein each hydrogen ion (e.g., -OH, -SH, etc.) of each internucleotide linkage (e.g., a native phosphate linkage, a phosphorothioate diester linkage, etc.) is replaced with a metal ion. In some embodiments, the provided salt is the full sodium salt. In some embodiments, the pharmaceutically acceptable salt provided is the full sodium salt. In some embodiments, the provided salt is a full sodium salt, wherein is natural phosphorusEach internucleotide linkage of the acid ester linkage (acid form-O-p (O) (oh) -O-) (if present) is present in its sodium salt form (-O-p (O) (ona) -O-) and each internucleotide linkage of the phosphorothioate diester linkage (acid form-O-p (O) (sh) -O-) (if present) is present in its sodium salt form (O-p (O) (sna) -O-).
In some embodiments, a provided compound (e.g., a provided oligonucleotide) has a purity of 60% -100%. In some embodiments, the purity is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the purity is at least 60%. In some embodiments, the purity is at least 70%. In some embodiments, the purity is at least 80%. In some embodiments, the purity is at least 85%. In some embodiments, the purity is at least 90%. In some embodiments, the purity is at least 91%. In some embodiments, the purity is at least 92%. In some embodiments, the purity is at least 93%. In some embodiments, the purity is at least 94%. In some embodiments, the purity is at least 95%. In some embodiments, the purity is at least 96%. In some embodiments, the purity is at least 97%. In some embodiments, the purity is at least 98%. In some embodiments, the purity is at least 99%. In some embodiments, the purity is at least 99.5%.
In some embodiments, a provided compound (e.g., a provided oligonucleotide) has a diastereomeric purity of 60% -100%. In some embodiments, the diastereomeric purity is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, a chiral element (e.g., a chiral center (carbon, phosphorus, etc.)) of a provided compound (e.g., a provided oligonucleotide) has a diastereomeric purity of 60% -100%. In some embodiments, a chiral element (e.g., a chiral center (carbon, phosphorus, etc.)) has a diastereomeric purity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the diastereomeric purity is at least 60%. In some embodiments, the diastereomeric purity is at least 70%. In some embodiments, the diastereomeric purity is at least 80%. In some embodiments, the diastereomeric purity is at least 85%. In some embodiments, the diastereomeric purity is at least 90%. In some embodiments, the diastereomeric purity is at least 91%. In some embodiments, the diastereomeric purity is at least 92%. In some embodiments, the diastereomeric purity is at least 93%. In some embodiments, the diastereomeric purity is at least 94%. In some embodiments, the diastereomeric purity is at least 95%. In some embodiments, the diastereomeric purity is at least 96%. In some embodiments, the diastereomeric purity is at least 97%. In some embodiments, the diastereomeric purity is at least 98%. In some embodiments, the diastereomeric purity is at least 99%. In some embodiments, the diastereomeric purity is at least 99.5%.
In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or more chiral elements of a provided compound each independently have a diastereomeric purity as described herein. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or more chiral carbon centers of a provided compound each independently have diastereomeric purity as described herein. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or more chiral phosphorus centers of a provided compound each independently have a diastereomeric purity as described herein.
In some embodiments, at least 5% -100% of all chiral elements of a provided compound each independently have a diastereomeric purity as described herein. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of all chiral elements of a provided compound each independently have a diastereomeric purity as described herein. In some embodiments, at least 5% -100% of all chiral phosphorus centers of a provided compound each independently have a diastereomeric purity as described herein. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of all chiral phosphorus centers of a provided compound each independently have a diastereomeric purity as described herein.
In some embodiments, each chiral element independently has a diastereomeric purity as described herein. In some embodiments, each chiral center independently has a diastereomeric purity as described herein. In some embodiments, each chiral carbon center independently has a diastereomeric purity as described herein. In some embodiments, each chiral phosphorus center independently has a diastereomeric purity as described herein.
In some embodiments, provided compounds (e.g., oligonucleotides and/or compositions thereof) can modulate the activity and/or function of a C9orf72 target. In some embodiments, a C9orf72 target gene is a gene intended to alter the expression and/or activity of one or more C9orf72 gene products (e.g., RNA and/or protein products) with which it is associated. In many embodiments, it is intended to inhibit the C9orf72 target gene. Thus, when a C9orf72 oligonucleotide as described herein acts on a particular C9orf72 target gene, the presence and/or activity of one or more gene products of the C9orf72 gene is altered in the presence of the oligonucleotide compared to in the absence of the oligonucleotide.
In some embodiments, the C9orf72 target is a specific allele (e.g., pathological allele) intended to alter the expression and/or activity of one or more products (e.g., RNA and/or protein products) with which it is associated. In many embodiments, the C9orf72 target allele is an allele whose presence and/or expression is correlated with (e.g., associated with) the presence, incidence, and/or severity of one or more diseases and/or disorders (e.g., C9orf 72-associated disorders). Alternatively or additionally, in some embodiments, the C9orf72 target allele is an allele that is associated with an improvement in one or more aspects of the disease and/or disorder (e.g., delayed onset, reduced severity, response to other therapies, etc.) because of altered levels and/or activities of one or more gene products thereof. In some such embodiments, C9orf72 oligonucleotides and methods thereof as described herein can preferentially or specifically target a pathological allele relative to a non-pathological allele (e.g., one or more less/unrelated alleles). In some embodiments, the pathological allele of C9orf72 comprises repeat amplification, e.g., a hexanucleotide repeat amplification (HRE), e.g., more than about 30 and up to 500 or 1000 or more hexanucleotide repeat amplification.
In some embodiments, the C9orf72 target sequence is a sequence to which an oligonucleotide as described herein binds. In many embodiments, the C9orf72 target sequence is identical to or the corresponding complement of a provided oligonucleotide or contiguous residues therein (e.g., the provided oligonucleotide includes a target binding sequence identical to or the corresponding complement of the C9orf72 target sequence). In some embodiments, few differences/mismatches are tolerated between (the relevant part of) the oligonucleotide and its target sequence. In many embodiments, the C9orf72 target sequence is present within the C9orf72 target gene. In many embodiments, the C9orf72 target sequence is present within a transcript (e.g., mRNA and/or pre-mRNA) produced by the C9orf72 target gene. In some embodiments, the C9orf72 target sequence includes one or more allelic sites (i.e., the location within the C9orf72 target gene where allelic variation occurs). In some such embodiments, provided oligonucleotides bind preferentially or specifically to one allele relative to one or more other alleles.
In some embodiments, C9ORF72 (chromosome 9 open reading frame 72) is a gene or gene product thereof, also referred to as C9ORF72, C9, ALSFTD, FTDALS, FTDALSl, DENNL 72; external ID: MGI: 1920455 HomoloGene: 10137 GeneCards: c9orf 72. C9orf72 is also informally referred to as C9. C9orf72 ortholog: species: human Entrez: 203228, respectively; ensembl: ENSG 00000147894; UniProt: q96LT 7; refseq (mrna): NM _145005NM _001256054 NM _ 018325; RefSeq (protein): NP _001242983 NP _060795 NP _ 659442; position (UCSC): and (2) Chr 9: 27.55-27.57 Mb; species: mouse Entrez: 73205; ensembl: ensusg 00000028300; UniProt: q6DFW 0; refseq (mrna): NM-001081343; RefSeq (protein): NP-00107481; position (UCSC): chr 4: 35.19-35.23 Mb. Nucleotides encoding C9orf72 include, but are not limited to, GENBANK accession No. NM _ 001256054.1; GENBANK accession No. NT _ 008413.18; GENBANK accession number BQ 068108.1; GENBANK accession No. NM _ 018325.3; GENBANK accession number DN 993522.1; GENBANK accession No. NM _ 145005.5; GENBANK accession number DB 079375.1; GENBANK accession number BU 194591.1; sequence identifier 4141_014_ A5; a sequence identifier 4008_73_ a; and GENBANK accession No. NT _ 008413.18. C9orf72 is reported to be a 481 amino acid protein with a molecular weight of 54328Da, which can undergo post-translational modifications of ubiquitination and phosphorylation. It was reported that the expression level of C9orf72 may be highest in the central nervous system, and the protein is localized in the cytoplasm of neurons as well as presynaptic terminals. C9orf72 is reported to play a role in the regulation of endosomal and lysosomal trafficking, and has been shown to interact with RAB proteins involved in autophagy and endocytic transport. C9orf72 was reported to activate RAB5, RAB5 is the gtpase enzyme mediating early endosomal trafficking. Mutations in C9orf72 were reported to be associated with ALS and FTD. DeJesus-Hernandez et al 2011 Neuron 72: 245-256; renton et al 2011 Neuron 72: 257-; and Itzcovich et al 2016.neurobiol. aging. [ neurobiology and aging ] Vol 40, pp 192.e13-192.e 15. It has been reported that repeated amplifications of hexanucleotides (e.g., (GGGGCC) n) in C9orf72 may be present in individuals with neurological diseases such as C9orf 72-related disorders.
In some embodiments, C9orf72 is not capitalized and is denoted as C9orf 72.
In some embodiments, the C9orf72 oligonucleotide may comprise any of a variety of linkers, additional moieties (including but not limited to targeting moieties), and/or be chirally controlled, and/or have any of a variety of base sequences and/or chemical structures or forms as described herein.
Various linkers, carbohydrate moieties, and targeting moieties (including many known in the art) can be used in accordance with the present disclosure. In some embodiments, the carbohydrate moiety is a targeting moiety. In some embodiments, the targeting moiety is a carbohydrate moiety.
In some embodiments, the present disclosure provides chirally controlled oligonucleotides and oligonucleotide compositions. For example, in some embodiments, provided compositions contain a non-random level or a controlled level of one or more individual oligonucleotide types, wherein the oligonucleotide types are defined by: 1) a base sequence; 2) a skeletal linkage mode; 3) pattern of backbone chiral centers; and 4) patterns of backbone P-modification. In some embodiments, a particular oligonucleotide type may be defined by: 1A) base identity; 1B) a base modification pattern; 1C) a sugar modification mode; 2) a skeletal linkage mode; 3) pattern of backbone chiral centers; and 4) patterns of backbone P-modification. In some embodiments, oligonucleotides of the same oligonucleotide type are identical. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide composition of oligonucleotides, wherein the composition comprises a plurality of oligonucleotides at non-random or controlled levels, wherein the plurality of oligonucleotides share a common base sequence and comprise the same configuration of linkage phosphorus at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chiral internucleotide linkages (chirally controlled internucleotide linkages). In some embodiments, the predetermined level of oligonucleotide and/or the plurality of oligonucleotides provided, for example, has the formula O-I, Ac-[-LM-(RD)a]b、[(Ac)a-LM]b-RD、(Ac)a-LM-(Ac)bOr (A)c)a-LM-(RD)bThose oligonucleotides of (a), comprise 1-30 chirally controlled internucleotide linkages. In some embodiments, provided C9orf72 oligonucleotides comprise 2-30 chiral controlled internucleotide linkages. In some embodiments, provided oligonucleotides comprise 5-30 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotides comprise 10-30 chirally controlled internucleotide linkages. In some embodimentsThe provided oligonucleotides comprise 1 chirally controlled internucleotide linkage. In some embodiments, provided oligonucleotides comprise 2 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotides comprise 3 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotides comprise 4 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotides comprise 5 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotides comprise 6 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotides comprise 7 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotides comprise 8 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotides comprise 9 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotides comprise 10 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotides comprise 11 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotides comprise 12 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotides comprise 13 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotides comprise 14 chirally controlled internucleotide linkages. In some embodiments, provided oligonucleotides have 15 chirality controlled internucleotide linkages. In some embodiments, provided oligonucleotides have 16 chirality controlled internucleotide linkages. In some embodiments, provided oligonucleotides have 17 chirality controlled internucleotide linkages. In some embodiments, provided oligonucleotides have 18 chirality controlled internucleotide linkages. In some embodiments, provided oligonucleotides have 19 chirality controlled internucleotide linkages. In some embodiments, provided oligonucleotides have 20 chirality controlled internucleotide linkages. In some embodiments, about 1% to 100% of all internucleotide linkages are chirally controlled internucleotide linkages. In some embodiments, the percentage is about 5% -100%. In some implementationsIn examples, the percentage is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 965, 96%, 98%, or 99%. In some embodiments, the percentage is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 965, 96%, 98%, or 99%.
In some embodiments, provided oligonucleotides are unimers. In some embodiments, provided oligonucleotides are P-modified unimers. In some embodiments, provided oligonucleotides are stereounimers. In some embodiments, provided oligonucleotides are stereounimers of configuration Rp in some embodiments, provided oligonucleotides are stereounimers of configuration Sp
In some embodiments, the provided oligonucleotides are alternators. In some embodiments, the provided oligonucleotides are P-modified alternators. In some embodiments, provided oligonucleotides are stereo-alternators.
In some embodiments, provided oligonucleotides are block entities. In some embodiments, provided oligonucleotides are P-modified blocks. In some embodiments, provided oligonucleotides are stereoblock.
In some embodiments, the provided oligonucleotides are gapmers.
In some embodiments, provided oligonucleotides are skips.
In some embodiments, provided oligonucleotides are semimers. In some embodiments, a semimer is an oligonucleotide whose sequence at the 5 'end or the 3' end region has a structural feature not possessed by the remainder of the oligonucleotide. In some embodiments, the 5 'end or the 3' end region has or comprises 2 to 20 nucleotides. In some embodiments, the structural feature is a base modification. In some embodiments, the structural feature is a sugar modification. In some embodiments, the structural feature is a P modification. In some embodiments, the structural feature is the stereotaxic nature of the chiral internucleotide linkageIn some embodiments, the structural feature is or comprises stereochemistry of base modifications, sugar modifications, P modifications, or chiral internucleotide linkages, or combinations thereof.A semimer is an oligonucleotide in which each sugar moiety of the 5 'end region shares a common modification.A semimer is an oligonucleotide in which each sugar moiety of the 3' end region shares a common modification.A common sugar modification of the 5 'or 3' end region is not shared by any other sugar moiety in the oligonucleotideTMOr ENATMBicyclic sugar modified nucleoside) and the other terminal region comprises the sequence of a nucleoside having a different sugar moiety, such as a substituted or unsubstituted 2' -O-alkyl sugar modified nucleoside, a bicyclic sugar modified nucleoside, or a natural nucleoside. In some embodiments, provided oligonucleotides are combinations of one or more of a monomer, an altemative, a block, a notch, a semimer, and a skip. In some embodiments, provided oligonucleotides are combinations of one or more of monomers, alternators, segregants, gapmers, and skips. For example, in some embodiments, provided oligonucleotides are alternators and gapmers. In some embodiments, the provided nucleotides are notch and skip. One skilled in the chemical and synthetic arts will recognize that many other combinations of modes are available and are limited only by the commercial availability and/or synthetic feasibility of the components required for synthesizing the provided oligonucleotides according to the methods of the present disclosure. In some embodiments, the semi-polymeric structure provides advantageous benefits. In some embodiments, provided oligonucleotides are 5 'semimers comprising a modified sugar moiety in the 5' terminal sequence. In some embodiments, provided oligonucleotides are 5 ' semimers comprising a modified 2 ' sugar moiety in the 5 ' terminal sequence.
In some embodiments, provided oligonucleotides comprise one or more optionally substituted nucleotides. In some embodiments, provided oligonucleotides comprise one or more modified nucleotides. In some embodiments, provided oligonucleotides comprise one or more optionally substituted nucleosides. In some embodiments, provided oligonucleotides comprise one or more modified nucleosides. In some embodiments, provided oligonucleotides comprise one or more optionally substituted LNAs.
In some embodiments, provided oligonucleotides comprise one or more optionally substituted nucleobases. In some embodiments, provided oligonucleotides comprise one or more optionally substituted natural nucleobases. In some embodiments, provided oligonucleotides comprise one or more optionally substituted modified nucleobases. In some embodiments, provided oligonucleotides comprise one or more 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytosine, or 5-carboxycytosine. In some embodiments, provided oligonucleotides comprise one or more 5-methylcytidines.
In some embodiments, provided oligonucleotides, for example, of formula O-I, Ac-[-LM-(RD)a]b、[(Ac)a-LM]b-RD、(Ac)a-LM-(Ac)bOr (A)c)a-LM-(RD)bEach nucleobase of one of (a) is independently an optionally substituted or protected nucleobase of adenine, cytosine, guanosine, thymine or uracil. In some embodiments, each BA is independently an optionally substituted or protected nucleobase of adenine, cytosine, guanosine, thymine or uracil. As will be appreciated by those skilled in the art, a variety of protected nucleobases may be used in accordance with the present disclosure, including those widely known in the art, such as those used in oligonucleotide preparation (e.g., the protected nucleobases in WO/2010/064146, WO/2011/005761, WO/2013/012758, WO/2014/010250, US2013/0178612, WO/2014/012081, WO/2015/107425, WO2017/015555, and WO2017/062862, willThe protected nucleobases in each of the documents are incorporated herein by reference).
In some embodiments, provided oligonucleotides comprise one or more optionally substituted sugars. In some embodiments, provided oligonucleotides comprise one or more optionally substituted sugars present in naturally occurring DNA and RNA. In some embodiments, provided oligonucleotides comprise one or more optionally substituted ribose or deoxyribose sugars. In some embodiments, provided oligonucleotides comprise one or more optionally substituted ribose or deoxyribose sugars, wherein one or more hydroxyl groups of the ribose or deoxyribose moiety are optionally and independently halogen, R ', -N (R')2-OR ' OR-SR ', wherein each R ' is independently described in the disclosure. In some embodiments, provided oligonucleotides comprise one or more optionally substituted deoxyribose sugars, wherein the 2 ' position of the deoxyribose sugar is optionally and independently halogen, R ', -N (R ')2-OR ' OR-SR ', wherein each R ' is independently described in the disclosure. In some embodiments, provided oligonucleotides comprise one or more optionally substituted deoxyribose sugars, wherein the 2' position of the deoxyribose sugar is optionally and independently substituted with a halogen. In some embodiments, provided oligonucleotides comprise one or more optionally substituted deoxyribose sugars, wherein the 2' position of the deoxyribose sugar is optionally and independently substituted with one or more-F halogen. In some embodiments, provided oligonucleotides comprise one OR more optionally substituted deoxyribose sugars, wherein the 2 ' position of the deoxyribose sugar is optionally and independently substituted with-OR ', wherein each R ' is independently described in the present disclosure. In some embodiments, provided oligonucleotides comprise one OR more optionally substituted deoxyribose sugars, wherein the 2 ' position of the deoxyribose sugar is optionally and independently substituted with-OR ', wherein each R ' is independently an optionally substituted C1-C6An aliphatic group. In some embodiments, provided oligonucleotides comprise one OR more optionally substituted deoxyribose sugars, wherein the 2 ' position of the deoxyribose sugar is optionally and independently substituted with-OR ', wherein each R ' is independently optionally substitutedC1-C6An alkyl group. In some embodiments, provided oligonucleotides comprise one or more optionally substituted deoxyribose sugars, wherein the 2' position of the deoxyribose sugar is optionally and independently substituted with-OMe. In some embodiments, provided oligonucleotides comprise one or more optionally substituted deoxyribose sugars, wherein the 2' position of the deoxyribose sugar is optionally and independently substituted with-O-methoxyethyl.
In some embodiments, the provided oligonucleotides are single stranded oligonucleotides. In some embodiments, the provided single-stranded C9orf72 oligonucleotide further comprises one or more additional strands that are partially or fully complementary to the single-stranded C9orf72 oligonucleotide.
In some embodiments, the provided oligonucleotides are mixed oligonucleotide strands. In certain embodiments, the provided oligonucleotides are partially hybrid oligonucleotide strands. In certain embodiments, the provided oligonucleotides are fully hybrid oligonucleotide strands. In certain embodiments, the provided oligonucleotides are double-stranded oligonucleotides. In certain embodiments, the provided oligonucleotides are triplex oligonucleotides (e.g., triplexes).
In some embodiments, provided C9orf72 oligonucleotides are chimeric. For example, in some embodiments, provided oligonucleotides (e.g., C9orf72 oligonucleotides whose base sequences comprise, consist of, or comprise a portion of the base sequences of C9orf72 oligonucleotides disclosed herein) are DNA-RNA chimeras, DNA-LNA chimeras, chimeras comprising any two or more of DNA, RNA, LNA, 2' modified sugar, and the like.
In some embodiments, the C9orf72 oligonucleotide may comprise the chemical structure described in WO 2012/030683.
In some embodiments, provided oligonucleotides are therapeutic agents.
In some embodiments, provided oligonucleotides include nucleic acid analogs such as GNA, LNA, PNA, TNA, F-HNA (F-THP or 3' -fluorotetrahydropyran), MNA (a mannitol nucleic acid, such as Leumann 2002 bioorg. Med. chem. [ J. Bioorganic and medicinal Chemicals ] 10: 841-854), ANA (anitol nucleic acid), and N-morpholino.
In some embodiments, provided oligonucleotides are about 2-500 nucleotide units in length. In some embodiments, provided oligonucleotides are about 5-500 nucleotide units in length. In some embodiments, provided oligonucleotides are about 10-50 nucleotide units in length. In some embodiments, provided oligonucleotides are about 15-50 nucleotide units in length. In some embodiments, each nucleotide unit independently comprises a heteroarylnucleobase unit (e.g., adenine, cytosine, guanosine, thymine, and uracil, each of which is optionally and independently substituted or protected), a sugar unit comprising a 5-to 10-membered heterocyclic ring, and an internucleotide linkage having the structure of formula I.
In some embodiments, provided oligonucleotides are about 15 to about 30 nucleotide units in length. In some embodiments, provided oligonucleotides are about 10 to about 25 nucleotide units in length. In some embodiments, provided oligonucleotides are about 15 to about 22 nucleotide units in length. In some embodiments, provided oligonucleotides are about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide units in length.
In some embodiments, the oligonucleotide is at least 15 nucleotide units in length. In some embodiments, the oligonucleotide is at least 16 nucleotide units in length. In some embodiments, the oligonucleotide is at least 17 nucleotide units in length. In some embodiments, the oligonucleotide is at least 18 nucleotide units in length. In some embodiments, the oligonucleotide is at least 19 nucleotide units in length. In some embodiments, the oligonucleotide is at least 20 nucleotide units in length. In some embodiments, the oligonucleotide is at least 21 nucleotide units in length. In some embodiments, the oligonucleotide is at least 22 nucleotide units in length. In some embodiments, the oligonucleotide is at least 23 nucleotide units in length. In some embodiments, the oligonucleotide is at least 24 nucleotide units in length. In some embodiments, the oligonucleotide is at least 25 nucleotide units in length. In some other embodiments, the oligonucleotide is at least 30 nucleotide units in length. In some other embodiments, the oligonucleotide is a double helix of a complementary strand that is at least 18 nucleotide units in length. In some other embodiments, the oligonucleotide is a double helix of a complementary strand that is at least 21 nucleotide units in length.
In some embodiments, oligonucleotides of one oligonucleotide type that are characterized by 1) a common base sequence and length, 2) a common pattern of backbone linkages, and 3) a common pattern of backbone chiral centers have the same chemical structure. For example, they have the same base sequence, the same nucleoside modification pattern, the same backbone linkage pattern (i.e., internucleotide linkage type pattern, e.g., phosphate, phosphorothioate, etc.), the same backbone chiral center pattern (i.e., bonded phosphorus stereochemistry (Rp/Sp) pattern), and the same backbone phosphorus modification pattern (e.g., "XLR" in formula I)1"mode of group").
Oligonucleotides
In some embodiments, the provided C9orf72 oligonucleotides can direct a decrease in expression, level, and/or activity of a C9orf72 target gene or gene product thereof. In some embodiments, provided C9orf72 oligonucleotides can direct a reduction in expression, level, and/or activity of a C9orf72 target gene or gene product thereof, and have a base sequence consisting of, comprising, or comprising a portion of (e.g., a stretch of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more consecutive bases) the base sequence of any of the C9orf72 oligonucleotides disclosed herein, and which comprise at least one non-naturally occurring modification of a base, a sugar, and/or an internucleotide linkage.
In some embodiments, provided compositions comprise an oligonucleotide. In some embodiments, provided oligonucleotides comprise one or more carbohydrate moieties. In some embodiments, provided oligonucleotides comprise one or more targeting moieties. Non-limiting examples of additional chemical moieties that can be conjugated to the oligonucleotide are shown in example 1.
In some embodiments, the provided oligonucleotides can direct a decrease in the expression, level, and/or activity of the C9orf72 target gene or gene product thereof. In some embodiments, the provided oligonucleotides can direct a decrease in the expression, level, and/or activity of the C9orf72 target gene or gene product thereof via RNase H-mediated knock down. In some embodiments, provided oligonucleotides can direct a decrease in the expression, level, and/or activity of a C9orf72 target gene or gene product thereof by spatially blocking translation after binding to the C9orf72 target gene mRNA and/or by altering or interfering with mRNA splicing. In some embodiments, the C9orf72 target gene comprises a hexanucleotide repeat amplification.
In some embodiments, C9orf72 oligonucleotides include nucleic acids (including antisense compounds), including but not limited to antisense oligonucleotides (ASOs), oligonucleotides, double-stranded and single-stranded sirnas; and the C9orf72 oligonucleotide can be co-administered with or used as part of a treatment regimen with: aptamers, antibodies, peptides, small molecules, and/or other agents capable of inhibiting the expression of: c9orf72 antisense transcript or gene and/or expression product or gene product thereof; or increasing the expression, activity and/or level of a gene or gene product comprising a repeatedly amplified C9orf72 transcript or gene product thereof; or a gene or gene product associated with a C9orf 72-related disorder.
In some embodiments, provided oligonucleotides capable of directing a reduction in expression, level, and/or activity of a C9orf72 target gene or gene product thereof have a base sequence (or portion thereof), a chemical modification pattern (or portion thereof), a structural element or portion or form or portion thereof described herein. In some embodiments, provided oligonucleotides capable of directing a reduction in expression, level, and/or activity of a C9orf72 target gene or gene product thereof have the base sequence (or portion thereof), chemical modification pattern (or portion thereof), form, or structural element or form or portion thereof described herein of any of the oligonucleotides disclosed herein, e.g., in table 1A or figures, or elsewhere herein.
In some embodiments, the C9orf72 oligonucleotide can hybridize to a C9orf72 nucleic acid derived from either DNA strand. In some embodiments, the C9orf72 oligonucleotide can hybridize to the C9orf72 antisense or sense transcript. In some embodiments, the C9orf72 oligonucleotide can hybridize to C9orf72 nucleic acid at any stage of RNA processing, including but not limited to pre-mRNA or mature mRNA. In some embodiments, the C9orf72 oligonucleotide can hybridize to any element of the C9orf72 nucleic acid or its complementary sequence, including but not limited to: promoter region, enhancer region, transcription termination region, translation initiation signal, translation termination signal, coding region, non-coding region, exon, intron, 5 'UTR, 3' UTR, repeat region, hexanucleotide repeat amplification, splice junction, intron/exon or exon/intron junction of C9orf72 nucleic acid, exon: exon splicing junctions, exon splicing quiescence (ESS), Exon Splicing Enhancer (ESE), exon 1a, exon 1b, exon 1c, exon 1d, exon 1e, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, or intron 10. Introns alternate with exons; intron 1 is between exon 1 (or 1a or 1b or 1c, etc.) and exon 2; intron 2 is between exon 2 and exon 3; and so on. A graphical representation of the location of exons and introns in transcripts of C9orf72 variants is shown, for example, in the literature of WO 2014/062691.
In some embodiments, the C9orf72 sequence is represented as:
in some embodiments, the sequence of exon 1a is represented as SEQ ID NO: nt 1137-1216 of 1; exon 1b is represented as SEQ ID NO: 1510-1572 of 1; exon 1c is represented as SEQ ID NO: 1137-1294 of 1; exon 1d is represented as SEQ ID NO: 1241-1279 of 1; and exon 1e is represented as SEQ ID NO: 1135 and 1169 of 1. In some embodiments, intron 1 is represented by SEQ ID NO: 1 (if the transcript comprises exon 1a), 1573-7838(1b), 1295-7838(1c), 1280-7838(1d) or 1170-7838(1 e). In some embodiments, the sequence of exon 2 is represented as SEQ ID NO: nt 7839-8326 of 1; exon 3 is represented as SEQ ID NO: 9413-; exon 4 is represented as SEQ ID NO: 12527-12622 of 1; exon 5 is represented as SEQ ID NO: 13354 of 1 and 13418; exon 6 is represented as SEQ ID NO: 14704 — 14776 of 1; exon 7 is represented as SEQ ID NO: 16396 and 16512 of 1; exon 8 is represented as SEQ ID NO: 18207-18442 of 1; exon 9 is represented as SEQ ID NO: 24296-; exon 10 is represented as SEQ ID NO: 26337-26446 of 1; and exon 11 is represented as SEQ ID NO: 26581-28458 of 1. In some embodiments, the intron is located between the exons. The portion upstream (5 ') of exon 1a, 1b, 1c, 1d or 1e comprises the 5' -UTR. The portion downstream (3 ') of exon 11 is the 3' -UTR.
In some embodiments, the C9orf72 oligonucleotide recognizes a site near repeat amplification within intron 1 of C9orf72 and is selected from: WV-6967, WV-3690, WV-6976, WV-7002, WV-6970, WV-3689, WV-6960, WV-7001, WV-6974, WV-6978, WV-6952, WV-6989, WV-3704, WV-7007, WV-7004, WV-6951, WV-6474, WV-3688, WV-7006, WV-6977, WV-6955, WV-6995, WV-6972, WV-7003, WV-6989, WV-6996, WV-7005, WV-6989, WV-6971, WV-6985, WV-6488, WV-6489, WV-6989, WV-6980, WV-6981, or an oligonucleotide having the same base sequence as any of these nucleotides. In some embodiments, the C9orf72 oligonucleotide recognizes a site within exon 1a of C9orf72 and is selected from: WV-3677, WV-6940, WV-3683, WV-6931, WV-3679, WV-6927, WV-6922, WV-6937, WV-6926, WV-3685, WV-6930, WV-6932, WV-6928, WV-6933, WV-6936, WV-7027, WV-3678, WV-8114, WV-8122, WV-8311, WV-8315, WV-8312, WV-8313, WV-8314, WV-8316, WV-8317, or WV-8318, or any oligonucleotide having the same base sequence as any of these oligonucleotides. In some embodiments, the C9orf72 oligonucleotide recognizes a site within a C9orf72 Antisense (AS) transcript and is selected from: WV-3723, WV-3737, WV-3719, WV-3730, WV-3722, WV-3743, WV-3745, WV-3739, WV-3724, WV-3732, WV-3734, WV-3733, WV-3720, WV-3721, WV-3731, or any oligonucleotide having the same base sequence of any of these oligonucleotides. In some embodiments, the C9orf72 oligonucleotide recognizes a site within the C9orf72 exon 2 transcript and is selected from: WV-3662 and WV-7118, or any oligonucleotide having the same base sequence as any of these oligonucleotides. In some embodiments, the C9orf72 oligonucleotide can hybridize to a portion of the C9orf72 sequence represented by GENBANK accession No. NT _008413.18, or a complement thereof. In some embodiments, the C9orf72 oligonucleotide can hybridize to a portion of the C9orf72 pre-mRNA represented by the region beginning in the region from the start of exon 1a to the start of exon 1 b. In some embodiments, the C9orf72 oligonucleotide can hybridize to a portion of the C9orf72 pre-mRNA represented by the region beginning in the region from the end site of exon 1a to the start site of exon 1 b. In some embodiments, the c9orf72 oligonucleotide recognizes a site that spans the junction between an intron and an exon.
In some embodiments, the c9orf72 oligonucleotide spans the junction between exon 1b and intron 1. In some embodiments, the c9orf72 oligonucleotide spans the junction between exon 1b and intron 1 and has a base sequence of 15 contiguous bases of sequence CCTCACTCACCCACTCGCCA, comprising sequence CCTCACTCACCCACTCGCCA, or comprising sequence CCTCACTCACCCACTCGCCA.
Without wishing to be bound by any particular scientific theory, the present disclosure indicates that sequence CCTCACTCACCCACTCGCCA spans the junction of the reported exon 1b of c9orf72 mRNA variant 2 or V2 (which does not have a six nucleotide repeat) with intron 1 and can prevent an oligonucleotide whose sequence is sequence CCTCACTCACCCACTCGCCA, comprises sequence CCTCACTCACCCACTCGCCA, or comprises 15 contiguous bases of sequence CCTCACTCACCCACTCGCCA from binding to this site by a splicing machinery. This sequence is present in c9orf72 mRNA variants V1, V2 and V3, but the sequence is sequence CCTCACTCACCCACTCGCCA, an oligonucleotide comprising sequence CCTCACTCACCCACTCGCCA or comprising 15 contiguous bases of sequence CCTCACTCACCCACTCGCCA is particularly effective in degrading disease-related variants V1 and V3 relative to non-disease-related V2. Without wishing to be bound by any particular theory, the present disclosure suggests that sequence CCTCACTCACCCACTCGCCA is intermediate to the reported introns in V1 and V3; sequence CCTCACTCACCCACTCGCCA spans the junction of the reported exon (1b) and intron (1) of V2 and can be sterically blocked from access to this site by a splicing machinery. In some embodiments, the C9orf72 oligonucleotide comprises a base sequence that is complementary to a 5' branching site at an intron-exon junction. In some embodiments, the C9orf72 oligonucleotide comprises a sequence complementary to the 5' branching site at the junction of exon 1 of C9orf72 and intron 1 of C9orf 72. In some embodiments, the 5' branching site at the junction of exon 1 and intron 1 of C9orf72 comprises the base sequence GTGAGT. In some embodiments, the C9orf72 oligonucleotide comprises a base sequence complementary to GTGAGT. In some embodiments, the oligonucleotide is capable of preferentially reducing the expression, level, and/or activity of a corresponding disease-associated allele of a gene or its gene product relative to the wild-type allele of the gene or its gene product, wherein the oligonucleotide has a base sequence that is complementary to both the disease-associated allele and the wild-type allele, and wherein the oligonucleotide binding site in the mRNA or DNA of the disease-associated allele has less access to the oligonucleotide than the oligonucleotide binding site in the mRNA or DNA expressing the wild-type allele. In some embodiments, accessibility of the oligonucleotide to a binding site in mRNA or DNA of a disease-associated allele is reduced due to the binding of splice mechanisms and/or other nucleic acids or proteins to mRNA or DNA of the disease-associated allele. In some embodiments, the disclosure pertains to: an oligonucleotide capable of preferentially reducing (or knocking down) the expression, level and/or activity of a corresponding mutant or disease-associated allele of a gene or gene product thereof relative to the wild-type or non-disease-associated allele of the gene or gene product thereof, wherein the oligonucleotide has a base sequence that is complementary to both the mutant or disease-associated allele and the wild-type or non-disease-associated allele, and wherein an oligonucleotide binding site (e.g., a sequence complementary to the oligonucleotide) in a nucleic acid of the mutant or disease-associated allele (e.g., chromosomal DNA, mRNA, pre-mRNA, etc.) has less access to the oligonucleotide (e.g., due to increased binding by a splicing machinery and/or other nucleic acids or proteins) than an oligonucleotide binding site in a nucleic acid of the wild-type or non-disease-associated allele.
In some embodiments, the C9orf72 oligonucleotide can hybridize to a portion of the C9orf72 pre-mRNA, nucleosides 27535000 through 27565000, or the complement thereof, represented by GENBANK accession No. NT _ 008413.18.
In some embodiments, the C9orf72 oligonucleotide can hybridize to an intron. In some embodiments, the C9orf72 oligonucleotide can hybridize to an intron comprising a hexanucleotide repeat.
In some embodiments, the C9orf72 oligonucleotide hybridizes to all C9orf72 variants derived from the sense strand. In some embodiments, antisense oligonucleotides described herein selectively hybridize to C9orf72 variants derived from the sense strand, including but not limited to variants comprising repeated amplifications of hexanucleotides. In some embodiments, the hexanucleotide repeat amplification comprises at least 24 repeats of any hexanucleotide. In some embodiments, the hexanucleotide repeat amplification comprises at least 30 repeats of any hexanucleotide. In some embodiments, the hexanucleotide repeat amplification comprises at least 50 repeats of any one of the hexanucleotides. In some embodiments, the hexanucleotide repeat amplification comprises at least 100 repeats of any one of the hexanucleotides. In some embodiments, the hexanucleotide repeat amplification comprises at least 200 repeats of any hexanucleotide. In some embodiments, the hexanucleotide repeat amplification comprises anyAt least 500 repeats of a hexanucleotide. In some embodiments, the hexanucleotide is GGGGCC, gggggggg, GGGGGC, GGGGCG, CCCCGG, ccccccc, GCCCCC, and/or CGCCCC. In some embodiments, the hexanucleotide GGGGGGCC is referred to as GGGGCCexp or (GGGGCC)nOr a repeat of the hexanucleotide GGGGCC.
In some embodiments, the C9orf72 target of the C9orf72 oligonucleotide is C9orf72 RNA that is not mRNA. In some embodiments, a provided oligonucleotide, e.g., a first plurality of oligonucleotides, in a provided composition comprises base modifications, sugar modifications, and/or internucleotide linkage modifications. In some embodiments, provided oligonucleotides comprise base modifications and sugar modifications. In some embodiments, provided oligonucleotides comprise base modifications and internucleotide linkage modifications. In some embodiments, provided oligonucleotides comprise sugar modifications and internucleotide modifications. In some embodiments, provided compositions comprise base modifications, sugar modifications, and internucleotide linkage modifications. Example chemical modifications such as base modifications, sugar modifications, internucleotide linkage modifications, and the like are widely known in the art and include, but are not limited to, those described in the present invention. In some embodiments, the modified base is substituted A, T, C, G or U. In some embodiments, the sugar modification is a 2' modification. In some embodiments, the 2' modification is a 2-F modification. In some embodiments, the 2 '-modification is 2' -OR1. In some embodiments, the 2 'modification is 2' -OR1Wherein R is1Is an optionally substituted alkyl group. In some embodiments, the 2 'modification is 2' -OMe. In some embodiments, the 2 'modification is 2' -MOE. In some embodiments, the modified sugar moiety is a bridged bicyclic or polycyclic ring. In some embodiments, the modified sugar moiety is a bridged bicyclic or polycyclic ring having 5-20 ring atoms, wherein one or more ring atoms are optionally and independently a heteroatom. Exemplary ring structures are widely known in the art, such as those found in BNAs, LNAs, and the like. In some embodiments, provided oligonucleotides comprise one or more modified internucleotide linkages and one or more natural phosphate linkages. In some embodimentsOligonucleotides and compositions thereof comprising modified internucleotide linkages and natural phosphate linkages provide improved properties, such as activity, and the like. In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the modified internucleotide linkage is a substituted phosphorothioate linkage.
In some embodiments, the disclosure provides a stereorandom oligonucleotide whose base sequence is, comprises, or comprises a portion of: the base sequence of any of the oligonucleotides described herein. In some embodiments, a portion of the base sequence is at least 15 consecutive bases thereof. In some embodiments, the disclosure provides an oligonucleotide whose base sequence is, comprises, or comprises a portion of: a base sequence of any of the oligonucleotides described herein; wherein the oligonucleotide comprises one or more sterically random internucleotide linkages. In some embodiments, the disclosure provides an oligonucleotide whose base sequence is, comprises, or comprises a portion of: a base sequence of any of the oligonucleotides described herein; wherein the oligonucleotide comprises one or more sterically random phosphorothioate internucleotide linkages.
In some embodiments, oligonucleotide properties can be modulated by optimizing stereochemistry (pattern of backbone chiral centers) and chemical modifications (modifications of bases, sugars, and/or internucleotide linkages) or patterns thereof.
In some embodiments, the pattern of backbone chiral centers in the C9orf72 oligonucleotide provides increased stability. In some embodiments, the backbone chiral center pattern provides unexpectedly increased activity. In some embodiments, the backbone chiral center pattern provides increased stability and activity. In some embodiments, the pattern of backbone chiral centers provides unexpectedly increased binding to certain proteins. In some embodiments, the pattern of backbone chiral centers provides unexpectedly enhanced delivery.
In some embodiments, the disclosure relates to a c9orf72 oligonucleotide, wherein the oligonucleotide comprises a backbone comprising at least one chiral center. In some embodiments, the disclosure relates to a c9orf72 oligonucleotide, wherein said oligonucleotide comprises a backbone comprising at least one chiral center that is a phosphorothioate in the Rp or Sp configuration.
In some embodiments, the C9orf72 oligonucleotide has a pattern of backbone chiral centers.
In some embodiments, the backbone chiral center pattern of the provided oligonucleotides or regions thereof (e.g., cores) comprises or is (Sp) m (Rp) n, (Rp) n (Sp) m, (Np) t [ (Op) n (Sp) m ] y, (Sp) t [ (Op) n (Sp) m ] y, (Np) t [ (Rp) n (Sp) m ] y, or (Sp) t [ (Rp) n (Sp) m ] y, wherein the variables are as described in the present disclosure. In some embodiments, y is 1. In some embodiments, the backbone chiral center pattern comprises or is (Sp) m (Rp) n, (Rp) n (Sp) m, (Np) t (Rp) n (Sp) m, (Sp) t (Rp) n (Sp) m, (Np) t [ (Rp) n (Sp) m ]2, (Sp) t [ (Rp) n (Sp) m ]2, (Np) t (Op) n (Sp) m, (Sp) t (Op) n (Sp) m, (Np) t [ (Op) n (Sp) m ]2, or (Sp) t [ (Op) n (Sp) m ] 2. In some embodiments, y is 2. In some embodiments, the pattern is (Np) t (Op/Rp) n (sp) m. In some embodiments, the pattern is (Np) t (Op/Rp) n (sp)1-5(Op/Rp) n (sp) m. In some embodiments, the pattern is (Np) t (Op/Rp) n (sp)2-5(Op/Rp) n (sp) m. In some embodiments, the pattern is (Np) t (Op/Rp) n (sp)2(Op/Rp) n (sp) m. In some embodiments, the pattern is (Np) t (Op/Rp) n (sp)3(Op/Rp) n (sp) m. In some embodiments, the pattern is (Np) t (Op/Rp) n (sp)4(Op/Rp) n (sp) m. In some embodiments, the pattern is (Np) t (Op/Rp) n (sp)5(Op/Rp) n (sp) m. In some embodiments, Np is Sp. In some embodiments, (Op/Rp) is Op. In some embodiments, (Op/Rp) is Rp. In some embodiments, Np is Sp and (Op/Rp) is Rp. In some embodiments, Np is Sp and (Op/Rp) is Op. In some embodiments, Np is Sp and at least one (Op/Rp) is Rp and at least one (Op/Rp) is Op. In some embodiments, the backbone chiral center pattern comprises or is (Rp) n (Sp) m, (Np) t (Rp) n (Sp) m, or (Sp) t (Rp) n (Sp) m, wherein m > 2. In some embodiments, the pattern of backbone chiral centers comprises or is (Rp) n (Sp) m, (Np) t (Rp) n (Sp) m, or (Sp) t (Rp) n (Sp) m, wherein n is 1, at least one t > 1, and at least one m > 2. In some embodiments, at least one n is 1, at least one t is not less than 1, and at least one m is not less than 2. In some embodiments, at least one n is 1, at least one t is not less than 2, and at least one m is not less than 3. In some embodiments, each n is 1. In some embodiments, at least one t > 1. In some embodiments, at least one t > 2. In some embodiments, at least one t > 3. In some embodiments, at least one t > 4. In some embodiments, at least one m > 1. In some embodiments, at least one m > 2. In some embodiments, at least one m > 3. In some embodiments, at least one m > 4. In some embodiments, the pattern of backbone chiral centers comprises one or more achiral native phosphate linkages. In some embodiments, the sum of m, t, and n (or the sum of m and n in the absence of t in a mode) is not less than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the sum is 5. In some embodiments, the sum is 6. In some embodiments, the sum is 7. In some embodiments, the sum is 8. In some embodiments, the sum is 9. In some embodiments, the sum is 10. In some embodiments, the sum is 11. In some embodiments, the sum is 12. In some embodiments, the sum is 13. In some embodiments, the sum is 14. In some embodiments, the sum is 15.
In some embodiments, the nucleotide unit comprising Op is Nu as described in the present disclosureO. For example, in some embodiments, NuOComprising a 5' substitution/modification as described in the present disclosure, e.g., -C (R) as described in the present disclosure5s)2-. In some embodiments, -C (R)5s)2Is 5MRd as described in this disclosure. In some embodiments, -C (R)5s)2Is 5MSd as described in this disclosure.
In some embodiments, the pattern of backbone chiral centers comprises or is (Rp) n (sp) m. In some embodiments, the pattern of backbone chiral centers comprises or is (Sp) t (Rp) n. In some embodiments, the pattern of backbone chiral centers comprises or is (Np) t (rp) n (sp) m. In some embodiments, the pattern of backbone chiral centers comprises or is (Sp) t (Sp) m, optionally with n achiral phosphodiester internucleotide linkages and/or sterically random (achiral controlled) chiral internucleotide linkages between the segment having (Sp) t and the segment having (Sp) m. In some embodiments, there are n achiral phosphodiester internucleotide linkages between the two. In some embodiments, there are n sterically random chiral internucleotide linkages between the two. In some embodiments, the pattern of backbone chiral centers comprises or is (Sp) t (Rp) n (Sp) m. In some embodiments, t and m are each independently equal to or greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
In some embodiments, the common pattern of backbone chiral centers in the provided oligonucleotides comprises pattern io-is-io-is-io、io-is-is-is-io、io-is-is-is-io-is、is-io-is-io、is-io-is-io、is-io-is-io-is、is-io-is-io-is-io、is-io-is-io-is-io-is-io、is-io-is-is-is-io、is-is-io-is-is-is-io-is-is、is-is-is-io-is-io-is-is-is、is-is-is-is-io-is-io-is-is-is-is、is-is-is-is-is、is-is-is-is-is-is、is-is-is-is-is-is-is、is-is-is-is-is-is-is-is、is-is-is-is-is-is-is-is-isOr ir-ir-irWherein isRepresents an internucleotide linkage in the Sp configuration; i.e. ioRepresents an achiral internucleotide linkage; and i isrRepresents an internucleotide linkage in the Rp configuration.
In some embodiments, the common pattern of backbone chiral centers (e.g., a C9orf72 oligonucleotide or a backbone chiral center pattern in a core or a wing or both wings thereof) comprises a pattern OSOSO, OSSSO, osssosos, SOSO, SOSOs, sosso, SOSOSOSO, sosssoso, ssossssoss, sssoss, ssssososs, sssssssssssssssss, ssssssssssssssssssssssssssssssssssssss, sssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssss, sssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssss, or RRR, wherein S represents a phosphorothioate in Sp configuration, O represents a phosphodiester, and.
In some embodiments, the achiral center is a phosphodiester-linked phosphorus linkage. In some embodiments, the chiral center in the Sp configuration is a phosphorothioate-linked phosphorus linkage. In some embodiments, the chiral center in the Rp configuration is a phosphorothioate-linked phosphorane.
In some embodiments, 5% or more of the internucleotide linkages in the provided oligonucleotides are modified internucleotide linkages. In some embodiments, 10% or more of the internucleotide linkages in the provided oligonucleotides are modified internucleotide linkages. In some embodiments, 15% or more of the internucleotide linkages in the provided oligonucleotides are modified internucleotide linkages. In some embodiments, 20% or more of the internucleotide linkages in the provided oligonucleotides are modified internucleotide linkages. In some embodiments, 25% or more of the internucleotide linkages in the provided oligonucleotides are modified internucleotide linkages. In some embodiments, 30% or more of the internucleotide linkages in the provided oligonucleotides are modified internucleotide linkages. In some embodiments, 35% or more of the internucleotide linkages in the provided oligonucleotides are modified internucleotide linkages. In some embodiments, 40% or more of the internucleotide linkages in the provided oligonucleotides are modified internucleotide linkages.
In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% as a result of the administration of the oligonucleotide. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% in total due to oligonucleotide-directed RNase H mediated knockdown. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% as a result of administering the oligonucleotide in one or more cells in vitro. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% due to oligonucleotide-directed RNaseH-mediated knockdown in one or more cells in vitro. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% as a result of administering the oligonucleotide at a concentration of 25nm or less in one or more cells in vitro. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% as a result of administering the oligonucleotide at a concentration of 10nm or less in one or more cells in vitro. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% as a result of administering the oligonucleotide at a concentration of 5nm or less in one or more cells in vitro. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% due to oligonucleotide-directed RNase H-mediated knockdown at a concentration of 5nm or less in one or more cells in vitro. In some embodiments, the one or more cells are one or more mammalian cells. In some embodiments, the one or more cells are one or more human cells. In some embodiments, the one or more cells are one or more hepatocytes. In some embodiments, the one or more cells are one or more Huh7 or Hep3B cells. In some embodiments, a concentration of 25nM or less of the C9orf72 oligonucleotide is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 20% in one or more cells in vitro. In some embodiments, a concentration of 25nM or less of the C9orf72 oligonucleotide is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 30% in one or more cells in vitro. In some embodiments, a concentration of 25nM or less of the C9orf72 oligonucleotide is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 40% in one or more cells in vitro. In some embodiments, a concentration of 25nM or less of the C9orf72 oligonucleotide is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 50% in one or more cells in vitro. In some embodiments, a concentration of 25nM or less of the C9orf72 oligonucleotide is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 60% in one or more cells in vitro. In some embodiments, a concentration of 25nM or less of the C9orf72 oligonucleotide is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 70% in one or more cells in vitro. In some embodiments, a concentration of 25nM or less of the C9orf72 oligonucleotide is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 80% in one or more cells in vitro. In some embodiments, a concentration of 25nM or less of the C9orf72 oligonucleotide is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 90% in one or more cells in vitro. In some embodiments, an oligonucleotide at a concentration of 25nM or less is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 20% in one or more cells in vitro. In some embodiments, an oligonucleotide at a concentration of 25nM or less is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 30% in one or more cells in vitro. In some embodiments, an oligonucleotide at a concentration of 25nM or less is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 40% in one or more cells in vitro. In some embodiments, an oligonucleotide at a concentration of 25nM or less is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 50% in one or more cells in vitro. In some embodiments, an oligonucleotide at a concentration of 25nM or less is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 60% in one or more cells in vitro. In some embodiments, an oligonucleotide at a concentration of 25nM or less is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 70% in one or more cells in vitro. In some embodiments, an oligonucleotide at a concentration of 25nM or less is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 80% in one or more cells in vitro. In some embodiments, an oligonucleotide at a concentration of 25nM or less is capable of reducing the expression or level of the C9orf72 target gene or gene product thereof by at least about 90% in one or more cells in vitro. In some embodiments, IC50 is an inhibitory concentration that reduces the expression or level of a C9orf72 target gene or gene product thereof by 50% in one or more cells in vitro.
In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, whose backbone chiral center pattern comprises (Sp) mRp or rp (Sp) m. In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, the backbone of whichThe chiral center pattern comprises Rp (Sp) m. In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, whose backbone chiral center pattern comprises (Sp) mRp. In some embodiments, m is 2. In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, whose backbone chiral center pattern comprises rp (sp)2. In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, having a backbone chiral center pattern comprising (Sp)2Rp(Sp)2. In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, whose backbone chiral center pattern comprises (Rp)2Rp(Sp)2. In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type having a backbone chiral center pattern comprising rpsprp (sp)2. In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type having a pattern of backbone chiral centers comprising sprprp (sp)2. In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, having a backbone chiral center pattern comprising (Sp)2Rp。
As defined herein, m is 1 to 50. In some embodiments, m is 1. In some embodiments, m is 2-50. In some embodiments, m is 2, 3, 4, 5, 6, 7, or 8. In some embodiments, m is 3, 4, 5, 6, 7, or 8. In some embodiments, m is 4, 5, 6, 7, or 8. In some embodiments, m is 5, 6, 7, or 8. In some embodiments, m is 6, 7, or 8. In some embodiments, m is 7 or 8. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 11. In some embodiments, m is 12. In some embodiments, m is 13. In some embodiments, m is 14. In some embodiments, m is 15. In some embodiments, m is 16. In some embodiments, m is 17. In some embodiments, m is 18. In some embodiments, m is 19. In some embodiments, m is 20. In some embodiments, m is 21. In some embodiments, m is 22. In some embodiments, m is 23. In some embodiments, m is 24. In some embodiments, m is 25. In some embodiments, m is greater than 25.
In some embodiments, the repeating pattern is (Sp) m (rp) n, wherein n is 1-10, and m is independently described in the present disclosure. In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, whose backbone chiral center pattern comprises (Sp) m (rp) n. In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, whose backbone chiral center pattern comprises (Sp) m (rp) n. In some embodiments, the repeating pattern is (Rp) n (sp) m, where n is 1-10, and m is independently described in the present disclosure. In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, whose backbone chiral center pattern comprises (Rp) n (sp) m. In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, whose backbone chiral center pattern comprises (Rp) n (sp) m. In some embodiments, (Rp) n (Sp) m is (Rp) (Sp)2. In some embodiments, (Sp) n (Rp) m is (Sp)2(Rp)。
In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, having a backbone chiral center pattern comprising (Sp) m (rp) n (Sp) t. In some embodiments, the repeating pattern is (Sp) m (rp) n (Sp) t, wherein n is 1-10, t is 1-50, and m is as described in the present disclosure. In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, having a backbone chiral center pattern comprising (Sp) m (rp) n (Sp) t. In some embodiments, the repeating pattern is (Sp) t (rp) n (Sp) m, wherein n is 1-10, t is 1-50, and m is as described in the present disclosure. In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, having a backbone chiral center pattern comprising (Sp) t (rp) n (Sp) m. In some embodiments, the disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, having a backbone chiral center pattern comprising (Sp) t (rp) n (Sp) m.
In some embodiments, the repeating pattern is (Np) t (Rp) n (Sp) m, wherein n is 1-10, t is 1-50, Np is independently Rp or Sp, and m is as described in the present disclosure. In some embodiments, the present disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type having a pattern of backbone chiral centers comprising (Np) t (rp) n (sp) m. In some embodiments, the present disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type having a pattern of backbone chiral centers comprising (Np) t (rp) n (sp) m. In some embodiments, the repeating pattern is (Np) m (Rp) n (Sp) t, wherein n is 1-10, t is 1-50, Np is independently Rp or Sp, and m is as described in the present disclosure. In some embodiments, the present disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, having a backbone chiral center pattern comprising (Np) m (rp) n (sp) t. In some embodiments, the present disclosure provides a C9orf72 oligonucleotide of the oligonucleotide type, having a backbone chiral center pattern comprising (Np) m (rp) n (sp) t. In some embodiments, Np is Rp. In some embodiments, Np is Sp. In some embodiments, all Np are the same. In some embodiments, all Np are Sp. In some embodiments, at least one Np is different from another Np. In some embodiments, t is 2.
As defined herein, n is 1-10. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n is 1. In some embodiments, n is 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n is 3, 4, 5, 6, 7, or 8. In some embodiments, n is 4, 5, 6, 7, or 8. In some embodiments, n is 5, 6, 7, or 8. In some embodiments, n is 6, 7, or 8. In some embodiments, n is 7 or 8. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.
As defined herein, t is 1-50. In some embodiments, t is 1. In some embodiments, t is 2-50. In some embodiments, t is 2, 3, 4, 5, 6, 7, or 8. In some embodiments, t is 3, 4, 5, 6, 7, or 8. In some embodiments, t is 4, 5, 6, 7, or 8. In some embodiments, t is 5, 6, 7, or 8. In some embodiments, t is 6, 7, or 8. In some embodiments, t is 7 or 8. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4. In some embodiments, t is 5. In some embodiments, t is 6. In some embodiments, t is 7. In some embodiments, t is 8. In some embodiments, t is 9. In some embodiments, t is 10. In some embodiments, t is 11. In some embodiments, t is 12. In some embodiments, t is 13. In some embodiments, t is 14. In some embodiments, t is 15. In some embodiments, t is 16. In some embodiments, t is 17. In some embodiments, t is 18. In some embodiments, t is 19. In some embodiments, t is 20. In some embodiments, t is 21. In some embodiments, t is 22. In some embodiments, t is 23. In some embodiments, t is 24. In some embodiments, t is 25. In some embodiments, t is greater than 25.
In some embodiments, at least one of m and t is greater than 2. In some embodiments, at least one of m and t is greater than 3. In some embodiments, at least one of m and t is greater than 4. In some embodiments, at least one of m and t is greater than 5. In some embodiments, at least one of m and t is greater than 6. In some embodiments, at least one of m and t is greater than 7. In some embodiments, at least one of m and t is greater than 8. In some embodiments, at least one of m and t is greater than 9. In some embodiments, at least one of m and t is greater than 10. In some embodiments, at least one of m and t is greater than 11. In some embodiments, at least one of m and t is greater than 12. In some embodiments, at least one of m and t is greater than 13. In some embodiments, at least one of m and t is greater than 14. In some embodiments, at least one of m and t is greater than 15. In some embodiments, at least one of m and t is greater than 16. In some embodiments, at least one of m and t is greater than 17. In some embodiments, at least one of m and t is greater than 18. In some embodiments, at least one of m and t is greater than 19. In some embodiments, at least one of m and t is greater than 20. In some embodiments, at least one of m and t is greater than 21. In some embodiments, at least one of m and t is greater than 22. In some embodiments, at least one of m and t is greater than 23. In some embodiments, at least one of m and t is greater than 24. In some embodiments, at least one of m and t is greater than 25.
In some embodiments, each of m and t is greater than 2. In some embodiments, each of m and t is greater than 3. In some embodiments, each of m and t is greater than 4. In some embodiments, each of m and t is greater than 5. In some embodiments, each of m and t is greater than 6. In some embodiments, each of m and t is greater than 7. In some embodiments, each of m and t is greater than 8. In some embodiments, each of m and t is greater than 9. In some embodiments, each of m and t is greater than 10. In some embodiments, each of m and t is greater than 11. In some embodiments, each of m and t is greater than 12. In some embodiments, each of m and t is greater than 13. In some embodiments, each of m and t is greater than 14. In some embodiments, each of m and t is greater than 15. In some embodiments, each of m and t is greater than 16. In some embodiments, each of m and t is greater than 17. In some embodiments, each of m and t is greater than 18. In some embodiments, each of m and t is greater than 19. In some embodiments, each of m and t is greater than 20.
In some embodiments, the sum of m and t is greater than 3. In some embodiments, the sum of m and t is greater than 4. In some embodiments, the sum of m and t is greater than 5. In some embodiments, the sum of m and t is greater than 6. In some embodiments, the sum of m and t is greater than 7. In some embodiments, the sum of m and t is greater than 8. In some embodiments, the sum of m and t is greater than 9. In some embodiments, the sum of m and t is greater than 10. In some embodiments, the sum of m and t is greater than 11. In some embodiments, the sum of m and t is greater than 12. In some embodiments, the sum of m and t is greater than 13. In some embodiments, the sum of m and t is greater than 14. In some embodiments, the sum of m and t is greater than 15. In some embodiments, the sum of m and t is greater than 16. In some embodiments, the sum of m and t is greater than 17. In some embodiments, the sum of m and t is greater than 18. In some embodiments, the sum of m and t is greater than 19. In some embodiments, the sum of m and t is greater than 20. In some embodiments, the sum of m and t is greater than 21. In some embodiments, the sum of m and t is greater than 22. In some embodiments, the sum of m and t is greater than 23. In some embodiments, the sum of m and t is greater than 24. In some embodiments, the sum of m and t is greater than 25.
In some embodiments, n is 1, and at least one of m and t is greater than 1. In some embodiments, n is 1, and m and t are each independently greater than 1. In some embodiments, m > n and t > n. In some embodiments, (Sp) m (Rp) n (Sp) t is (Sp)2Rp(Sp)2. In some embodiments, (Sp) t (Rp) n (Sp) m is (Sp)2Rp(Sp)2. In some embodiments, (Sp) t (Rp) n (Sp) m is SpRp (Sp)2. In some embodiments, (Np) t (rp) N (sp) m is (N)]p) tRP (Sp) m. In some embodiments, (Np) t (Rp) n (Sp) m is (Np)2Rp (Sp) m. In some embodiments, (Np) t (Rp) n (sp) m is (Rp)2Rp (Sp) m. In some embodiments, (Np) t (Rp) n (Sp) m is (Sp)2Rp (Sp) m. In some embodiments, (Wp) t (rp) n (sp) m is rpsprp (sp) m. In some embodiments, (Np) t (rp) n (sp) m is sprrp (sp) m.
In some embodiments, (Sp) t (rp) n (Sp) m is sprpsp. In some embodiments, (Sp) t (Rp) n (Sp) m is (Sp)2Rp(Sp)2. In some embodiments, (Sp) t (Rp) n (Sp) m is (Sp)3Rp(Sp)3. In some embodiments, (Sp) t (Rp) n (Sp) m is (Sp)4Rp(Sp)4. In some embodiments, (Sp) t (Rp) n (Sp) m is (Sp) tRP (Sp)5. In some embodiments, (Sp) t (Rp) n (Sp) m is SpRp (Sp)5. In some embodiments, (Sp) t (Rp) n (Sp) m is (Sp)2Rp(Sp)5. In some embodiments, (Sp) t (Rp) n (Sp) m is (Sp)3Rp(Sp)5. In some embodiments, (Sp) t (Rp) n (Sp) m is (Sp)4Rp(Sp)5. In some embodiments, (Sp) t (Rp) n (Sp) m is (Sp)5Rp(Sp)5。
In some embodiments, provided oligonucleotides are block entities. In some embodiments, the provided oligonucleotides are alternators. In some embodiments, provided oligonucleotides are alternators comprising alternating blocks. In some embodiments, a block or an alternating may be defined by chemical modifications (including the presence or absence) or patterns thereof, such as base modifications, sugar modifications, internucleotide linkage modifications, stereochemistry, and the like. Example chemical modifications, stereochemistry and patterns thereof of blocks and/or alternating units include, but are not limited to, those described in the present invention, such as those described for oligonucleotides and the like. In some embodiments, the block comprises the modes of.. SS.. RR.. SS.. RR.. In some embodiments, the alternators comprise a pattern of SRSRSRSRs.
In some embodiments, provided backbone chiral center patterns comprise repeating (Sp) m (Rp) n, (Rp) n (Sp) m, (Np) t (Rp) n (Sp) m, or (Sp) t (Rp) n (Sp) m units. In some embodiments, the repeat unit is (Sp) m (rp) n. In some embodiments, the repeat unit is SpRp. In some embodiments, the repeat unit is SpSpRp. In some embodiments, the repeat unit is SpRpRp. In some embodiments, the repeat unit is rprprpsp. In some embodiments, the repeat unit is (Rp) n (sp) m. In some embodiments, the repeat unit is (Np) t (rp) n (sp) m. In some embodiments, the repeat unit is (Sp) t (rp) n (Sp) m.
In some embodiments, provided backbone chiral center patterns are or comprise (Rp/Sp) x- (all Rp or all Sp) - (Rp/Sp) y. In some embodiments, provided backbone chiral center patterns are or comprise (Rp/Sp) - (all Rp or all Sp) - (Rp/Sp). In some embodiments, provided backbone chiral center patterns are or comprise (Rp) x- (all Sp) - (Rp) y. In some embodiments, provided backbone chiral center patterns are or comprise (Rp) - (all Sp) - (Rp). In some embodiments, provided backbone chiral center patterns are or comprise (Sp) x- (all Rp) - (Sp) y. In some embodiments, provided backbone chiral center patterns are or comprise (Sp) - (all Rp) - (Sp). In some embodiments, provided backbone chiral center patterns are or comprise (Rp/Sp) x- (repeats (Sp) m (Rp) n) - (Rp/Sp) y. In some embodiments, provided backbone chiral center patterns are or comprise (Rp/Sp) - (repeating (Sp) m (Rp) n) - (Rp/Sp). In some embodiments, provided backbone chiral center patterns are or comprise (Rp/Sp) x- (repeating SpSpRp) - (Rp/Sp) y. In some embodiments, provided backbone chiral center patterns are or comprise (Rp/Sp) - (repetitive SpSpRp) - (Rp/Sp).
In some embodiments, provided oligonucleotides comprise any stereochemical pattern or any sugar modification described herein.
In some embodiments, the modified sugar moiety comprises a 2' -modification. In some embodiments, the modified sugar moiety comprises a 2' -modification. In some embodiments, the 2 '-modification is 2' -OR1. In some embodiments, the 2 '-modification is 2' -OMe. In some embodiments, the 2 '-modification is 2' -MOE. In some embodiments, the 2' -modification is a LNA sugar modification. In some embodiments, the 2 '-modification is 2' -F. In some embodiments, each sugar modification is independently a 2' -modification. In some embodiments, each sugar modification is independently 2' -OR1Or 2' -F. In some embodiments, each sugar modification is independently 2' -OR1Or 2' -F, wherein R1Is optionally substituted C1-6An alkyl group. In some embodiments, each sugar modification is independently 2' -OR1Or 2 '-F, at least one of which is 2' -F. In some embodiments, each sugar modification is independently 2' -OR1Or 2' -F, wherein R1Is optionally substituted C1-6Alkyl, and at least one of them is 2' -OR1. In some embodiments, each sugar modification is independently 2' -OR1OR 2 ' -F, at least one of which is 2 ' -F and at least one of which is 2 ' -OR1. In some embodiments, each sugar modification is independently 2' -OR1Or 2' -F, wherein R1Is optionally substituted C1-6Alkyl groups, and at least one of which is 2 '-F and at least one is 2' -OR1。
In some embodiments, the disclosure providesFor which 5% or more of the sugar moieties in the oligonucleotide are modified. In some embodiments, 10% or more of the sugar moieties in a provided oligonucleotide are modified. In some embodiments, 15% or more of the sugar moieties in a provided oligonucleotide are modified. In some embodiments, 20% or more of the sugar moieties in a provided oligonucleotide are modified. In some embodiments, 25% or more of the sugar moieties in a provided oligonucleotide are modified. In some embodiments, 30% or more of the sugar moieties in a provided oligonucleotide are modified. In some embodiments, provided oligonucleotides have 35% or more of the sugar moieties modified. In some embodiments, provided oligonucleotides have 40% or more of the sugar moieties modified. In some embodiments, provided oligonucleotides have 45% or more of the sugar moieties modified. In some embodiments, 50% or more of the sugar moieties in a provided oligonucleotide are modified. In some embodiments, provided oligonucleotides have 55% or more of the sugar moieties modified. In some embodiments, provided oligonucleotides have 60% or more of the sugar moieties modified. In some embodiments, provided oligonucleotides have 65% or more of the sugar moieties modified. In some embodiments, 70% or more of the sugar moieties in a provided oligonucleotide are modified. In some embodiments, provided oligonucleotides have 75% or more of the sugar moieties modified. In some embodiments, an oligonucleotide is provided in which 80% or more of the sugar moieties are modified. In some embodiments, provided oligonucleotides have 85% or more of the sugar moieties modified. In some embodiments, 90% or more of the sugar moieties in a provided oligonucleotide are modified. In some embodiments, provided oligonucleotides have 95% or more of the sugar moieties modified. In some embodiments, each sugar moiety of the provided oligonucleotides is modified. In some embodiments, the modified sugar moiety comprises a 2' -modification. In some embodiments, the modified sugar moiety comprises a 2' -modification. In some embodiments, the 2 '-modification is 2' -OR1. In some embodiments, the 2 '-modification is 2' -OMe. In some embodiments, the 2 '-modification is 2' -MOE. In some embodiments, the 2' -modification is a LNA sugar modification. In some embodimentsThe 2 '-modification is 2' -F. In some embodiments, each sugar modification is independently a 2' -modification. In some embodiments, each sugar modification is independently 2' -OR1Or 2' -F. In some embodiments, each sugar modification is independently 2' -OR1Or 2' -F, wherein R1Is optionally substituted C1-6An alkyl group. In some embodiments, each sugar modification is independently 2' -OR1Or 2 '-F, at least one of which is 2' -F. In some embodiments, each sugar modification is independently 2' -OR1Or 2' -F, wherein R1Is optionally substituted C1-6Alkyl, and at least one of them is 2' -OR1. In some embodiments, each sugar modification is independently 2' -OR1OR 2 ' -F, at least one of which is 2 ' -F and at least one of which is 2 ' -OR1. In some embodiments, each sugar modification is independently 2' -OR1Or 2' -F, wherein R1Is optionally substituted C1-6Alkyl groups, and at least one of which is 2 '-F and at least one is 2' -OR1。
In some embodiments, a nucleoside comprising a 2' -modification is followed by a modified internucleotide linkage. In some embodiments, the nucleoside comprising a 2' -modification is preceded by a modified internucleotide linkage. In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate. In some embodiments, the chiral internucleotide linkage is Sp. In some embodiments, the nucleoside comprising the 2' modification is followed by an Sp chiral internucleotide linkage. In some embodiments, the nucleoside comprising a 2' -F is followed by an Sp chiral internucleotide linkage. In some embodiments, the nucleoside comprising the 2' modification is preceded by an Sp chiral internucleotide linkage. In some embodiments, the nucleoside comprising 2' -F is preceded by an Rp chiral internucleotide linkage. In some embodiments, the chiral internucleotide linkage is Rp. In some embodiments, the nucleoside comprising the 2' -modification is followed by an Rp chiral internucleotide linkage. In some embodiments, the nucleoside comprising 2' -F is followed by an Rp chiral internucleotide linkage. In some embodiments, the nucleoside comprising the 2' -modification is preceded by an Rp chiral internucleotide linkage. In some embodiments, the nucleoside comprising 2' -F is preceded by an Rp chiral internucleotide linkage.
The provided oligonucleotides can comprise various numbers of natural phosphate linkages. In some embodiments, provided oligonucleotides do not comprise a natural phosphate linkage. In some embodiments, provided oligonucleotides comprise a natural phosphate linkage. In some embodiments, provided oligonucleotides comprise 1 to 30 or more natural phosphate linkages. In some embodiments, provided oligonucleotides comprise about 25 or more consecutive modified sugar moieties. In some embodiments, provided oligonucleotides comprise about 1 to 20 or more consecutive modified sugar moieties. In some embodiments, provided oligonucleotides comprise up to about 5% to 90% unmodified sugar moieties. In some embodiments, each sugar moiety of the first plurality of oligonucleotides is independently modified. In some embodiments, the provided oligonucleotides are capable of directing a decrease in the expression, level, and/or activity of a C9orf72 target gene or gene product thereof.
In some embodiments, each of the first plurality of oligonucleotides comprises one or more modified sugar moieties and modified internucleotide linkages. In some embodiments, each of the first plurality of oligonucleotides comprises two or more modified sugar moieties. In some embodiments, each of the first plurality of oligonucleotides comprises three or more modified sugar moieties. In some embodiments, each of the first plurality of oligonucleotides comprises four or more modified sugar moieties. In some embodiments, each of the first plurality of oligonucleotides comprises five or more modified sugar moieties. In some embodiments, each of the first plurality of oligonucleotides comprises ten or more modified sugar moieties. In some embodiments, each of the first plurality of oligonucleotides comprises about 15 or more modified sugar moieties. In some embodiments, each of the first plurality of oligonucleotides comprises about 20 or more modified sugar moieties. In some embodiments, each of the first plurality of oligonucleotides comprises about 25 or more modified sugar moieties.
In some embodiments, each of the first plurality of oligonucleotides comprises two or more modified internucleotide linkages. In some embodiments, each of the first plurality of oligonucleotides comprises three or more modified internucleotide linkages. In some embodiments, each of the first plurality of oligonucleotides comprises four or more modified internucleotide linkages. In some embodiments, each of the first plurality of oligonucleotides comprises five or more modified internucleotide linkages. In some embodiments, each of the first plurality of oligonucleotides comprises ten or more modified internucleotide linkages. In some embodiments, each of the first plurality of oligonucleotides comprises about 15 or more modified internucleotide linkages. In some embodiments, each of the first plurality of oligonucleotides comprises about 20 or more modified internucleotide linkages. In some embodiments, each of the first plurality of oligonucleotides comprises about 25 or more modified internucleotide linkages.
In some embodiments, about 5% of the internucleotide linkages in each of the first plurality of oligonucleotides are modified internucleotide linkages. In some embodiments, about 10% of the internucleotide linkages in each of the first plurality of oligonucleotides are modified internucleotide linkages. In some embodiments, about 20% of the internucleotide linkages in each of the first plurality of oligonucleotides are modified internucleotide linkages. In some embodiments, about 30% of the internucleotide linkages in each of the first plurality of oligonucleotides are modified internucleotide linkages. In some embodiments, about 40% of the internucleotide linkages in each of the first plurality of oligonucleotides are modified internucleotide linkages. In some embodiments, about 50% of the internucleotide linkages in each of the first plurality of oligonucleotides are modified internucleotide linkages. In some embodiments, about 60% of the internucleotide linkages in each of the first plurality of oligonucleotides are modified internucleotide linkages. In some embodiments, about 70% of the internucleotide linkages in each of the first plurality of oligonucleotides are modified internucleotide linkages. In some embodiments, about 80% of the internucleotide linkages in each of the first plurality of oligonucleotides are modified internucleotide linkages. In some embodiments, about 85% of the internucleotide linkages in each of the first plurality of oligonucleotides are modified internucleotide linkages. In some embodiments, about 90% of the internucleotide linkages in each of the first plurality of oligonucleotides are modified internucleotide linkages. In some embodiments, about 95% of the internucleotide linkages in each of the first plurality of oligonucleotides are modified internucleotide linkages.
In some embodiments, the provided chirality controlled C9orf72 oligonucleotide compositions are unexpectedly effective compared to reference conditions. In some embodiments, a desired biological effect (e.g., as measured by a reduced level of undesired mRNA, protein, etc.) may be enhanced by 5, 10, 15, 20, 25, 30, 40, 50, or 100-fold. In some embodiments, the alteration is measured by an increase in the level of the desired mRNA compared to a reference condition. In some embodiments, the alteration is measured by an undesirable decrease in mRNA level as compared to a reference condition. In some embodiments, the reference condition is the absence of oligonucleotide treatment. In some embodiments, the reference condition is a sterically random composition of oligonucleotides having the same base sequence and chemical modification.
In some embodiments, provided oligonucleotides comprise increased levels of one or more isotopes. In some embodiments, provided oligonucleotides are labeled, for example, with one or more isotopes of one or more elements (e.g., hydrogen, carbon, nitrogen, etc.). In some embodiments, a provided oligonucleotide (e.g., a first plurality of oligonucleotides) in a provided composition comprises base modifications, sugar modifications, and/or internucleotide linkage modifications, wherein the oligonucleotide contains an enriched level of deuterium. In some embodiments, provided oligonucleotides are deuterium labeled (with-2H replacement-1H) In that respect In some embodiments, one or more of an oligonucleotide or any moiety conjugated to the oligonucleotide (e.g., targeting moiety, etc.)1H channel2Substitution of H for such oligonucleotides can be used in any of the compositions described hereinOr in a method.
In some embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides that:
1) has a common base sequence that is complementary to the C9orf72 target sequence in the transcript; and is
2) Comprising one or more modified sugar moieties and modified internucleotide linkages.
In some embodiments, each consecutive nucleoside unit independently precedes and/or follows the modified internucleotide linkage. In some embodiments, each consecutive nucleoside unit is independently before and/or after the phosphorothioate linkage. In some embodiments, each consecutive nucleoside unit independently precedes and/or follows a chirally controlled modified internucleotide linkage. In some embodiments, each consecutive nucleoside unit is independently before and/or after the chirally controlled phosphorothioate linkage. In some embodiments, the modified internucleotide linkage has the structure of formula I. In some embodiments, the modified internucleotide linkage has the structure of formula I-a.
In some embodiments, the modified internucleotide linkage has the structure of formula I. In some embodiments, the modified internucleotide linkage has the structure of formula I-a.
In some embodiments, the common base sequence and length may be referred to as a common base sequence. In some embodiments, oligonucleotides having a common base sequence may have the same pattern of nucleoside modifications, e.g., sugar modifications, base modifications, and the like. In some embodiments, the nucleoside modification pattern can be represented by a combination of position and modification. In some embodiments, the backbone linkage pattern comprises the position and type of each internucleotide linkage (e.g., phosphate, phosphorothioate, substituted phosphorothioate, etc.). The backbone chiral center pattern of the oligonucleotide can be named by a combination of bonded phosphorus stereochemistry (Rp/Sp) from 5 'to 3'. As exemplified above, the position of the achiral linkage can be obtained, for example, from a pattern of backbone linkages.
As understood by those of ordinary skill in the art, a stereorandom or racemic formulation of an oligonucleotide is prepared by non-stereoselective and/or low stereoselective coupling of nucleotide monomers, typically without the use of any chiral auxiliary agents, chiral modifying reagents, and/or chiral catalysts. In some embodiments, in substantially racemic (or chirally uncontrolled) oligonucleotide formulations, all or most of the coupling steps are not chirally controlled, as the coupling steps are not specifically performed to provide enhanced stereoselectivity. An exemplary substantially racemic formulation of the oligonucleotide is synthesized from commonly used phosphoramidite oligonucleotides (methods well known in the art) by sulfurizing phosphite triester with dithiotetraethylthiuram or (TETD) or 3H-1, 2-benzodithiol-3-one 1, 1-dioxide (BDTD). In some embodiments, a substantially racemic formulation of an oligonucleotide provides a substantially racemic oligonucleotide composition (or chiral uncontrolled oligonucleotide composition). In some embodiments, at least one coupling of nucleotide monomers has a diastereoselectivity of less than about 60: 40, 70: 30, 80: 20, 85: 15, 90: 10, 91: 9, 92: 8, 97: 3, 98: 2, or 99: 1. In some embodiments, at least two couplings of nucleotide monomers have a diastereoselectivity of less than about 60: 40, 70: 30, 80: 20, 85: 15, 90: 10, 91: 9, 92: 8, 97: 3, 98: 2, or 99: 1. In some embodiments, at least three couplings of nucleotide monomers have a diastereoselectivity of less than about 60: 40, 70: 30, 80: 20, 85: 15, 90: 10, 91: 9, 92: 8, 97: 3, 98: 2, or 99: 1. In some embodiments, at least four couplings of nucleotide monomers have a diastereoselectivity of less than about 60: 40, 70: 30, 80: 20, 85: 15, 90: 10, 91: 9, 92: 8, 97: 3, 98: 2, or 99: 1. In some embodiments, at least five of the couplings of nucleotide monomers have a diastereoselectivity of less than about 60: 40, 70: 30, 80: 20, 85: 15, 90: 10, 91: 9, 92: 8, 97: 3, 98: 2, or 99: 1. In some embodiments, each coupling of nucleotide monomers independently has a diastereoselectivity of less than about 60: 40, 70: 30, 80: 20, 85: 15, 90: 10, 91: 9, 92: 8, 97: 3, 98: 2, or 99: 1. In some embodiments, at least one internucleotide linkage in the stereorandom or racemic formulation has a diastereoselectivity of less than about 60: 40, 70: 30, 80: 20, 85: 15, 90: 10, 91: 9, 92: 8, 97: 3, 98: 2, or 99: 1. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 60: 40, 70: 30, 80: 20, 85: 15, 90: 10, 91: 9, 92: 8, 97: 3, 98: 2, or 99: 1. In some embodiments, at least three internucleotide linkages have a diastereoselectivity of less than about 60: 40, 70: 30, 80: 20, 85: 15, 90: 10, 91: 9, 92: 8, 97: 3, 98: 2, or 99: 1. In some embodiments, at least four internucleotide linkages have a diastereoselectivity of less than about 60: 40, 70: 30, 80: 20, 85: 15, 90: 10, 91: 9, 92: 8, 97: 3, 98: 2, or 99: 1. In some embodiments, at least five internucleotide linkages have a diastereoselectivity of less than about 60: 40, 70: 30, 80: 20, 85: 15, 90: 10, 91: 9, 92: 8, 97: 3, 98: 2, or 99: 1. In some embodiments, each internucleotide linkage independently has a diastereoselectivity of less than about 60: 40, 70: 30, 80: 20, 85: 15, 90: 10, 91: 9, 92: 8, 97: 3, 98: 2,. or 99: 1. In some embodiments, the diastereoselectivity is less than about 60: 40. In some embodiments, the diastereoselectivity is less than about 70: 30. In some embodiments, the diastereoselectivity is less than about 80: 20. In some embodiments, the diastereoselectivity is less than about 90: 10. In some embodiments, the diastereoselectivity is less than about 91: 9. In some embodiments, the diastereoselectivity is less than about 92: 8. In some embodiments, the diastereoselectivity is less than about 93: 7. In some embodiments, the diastereoselectivity is less than about 94: 6. In some embodiments, the diastereoselectivity is less than about 95: 5. In some embodiments, the diastereoselectivity is less than about 96: 4. In some embodiments, the diastereoselectivity is less than about 97: 3. In some embodiments, the diastereoselectivity is less than about 98: 2. In some embodiments, the diastereoselectivity is less than about 99: 1. In some embodiments, at least one coupling has a diastereoselectivity of less than about 90: 10. In some embodiments, at least two couplings have a diastereoselectivity of less than about 90: 10. In some embodiments, at least three couplings have a diastereoselectivity of less than about 90: 10. In some embodiments, at least four couplings have a diastereoselectivity of less than about 90: 10. In some embodiments, at least five couplings have a diastereoselectivity of less than about 90: 10. In some embodiments, each coupling independently has a diastereoselectivity of less than about 90: 10. In some embodiments, at least one internucleotide linkage has a diastereoselectivity of less than about 90: 10. In some embodiments, at least two internucleotide linkages have a diastereoselectivity of less than about 90: 10. In some embodiments, at least three internucleotide linkages have a diastereoselectivity of less than about 90: 10. In some embodiments, at least four internucleotide linkages have a diastereoselectivity of less than about 90: 10. In some embodiments, at least five internucleotide linkages have a diastereoselectivity of less than about 90: 10. In some embodiments, each internucleotide linkage independently has a diastereoselectivity of less than about 90: 10.
In some embodiments, chiral controlled internucleotide linkages (such as those of the oligonucleotides of the chiral controlled C9orf72 oligonucleotide composition) have a diastereoselectivity of 90: 10 or greater. In some embodiments, each chiral controlled internucleotide linkage (such as those of the oligonucleotides of the chiral controlled C9orf72 oligonucleotide composition) has a diastereoselectivity of 90: 10 or greater. In some embodiments, the selectivity is 91: 9or greater. In some embodiments, the selectivity is 92: 8 or greater. In some embodiments, the selectivity is 97: 3 or greater. In some embodiments, the selectivity is 94: 6 or greater. In some embodiments, the selectivity is 95: 5 or greater. In some embodiments, the selectivity is 96: 4 or greater. In some embodiments, the selectivity is 97: 3 or greater. In some embodiments, the selectivity is 98: 2 or greater. In some embodiments, the selectivity is 99: 1 or greater.
As understood by one of ordinary skill in the art, in some embodiments, the coupled or linked diastereoselectivity can be assessed by the diastereoselectivity of dimer formation under identical or comparable conditions, where the dimers have identical 5 '-and 3' -nucleosides and internucleotide linkages.
In some embodiments, the present disclosure provides chirally controlled (and/or stereochemically pure) oligonucleotide compositions comprising a first plurality of oligonucleotides defined by having:
1) a common base sequence and length;
2) a common backbone linkage pattern; and
3) a common pattern of backbone chiral centers, the composition being a substantially pure preparation of a single oligonucleotide in that at least about 10% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.
In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide composition of a first plurality of oligonucleotides, the composition enriched for oligonucleotides of a single oligonucleotide type relative to a substantially racemic formulation having the same oligonucleotides. In some embodiments, the disclosure provides a chirality controlled C9off72 oligonucleotide composition of a first plurality of oligonucleotides enriched in oligonucleotides sharing a single oligonucleotide type in the composition relative to a substantially racemic formulation with the same oligonucleotide:
1) a common base sequence and length;
2) a common backbone linkage pattern; and
3) common pattern of backbone chiral centers.
In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have the same structure.
In some embodiments, oligonucleotides of one oligonucleotide type have a common backbone phosphorus modification pattern and a common sugar modification pattern. In some embodiments, oligonucleotides of one oligonucleotide type have a common backbone phosphorus modification pattern and a common base modification pattern. In some embodiments, oligonucleotides of one oligonucleotide type have a common backbone phosphorus modification pattern and a common nucleoside modification pattern. In some embodiments, the oligonucleotides of one oligonucleotide type are identical.
In some embodiments, the C9orf72 oligonucleotide is a substantially pure preparation of an oligonucleotide type, and oligonucleotides in the composition that are not the oligonucleotide type are in the form of impurities during the preparation of the oligonucleotide type (and in some cases after certain purification steps).
In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of sugar modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers are the same.
In some embodiments, the oligonucleotides in the provided compositions have a common backbone phosphorus modification pattern. In some embodiments, the common base sequence is a base sequence of one oligonucleotide type. In some embodiments, the provided compositions are chirally controlled oligonucleotide compositions containing a first plurality of C9orf72 oligonucleotides of individual oligonucleotide types at non-random or controlled levels, wherein the oligonucleotide types are defined by:
1) a base sequence;
2) a skeletal linkage mode;
3) pattern of backbone chiral centers; and
4) pattern of backbone phosphorus modification.
As noted above and understood in the art, in some embodiments, the base sequence of an oligonucleotide can refer to the identity and/or modification state of nucleoside residues (e.g., nucleoside residues in the sugar and/or base composition, relative to standard naturally occurring nucleotides (e.g., adenine, cytosine, guanosine, thymine, and uracil)) in the oligonucleotide, and/or can refer to the hybridization characteristics (i.e., the ability to hybridize to a particular complementary residue) of such residues.
In some embodiments, a particular oligonucleotide type may be defined by:
1A) base identity;
1B) a pattern of base modifications;
1C) the mode of sugar modification;
2) a skeletal linkage mode;
3) pattern of backbone chiral centers; and
4) framework phosphorus modification mode.
Thus, in some embodiments, particular types of oligonucleotides may share the same bases, but differ in the pattern of base modifications and/or sugar modifications. In some embodiments, oligonucleotides of a particular type may share the same base and pattern of base modifications (including, e.g., the absence of base modifications), but differ in the pattern of sugar modifications.
In some embodiments, the purity of the C9orf72 oligonucleotide can be controlled by the stereoselectivity of each coupling step in its preparation. In some embodiments, the coupling step has a stereoselectivity (e.g., diastereoselectivity) of 60% (60% of the new internucleotide linkages formed by the coupling step have the expected stereochemistry). After such a coupling step, the new internucleotide linkage formed may be said to have a purity of 60%. In some embodiments, each coupling step has a stereoselectivity of at least 60%. In some embodiments, each coupling step has a stereoselectivity of at least 70%. In some embodiments, each coupling step has a stereoselectivity of at least 80%. In some embodiments, each coupling step has a stereoselectivity of at least 85%. In some embodiments, each coupling step has a stereoselectivity of at least 90%. In some embodiments, each coupling step has a stereoselectivity of at least 91%. In some embodiments, each coupling step has a stereoselectivity of at least 92%. In some embodiments, each coupling step has a stereoselectivity of at least 93%. In some embodiments, each coupling step has a stereoselectivity of at least 94%. In some embodiments, each coupling step has a stereoselectivity of at least 95%. In some embodiments, each coupling step has a stereoselectivity of at least 96%. In some embodiments, each coupling step has a stereoselectivity of at least 97%. In some embodiments, each coupling step has a stereoselectivity of at least 98%. In some embodiments, each coupling step has a stereoselectivity of at least 99%. In some embodiments, each coupling step has a stereoselectivity of at least 99.5%. In some embodiments, each coupling step has a stereoselectivity of almost 100%. In some embodiments, the coupling step has a stereoselectivity of almost 100%, because all detectable products from the coupling step have the expected stereoselectivity according to the analytical method (e.g., NMR, HPLC, etc.).
The present disclosure specifically contemplates that combinations of oligonucleotide structural elements (e.g., patterns of chemical modifications, backbone linkages, backbone chiral centers, and/or backbone phosphorus modifications) can provide unexpectedly improved properties, such as biological activity.
In some embodiments, provided chirally controlled (and/or stereochemically pure) formulations contain oligonucleotides that include one or more modified backbone linkages, bases, and/or sugars.
In some embodiments, provided chirally controlled (and/or stereochemically pure) formulations contain oligonucleotides having a common base sequence of at least 15 bases. In some embodiments, provided chirally controlled (and/or stereochemically pure) formulations contain oligonucleotides having a common base sequence of at least 16 bases. In some embodiments, provided chirally controlled (and/or stereochemically pure) formulations contain oligonucleotides having a common base sequence of at least 17 bases. In some embodiments, provided chirally controlled (and/or stereochemically pure) formulations contain oligonucleotides having a common base sequence of at least 18 bases. In some embodiments, provided chirally controlled (and/or stereochemically pure) formulations contain oligonucleotides having a common base sequence of at least 19 bases. In some embodiments, provided chirally controlled (and/or stereochemically pure) formulations contain oligonucleotides having a common base sequence of at least 20 bases. In some embodiments, provided chirally controlled (and/or stereochemically pure) formulations contain oligonucleotides having a common base sequence of at least 21 bases. In some embodiments, provided chirally controlled (and/or stereochemically pure) formulations contain oligonucleotides having a common base sequence of at least 22 bases. In some embodiments, provided chirally controlled (and/or stereochemically pure) formulations contain oligonucleotides having a common base sequence of at least 23 bases. In some embodiments, provided chirally controlled (and/or stereochemically pure) formulations contain oligonucleotides having a common base sequence of at least 24 bases. In some embodiments, provided chirally controlled (and/or stereochemically pure) formulations contain oligonucleotides having a common base sequence of at least 25 bases. In some embodiments, provided chirally controlled (and/or stereochemically pure) formulations contain oligonucleotides having a common base sequence of at least 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 bases.
In some embodiments, provided compositions comprise oligonucleotides comprising one or more residues modified at a sugar moiety. In some embodiments, provided compositions comprise oligonucleotides comprising one or more residues modified at the 2 'position of the sugar moiety (referred to herein as a "2' modification"). Examples of such modifications are described above and herein and include, but are not limited to, 2 ' -OMe, 2 ' -MOE, 2 ' -LNA, 2 ' -F, FRNA, FANA, 5 ' -vinyl, N-morpholinyl, S-cEt, and the like. In some embodiments, provided compositions comprise oligonucleotides comprising one or more 2' modified residues. For example, in some embodiments, provided oligonucleotides contain one or more residues that are 2 '-O-methoxyethyl (2' -MOE) -modified residues. In some embodiments, provided compositions comprise oligonucleotides that do not contain any 2' modifications. In some embodiments, provided compositions are oligonucleotides that do not contain any 2' -MOE residues. That is, in some embodiments, provided oligonucleotides are not MOE modified. Other example sugar modifications are described in the present disclosure.
In some embodiments, one or more is one. In some embodiments, one or more is two. In some embodiments, one or more is three. In some embodiments, one or more is four. In some embodiments, one or more is five. In some embodiments, one or more is six. In some embodiments, one or more is seven. In some embodiments, one or more is eight. In some embodiments, one or more is nine. In some embodiments, one or more is ten. In some embodiments, one or more is at least one. In some embodiments, one or more is at least two. In some embodiments, one or more is at least three. In some embodiments, one or more is at least four. In some embodiments, one or more is at least five. In some embodiments, one or more is at least six. In some embodiments, one or more is at least seven. In some embodiments, one or more is at least eight. In some embodiments, one or more is at least nine. In some embodiments, one or more is at least ten.
As understood by those of ordinary skill in the art, the provided oligonucleotide compositions and methods have a variety of uses known to those of ordinary skill in the art. Methods for evaluating the provided compositions and their properties and uses are also well known and practiced by those of ordinary skill in the art. Exemplary characteristics, uses and/or methods include, but are not limited to, those described in WO/2014/012081 and WO/2015/107425.
In some embodiments, the chiral internucleotide linkage has the structure of formula I. In some embodiments, the chiral internucleotide linkage is a phosphorothioate. In some embodiments, each chiral internucleotide linkage in a single oligonucleotide of a provided composition independently has the structure of formula I. In some embodiments, each chiral internucleotide linkage in a single oligonucleotide of a provided composition is a phosphorothioate.
In some embodiments, the oligonucleotides of the present disclosure comprise one or more modified sugar moieties. In some embodiments, the C9orf72 oligonucleotides of the present disclosure comprise one or more modified base moieties. Various modifications can be introduced into the sugars and/or moieties as known to those of ordinary skill in the art and described in the present disclosure. For example, in some embodiments, the modifications are those described in US 9006198, WO 2014/012081, and WO/2015/107425, the respective sugar and base modifications of which are incorporated herein by reference.
In some embodiments, the sugar isThe modification is a 2' -modification. Common 2 'modifications include, but are not limited to, 2' -OR1Wherein R is1Is not hydrogen. In some embodiments, the modification is 2' -OR, wherein R is optionally substituted aliphatic. In some embodiments, the modification is 2' -OMe. In some embodiments, the modification is 2' -O-MOE. In some embodiments, the present disclosure demonstrates that inclusion and/or location of particular chirally pure internucleotide linkages can provide stability improvements comparable to or better than those achieved by use of modified backbone linkages, bases, and/or sugars. In some embodiments, provided single oligonucleotides of provided compositions do not have modifications on the sugar. In some embodiments, a provided single oligonucleotide of a provided composition does not have a modification at the 2 'position of the sugar (i.e., the two groups at the 2' position are-H/-H or-H/-OH). In some embodiments, provided single oligonucleotides of provided compositions do not have any 2' -MOE modifications.
In some embodiments, the 2 '-modification is-O-L-or-L-, which links the 2' -carbon of the sugar moiety to another carbon of the sugar moiety. In some embodiments, the 2 ' -modification is-O-L-or-L-, which links the 2 ' -carbon of the sugar moiety to the 4 ' -carbon of the sugar moiety. In some embodiments, the 2' -modification is S-cEt. In some embodiments, the modified sugar moiety is an LNA moiety.
In some embodiments, the 2' -modification is-F. In some embodiments, the 2' -modification is FANA. In some embodiments, the 2' -modification is FRNA.
In some embodiments, the sugar modification is a 5 ' -modification, such as R-5 ' -Me, S-5 ' -Me, and the like.
In some embodiments, the sugar modification alters the size of the sugar ring. In some embodiments, the sugar modification is a sugar moiety in FHNA.
In some embodiments, the sugar modification replaces the sugar moiety with another cyclic or acyclic moiety. Examples of such moieties are widely known in the art, including but not limited to those moieties used in morpholino (optionally with its phosphorodiamidite linkage), diol nucleic acids, and the like.
In some embodiments, a single oligonucleotide in a provided composition has at least about 25% internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide in a provided composition has at least about 30% internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide in a provided composition has at least about 35% internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide in a provided composition has at least about 40% internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide in a provided composition has at least about 45% internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide in a provided composition has at least about 50% internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide in a provided composition has at least about 55% internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide in a provided composition has at least about 60% internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide in a provided composition has at least about 65% internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide in a provided composition has at least about 70% internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide in a provided composition has at least about 75% internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide in a provided composition has at least about 80% internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide in a provided composition has at least about 85% internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide in a provided composition has at least about 90% internucleotide linkages in the Sp configuration.
In some embodiments, the C9orf72 oligonucleotide is or comprises a C9orf72 oligonucleotide selected from the group consisting of: WV-3536, WV-3537, WV-3538, WV-3539, WV-3540, WV-3541, WV-3542, WV-3561, WV-3562, WV-3563, WV-3564, WV-3565, WV-3566, WV-3567, WV-3568, WV-3569, WV-3570, WV-3571, WV-3572, WV-3573, WV-3574, WV-3575, WV-3576, WV-3577, WV-3578, WV-3579, WV-3580, WV-3581, WV-3582, WV-3583, WV-3584, WV-3585, WV-3586, WV-3587, WV-3588, WV-3589, WV-3590, WV-3592, WV-3594, WV-3596, WV-3595, WV-3576, WV-3595, WV-, WV-3597, WV-3598, WV-3599, WV-3600, WV-3601, WV-3602, WV-3603, WV-3604, WV-3605, WV-3606, WV-3607, WV-3608, WV-3609, WV-3610, WV-3611, WV-3612, WV-3613, WV-3614, WV-3615, WV-3616, WV-3617, WV-3618, WV-3619, WV-3620, WV-3621, WV-3622, WV-3623, WV-3624, WV-3625, WV-3626, WV-3627, WV-3628, WV-3629, WV-3630, WV-3631, WV-3632, WV-3633, WV-3635, WV-3638, WV-3637, WV-3638, WV-3635, WV-3619, WV-3634, WV-3635, WV-, WV-3640, WV-3641, WV-3642, WV-3643, WV-3644, WV-3645, WV-3646, WV-3647, WV-3648, WV-3649, WV-3650, WV-3651, WV-3652, WV-3653, WV-3654, WV-3655, WV-3656, WV-3657, WV-3658, WV-3659, WV-3660, WV-3661, WV-3662, WV-3663, WV-3664, WV-3665, WV-3666, WV-3667, WV-3668, WV-3669, WV-3670, WV-3671, WV-3672, WV-3673, WV-3674, WV-3675, WV-3676, WV-3678, WV-3679, WV-3680, WV-3678, WV-3680, WV-, WV-3683, WV-3684, WV-3685, WV-3686, WV-3687, WV-3688, WV-3689, WV-3690, WV-3691, WV-3692, WV-3693, WV-3694, WV-3695, WV-3696, WV-3697, WV-3698, WV-3699, WV-3700, WV-3701, WV-3702, WV-3703, WV-3704, WV-3705, WV-3706, WV-3707, WV-3708, WV-3709, WV-3710, WV-3711, WV-3712, WV-3713, WV-3714, WV-3715, WV-3716, WV-3717, WV-3718, WV-3719, WV-3723, WV-, WV-3726, WV-3727, WV-3728, WV-3729, WV-3730, WV-3731, WV-3732, WV-3733, WV-3734, WV-3735, WV-3736, WV-3737, WV-3738, WV-3739, WV-3740, WV-3741, WV-3742, WV-3743, WV-3744, WV-3745, WV-3746, WV-3747, WV-3748, WV-3749, WV-3750, WV-3751, WV-3752, WV-5905, WV-5906, WV-5907, WV-5908, WV-5909, WV-5910, WV-5911, WV-5912, WV-5913, WV-5914, WV-599, WV-5918, WV-5919, WV-379, WV-, WV-5921, WV-5922, WV-5923, WV-5924, WV-5925, WV-5926, WV-5927, WV-5928, WV-5929, WV-5930, WV-5931, WV-5932, WV-5933, WV-5934, WV-5935, WV-5936, WV-5937, WV-5938, WV-5939, WV-5940, WV-5941, WV-5942, WV-5943, WV-5944, WV-5945, WV-5946, WV-5947, WV-5948, WV-5949, WV-5950, WV-5951, WV-5952, WV-5953, WV-5954, WV-5955, WV-5956, WV-5957, WV-5958, WV-593, WV-, WV-5964, WV-5965, WV-5966, WV-5967, WV-5968, WV-5969, WV-5970, WV-5971, WV-5972, WV-5973, WV-5974, WV-5975, WV-5976, WV-5977, WV-5978, WV-5979, WV-5980, WV-5981, WV-5982, WV-5983, WV-5984, WV-5985, WV-5986, WV-5987, WV-5988, WV-5989, WV-5990, WV-5991, WV-5992, WV-5993, WV-5994, WV-5995, WV-5996, WV-5997, WV-5998, WV-5999, WV-5908, WV-6471, WV-593, WV-6474, WV-593, WV, WV-6476, WV-6477, WV-6478, WV-6479, WV-6480, WV-6481, WV-6482, WV-6483, WV-6484, WV-6485, WV-6486, WV-6487, WV-6488, WV-6489, WV-6490, WV-6491, WV-6492, WV-6831, WV-6832, WV-6833, WV-6834, WV-6835, WV-6836, WV-6837, WV-6838, WV-6839, WV-6840, WV-6841, WV-6842, WV-6843, WV-6844, WV-6845, WV-6846, WV-6847, WV-6478, WV-6859, WV-6850, WV-6853, WV-6844, WV-6845, WV-6856, WV-6854, WV-6856, WV-6857, WV-6858, WV-6853, WV-6844, WV-6854, WV-, WV-6857, WV-6858, WV-6859, WV-6860, WV-6861, WV-6862, WV-6863, WV-6864, WV-6865, WV-6866, WV-6867, WV-6868, WV-6869, WV-6870, WV-6871, WV-6872, WV-6873, WV-6874, WV-6875, WV-6876, WV-6877, WV-6878, WV-6879, WV-6880, WV-6881, WV-6882, WV-6883, WV-6884, WV-6885, WV-6886, WV-6887, WV-6888, WV-686889, WV-6890, WV-6891, WV-6892, WV-68594, WV-96, WV-97, WV-96, WV-6898, WV-6899, WV-6900, WV-6901, WV-6902, WV-6903, WV-6904, WV-6905, WV-6906, WV-6907, WV-6908, WV-6909, WV-6910, WV-6911, WV-6912, WV-6913, WV-6914, WV-6915, WV-6916, WV-6917, WV-6918, WV-6919, WV-6920, WV-6921, WV-6922, WV-6923, WV-6924, WV-6925, WV-6926, WV-6927, WV-6928, WV-6929, WV-6930, WV-69431, WV-6932, WV-6933, WV-6934, WV-35, WV-6936, WV-6937, WV-6938, WV-691, WV-6930, WV-6941, WV-6932, WV-6930, WV-6943, WV-6944, WV-6945, WV-6946, WV-6947, WV-6948, WV-6949, WV-6950, WV-6951, WV-6952, WV-6953, WV-6954, WV-6955, WV-6956, WV-6957, WV-6958, WV-6959, WV-696, WV-6963, WV-696, WV-6967, WV-6971, WV-6972, WV-6973, WV-6974, WV-6975, WV-6976, WV-6977, WV-6978, WV-6980, WV-6982, WV-6985, WV-6981, WV-6985, WV-6965, WV-6980, WV-6981, WV-, WV-6986, WV-69821, WV-6989, WV-6993, WV-6994, WV-6995, WV-6996, WV-6997, WV-6998, WV-6999, WV-7000, WV-7001, WV-7002, WV-7003, WV-7004, WV-7005, WV-7006, WV-7007, WV-7008, WV-7009, WV-7010, WV-7011, WV-7012, WV-7013, WV-7014, WV-7015, WV-7016, WV-7017, WV-7018, WV-7019, WV-7020, WV-7021, WV-7022, WV-7024, WV-7028, WV-7026, WV, WV-7029, WV-7030, WV-7031, WV-7032, WV-7033, WV-7034, WV-7035, WV-7036, WV-7037, WV-7038, WV-7039, WV-7040, WV-7041, WV-7042, WV-7043, WV-7044, WV-7045, WV-7046, WV-7047, WV-7048, WV-7049, WV-7050, WV-7051, WV-7052, WV-7053, WV-7054, WV-7055, WV-7056, WV-7057, WV-7058, WV-7059, WV-7060, WV-7061, WV-7062, WV-7063, WV-7064, WV-7065, WV-7066, WV-7070, WV-7069, WV-7071, WV-7069, WV-, WV-7072, WV-7073, WV-7074, WV-7075, WV-7076, WV-7077, WV-7078, WV-7079, WV-7080, WV-7081, WV-7082, WV-7083, WV-7084, WV-7085, WV-7086, WV-7087, WV-7088, WV-7089, WV-7090, WV-7091, WV-7092, WV-7093, WV-7094, WV-7095, WV-7096, WV-7097, WV-7098, WV-7099, WV-7100, WV-7101, WV-7102, WV-7103, WV-7117, WV-7118, WV-7119, WV-7120, WV-7121, WV-7122, WV-7127, WV-7126, WV-7127, WV-7126, WV-7026, WV-7094, WV-7095, WV-7096, WV-7080, WV-, WV-7128, WV-7129, WV-7130, WV-7131, WV-7132, WV-7405, WV-7434, WV-7435, WV-7601, WV-7602, WV-7603, WV-7604, WV-7605, WV-7606, WV-7657, WV-7658, WV-7659, WV-7773, WV-7774, WV-7775, WV-7866, WV-8005, WV-8006, WV-8007, WV-8008, WV-8009, WV-8010, WV-8011, WV-8012, WV-8114, WV-8115, WV-8116, WV-8117, WV-8118, WV-8119, WV-8120, WV-8121, WV-8122, WV-8126, WV-816, WV-8006, WV-8007, WV-819, WV-, WV-8128, WV-8129, WV-8311, WV-8312, WV-8313, WV-8314, WV-8315, WV-8316, WV-8317, WV-8318, WV-8319, WV-8320, WV-8321, WV-8322, WV-8329, WV-8444, WV-8445, WV-8446, WV-8447, WV-8452, WV-8453, WV-8454, WV-8455, WV-8456, WV-8457, WV-8458, WV-8459, WV-8460, WV-8461, WV-8462, WV-8463, WV-8464, WV-8465, WV-8466, WV-8467, WV-8468, WV-8469, WV-8470, WV-8472, WV-8473, WV-8471, WV-8465, WV-, WV-8476, WV-8477, WV-8547, WV-8548, WV-8549, WV-8550, WV-8551, WV-8568, WV-8569, WV-8594, WV-8595, WV-8691, WV-8692, WV-8693, WV-8694, WV-8695, WV-8696, WV-9062, WV-9063, WV-9228, WV-9285, WV-9286, WV-9380, WV-9381, WV-9394, WV-9395, WV-9396, WV-9397, WV-9398, WV-9399 and WV-9421, and any C9orf72 oligonucleotide described herein.
One of ordinary skill in the art reading this disclosure will appreciate that this disclosure does not specifically exclude the possibility that any C9orf72 oligonucleotide or other oligonucleotide labeled as an antisense oligonucleotide (ASO) described herein may also or alternatively function via another mechanism (e.g., utilizing RISC as ssRNAi); the invention also indicates that the various oligonucleotides can act via different mechanisms (using RNase H, spatially blocking translation or other post-transcriptional processes, altering the conformation of the C9orf72 target nucleic acid, etc.).
Chirally controlled oligonucleotides and chirally controlled oligonucleotide compositions
In some embodiments, provided C9orf72 oligonucleotides are capable of directing a decrease in expression, level, and/or activity of a C9orf72 target gene or gene product thereof. In some embodiments, provided C9orf72 oligonucleotides are capable of directing a reduction in the expression, level, and/or activity of a C9orf72 target gene or gene product thereof by spatially blocking translation after annealing to C9orf72 target gene mRNA and/or by altering or interfering with mRNA splicing. In some embodiments, the C9orf72 target gene comprises repeat amplification. In some embodiments, provided C9orf72 oligonucleotides are chirally controlled.
The present disclosure provides chirally controlled C9orf72 oligonucleotides and chirally controlled C9orf72 oligonucleotide compositions having high crude product purity and high diastereomeric purity. In some embodiments, the disclosure provides chirally controlled C9orf72 oligonucleotides and chirally controlled C9orf72 oligonucleotide compositions with high crude product purity. In some embodiments, the disclosure provides chirally controlled C9orf72 oligonucleotides and chirally controlled C9orf72 oligonucleotide compositions with high diastereomeric purity.
In some embodiments, a C9orf72 oligonucleotide is a substantially pure preparation of a certain C9orf72 oligonucleotide type, and oligonucleotides in the composition that are not the oligonucleotide type are in an impurity form during the preparation of the oligonucleotide type (in some cases after certain purification steps).
In some embodiments, the disclosure provides C9orf72 oligonucleotides comprising one or more diastereomerically pure internucleotide linkages with respect to chirally bound phosphorus. In some embodiments, the disclosure provides C9orf72 oligonucleotides comprising one or more diastereomerically pure internucleotide linkages having the structure of formula I. In some embodiments, the disclosure provides C9orf72 oligonucleotides comprising one or more diastereomerically pure internucleotide linkages and one or more phosphodiester linkages with respect to chirally bound phosphorus. In some embodiments, the disclosure provides C9orf72 oligonucleotides comprising one or more diastereomerically pure internucleotide linkages and one or more phosphodiester linkages having the structure of formula I. In some embodiments, the disclosure provides C9orf72 oligonucleotides comprising one or more diastereomerically pure internucleotide linkages and one or more phosphodiester linkages having the structure of formula I-C. In some embodiments, such oligonucleotides are prepared by forming diastereomerically pure internucleotide linkages that are predesigned relative to a chirally bound phosphorus by using stereoselective oligonucleotide synthesis as described herein. Example internucleotide linkages, including those having the structure of formula I, are described further below.
Internucleotide linkage
In some embodiments, provided C9orf72 oligonucleotides are capable of directing a decrease in expression, level, and/or activity of a C9orf72 target gene or gene product thereof. In some embodiments, provided C9orf72 oligonucleotides comprise any internucleotide linkage described herein or known in the art.
Non-limiting examples of internucleotide linkages or unmodified internucleotide linkages are phosphodiesters; non-limiting examples of modified internucleotide linkages include those in which one or more of the oxygens of the phosphodiester are replaced by sulfur (as in phosphorothioate), H, an alkyl, or another moiety or element (as non-limiting examples) that is not an oxygen. A non-limiting example of an internucleotide linkage is a moiety that does not contain phosphorus but is used to link two sugars. A non-limiting example of an internucleotide linkage is a moiety that does not contain a phosphorus but serves to link two sugars in the backbone of a C9orf72 oligonucleotide. Disclosed herein are additional non-limiting examples of nucleotides, modified nucleotides, nucleotide analogs, internucleotide linkages, modified internucleotide linkages, bases, modified bases, and base analogs, sugars, modified sugars, and sugar analogs, as well as nucleosides, modified nucleosides, and nucleoside analogs.
In certain embodiments, the internucleotide linkage has the structure of formula I:
wherein the variables are as defined and described below. In some embodiments, the linkage of formula I is chiral. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide comprising one or more modified internucleotide linkages of formula I. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising one or more modified internucleotide linkages of formula I, and wherein individual nuclei of formula I within the oligonucleotideThe internucleotide linkages differ with respect to each other-. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising one or more modified internucleotide linkages of formula I, and wherein individual internucleotide linkages of formula I within the oligonucleotide have different-X-L-R relative to each other1. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising one or more modified internucleotide linkages of formula I, and wherein the individual internucleotide linkages of formula I within the oligonucleotide have a different X relative to each other. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising one or more modified internucleotide linkages of formula I, and wherein individual internucleotide linkages of formula I within the oligonucleotide have different-L-R relative to each other1. In some embodiments, the chirally controlled C9orf72 oligonucleotide is a C9orf72 oligonucleotide in a provided composition with a particular oligonucleotide type. In some embodiments, the chirally controlled C9orf72 oligonucleotides are C9orf72 oligonucleotides in provided compositions having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, the chirally controlled C9orf72 oligonucleotide is a C9orf72 oligonucleotide in a chirally controlled composition having a particular oligonucleotide type, and the chirally controlled C9orf72 oligonucleotide has that type. In some embodiments, the chirally controlled C9orf72 oligonucleotide is a C9orf72 oligonucleotide in a provided composition comprising a non-random or controlled level plurality of oligonucleotides sharing a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers, and the chirally controlled C9orf72 oligonucleotide shares a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.
In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different stereochemistry and/or different P modifications relative to each other. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different stereochemistry relative to each other, and wherein at least a portion of the structure of the chirally controlled C9orf72 oligonucleotide is characterized by a repeating pattern of alternating stereochemistry.
In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different P modifications relative to each other that differ in their-XLR1The moieties having different X atoms, and/or-XLR thereof1The moieties having different L groups, and/or-XLR thereof1The moieties having different atoms of R1, wherein XLR1Is equal to X-L-R1And X, L and R1As defined in formula I as disclosed herein.
In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different stereochemistry and/or different P modifications relative to each other, and the oligonucleotide has a structure represented by the formula:
[SBn1RBn2SBn3RBn4...SBnxRBny]
wherein:
each RBIndependently represent a block of nucleotide units having the R configuration at the point of bonding to the phosphorus;
each SBIndependently represent a block of nucleotide units having an S configuration at the point of linkage to the phosphorus;
each of n1 through ny is zero or an integer, with the proviso that at least one odd n and at least one even n must not be zero, such that the oligonucleotide comprises at least two individual internucleotide linkages having different stereochemistry relative to each other; and is
Wherein the sum of n1 through ny is between 2 and 200, and in some embodiments, between a lower limit selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or greater numbers and an upper limit selected from the group consisting of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200, the upper limit being greater than the lower limit.
In some such embodiments, each n has the same value; in some embodiments, each even n has the same value as each other even n; in some embodiments, each odd n has the same value as each other odd n; in some embodiments, at least two even numbers n have different values from each other; in some embodiments, at least two odd numbers n have different values from each other.
In some embodiments, at least two adjacent n are equal to each other, such that the provided C9orf72 oligonucleotide comprises adjacent blocks of equal length S and R stereochemically linked. In some embodiments, provided C9orf72 oligonucleotides include repeating blocks of S and R stereochemically linked blocks of equal length. In some embodiments, provided C9orf72 oligonucleotides include repeating blocks of S and R stereochemically linked, wherein at least two such blocks have different lengths from each other; in some such embodiments, each S stereochemical block has the same length and has a different length than each R stereochemical length, which may optionally be the same length as each other.
In some embodiments, at least two non-adjacent n are equal to each other such that the provided C9orf72 oligonucleotide comprises at least two linked blocks of a first stereochemistry that are equal in length to each other and separated by a linked block of another stereochemistry, which may be the same or different in length from the block of the first stereochemistry.
In some embodiments, n associated with the provided linkage block at the end of the C9orf72 oligonucleotide is of the same length. In some embodiments, provided C9orf72 oligonucleotides contain end blocks with the same linkage stereochemistry. In some such embodiments, the end blocks are separated from each other by a mid-block having another bonded stereochemistry.
In some embodiments, provided is the formula [ S ]Bn1RBn2SBn3RBn4...SBnxRBny]The C9orf72 oligonucleotide (E) is a stereoblock. In some embodiments, provided is the formula [ S ]Bn1RBn2SBn3RBn4...SBnxRBny]The C9orf72 oligonucleotide (E) is a stereo-skip. In some embodiments, provided is the formula [ S ]Bn1RBn2SBn3RBn4...SBnxRBny]The C9orf72 oligonucleotide (E) is a stereo-alternating. In some embodiments, provided is the formula [ S ]Bn1RBn2SBn3RBn4...SBnxRBny]The C9orf72 oligonucleotide (E) is a gapmer.
In some embodiments, provided is the formula [ S ]Bn1RBn2SBn3RBn4...SBnxRBny]The C9orf72 oligonucleotide of (a) has any of the above-described patterns and further comprises a P modification pattern. For example, in some embodiments, the formula [ S ] is providedBn1RBn2SBn3RBn4...SBnxRBny]The C9orf72 oligonucleotide (C.sub.9) is a steric skip and a P-modified skip. In some embodiments, provided is the formula [ S ]Bn1RBn2SBn3RBn4...SBnxRBny]The C9orf72 oligonucleotide (see above) is a stereoblock and a P-modified alternating. In some embodiments, provided is the formula [ S ]Bn1RBn2SBn3RBn4...SBnxRBny]The C9orf72 oligonucleotide (see above) is a stereo-alternating and P-modified block.
In some embodiments, provided is the formula [ S ]Bn1RBn2SBn3RBn4...SBnxRBny]The C9orf72 oligonucleotide of (a) is a chirally controlled C9orf72 oligonucleotide comprising one or more modified internucleotide linkages independently having the structure of formula I:
wherein:
p is a symmetric phosphorus atom, or an asymmetric phosphorus atom which is Rp or Sp;
w is O, S or Se;
x, Y and Z are each independently-O-, -S-, -N (-L-R)1) -, or L;
l is a covalent bond or an optionally substituted straight or branched chain C1-C10Alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted group selected from: c1-C6Alkylene radical, C1-C6Alkenylene, -C ≡ C-, C1-C6Heteroaliphatic moiety, -C (R')2-、-Cy-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)-、-N(R′)C(O)O-、-OC(O)N(R′)-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-N(R′)S(O)2-, -SC (O) -, -C (O) S-, -OC (O) -, and-C (O) O-;
R1is halogen, R or optionally substituted C1-C50An aliphatic group wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from: c1-C6Alkylene radical, C1-C6Alkenylene, -C ≡ C-, C1-C6Heteroaliphatic moiety, -C (R')2-、-Cy-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)-、-N(R′)C(O)O-、-OC(O)N(R′)-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-N(R′)S(O)2- - -SC (O) - -, - -C (O) S- -, - -OC (O) - -and- -C (O) O- -
Each R' is independently-R, -C (O) R, -CO2R, or-SO2R, or
Two R' taken together with the intervening atoms form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring;
-Cy-is an optionally substituted divalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, and heterocyclylene;
each R is independently hydrogen or selected from C1-C6Optionally substituted groups of aliphatic, carbocyclic, aryl, heteroaryl and heterocyclic groups; and is
Each one of which isIndependently represents a linkage to a nucleoside.
In some embodiments, L is a covalent bond or an optionally substituted linear or branched C1-C10Alkylene, wherein one or more methylene units of L are optionally and independently replaced by: optionally substituted C1-C6Alkylene radical, C1-C6Alkenylene, -C ≡ C-, -C (R')2-、-Cy-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)-、-N(R′)C(O)O-、-OC(O)N(R′)-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-N(R′)S(O)2-, -SC (O) -, -C (O) S-, -OC (O) -, or-C (O) O-;
R1is halogen, R or optionally substituted C1-C50Aliphatic, wherein one or more methylene units are optionally and independently replaced by: optionally substituted C1-C6Alkylene radical, C1-C6Alkenylene, -C ≡ C-, -C (R')2-、-Cy-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)-、-N(R′)C(O)O-、-OC(O)N(R′)-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-N(R′)S(O)2-, -SC (O) -, -C (O) S-, -OC (O) -, or-C (O) O-;
each R' is independently-R, -C (O) R, -CO2R, or-SO2R, or
Two R' on the same nitrogen, taken together with the intervening atoms, form an optionally substituted heterocyclic or heteroaryl ring, or
Two R' on the same carbon, together with the intervening atoms, form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring;
-Cy-is an optionally substituted divalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, and heterocyclylene;
each R is independently hydrogen or an optionally substituted group selected from: c1-C6Aliphatic, phenyl, carbocyclyl, aryl, heteroaryl and heterocyclyl; and is
Each one of which isIndependently represents a linkage to a nucleoside. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises one or more modified internucleotide phosphorus linkages. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises, for example, a phosphorothioate or phosphorothioate triester linkage. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises a phosphorothioate triester linkage. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least two phosphorothioate triester linkages. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least three phosphorothioate triester linkages. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least four phosphorothioate triester linkages. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least five phosphorothioate triester linkages. Examples of such modified internucleotide phosphorus linkages are further described herein.
In some embodiments, the chirally controlled C9orf72 oligonucleotides comprise different internucleotide phosphorus linkages. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least one modified internucleotide linkage. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least one phosphorothioate triester linkage. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least two phosphorothioate triester linkages. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least three phosphorothioate triester linkages. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least four phosphorothioate triester linkages. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least five phosphorothioate triester linkages. Examples of such modified internucleotide phosphorus linkages are further described herein.
In some embodiments, the phosphorothioate triester linkage comprises a chiral auxiliary, for example to control the stereoselectivity of the reaction. In some embodiments, the phosphorothioate triester linkage does not comprise a chiral auxiliary. In some embodiments, phosphorothioate triester linkages are intentionally maintained until administration to a subject, and/or phosphorothioate triester linkages are intentionally maintained during administration to a subject.
In some embodiments, the chirally controlled C9orf72 oligonucleotide is attached to a solid support. In some embodiments, the chirally controlled C9orf72 oligonucleotide is cleaved from the solid support.
In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least two consecutive modified internucleotide linkages. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least two consecutive phosphorothioate triester internucleotide linkages.
In some embodiments, provided C9orf72 oligonucleotides or a core or wings or both wings thereof comprise a backbone linkage pattern. In some embodiments, the backbone linkage pattern is or comprises a sequence of any one of: XOXOO, OOOOOOOOO, OOOOOOOOOOO, OOOOOOOOOXOOXOOXOOXOOXOOXOOXOOXOXXOOXX, OOOOOXOOXXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXXXOOXXXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX, OXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX, OXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXXOXXXOXXXOXXXXXXXOXOXXXOXXXXXXXOXOXOXXXOXXXOXOXOXOXOXOXOXOXOXOXOXOXXXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXXOXOXOXOXOXOXXXOXXXOXOXOXOXOXOXOXOXOXOXXXOXXXOXXXOXXX, OXOXOXOXOXOXOXOXOXXXOXXXOXXXOXXXOXXXXXXXOXXXOXXXOXXXOXXXOXOXOXXXOXOXOXOXOXXXOXXXOXXXOXXX, OXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXXOXXXOXXXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXXOXXXOXXXOXXXXXXXXXXXXXOXXXOXXXOXXXOXOXOXOXXXXXXXXX, OXOXOXOXXXOXOXOXOXOXOXOXXXOXXXOXXXOXXXOXXXOXXXOXXXOXXXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXX, OXXXOXXXOXXXOXOXOXOXOXOXOXXXOXXXOXXXOXOXXXOXXXOXOXOXOXOXXXOXXXOXXXOXXXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXXOXXXOXOXOXXXOXXXOXOXOXOXOXXXOXXXOXXXOXOXOXOXOXOXXXOXXXOXOXXXOXXX, OXXXOXXXOXOXOXOXOXOXOXXXOXXXOXXXOXXXOXOXOXOXOXOXOXXXOXXXOXXXOXXXOXXXOXXXOXXXOXXXOXXXOXXXOXXX, OXXXOXXXOXXXOXOXOXOXOXOXXXOXXXOXXX, OXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXXOXOXOXOXOXOXOXOXOXOXOXXXOXXXOXOXOXOXOXOXOXOXOXXX, XXOXOXOXOXOXOXOXOXOXOXOXOXOXXXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXX, OXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXXOXOXOXOXOXXX, OXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXXOXXXOXOXOXOXXXOXXXXXXXXXXXXXXXXX, XX, XXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXXX, XXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOX, XXXXXXXXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOXOOOOOOOXXX, XXXXXXXXXXXXXXXXXXXXOXOXOOOXOXOXOXOXOXOXOXOXOOXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXOXOOOXOOOXOOOXOOOXOOOXOOOXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXOXOO, XXOXOXOOOXOOOXOO, XXXXXXXXXXOXOXOOOXOOOXOO, XXXXXXXXXXOXOXOXOXOXOXOOOXOOOXOXOXOO, XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXOXOXOXOXOXOXOXOXOXOXOOOXOXOXOXOXOXOXOXOO, XXOXOOOXOOOXOXOXOXOXOXOXOXOXOXOXOOOXOO, XXXXXXXXXXXXOXOXOOOXOOOXOOOXOXOXOO, XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXOXOOOXOOOXOO, XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXOXOO, XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXOXOO, XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX, wherein O represents a phosphodiester and X represents an internucleotide linkage or a modified internucleotide linkage that is not a phosphodiester; in some embodiments, the modified internucleotide linkage is phosphorothioate; in some embodiments, the modified internucleotide linkages are chirally controlled; in some embodiments, the modified internucleotide linkage is a chirally controlled phosphorothioate.
In some embodiments, the C9orf72 oligonucleotide may comprise any internucleotide linkage described herein or known in the art.
In some embodiments, the disclosure provides C9orf72 oligonucleotides comprising one or more modified internucleotide linkages independently having the structure of formula I disclosed herein. In some embodiments, the modified internucleotide linkage is a phosphorothioate. Examples of internucleotide linkages having the structure of formula I are widely known in the art.
In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein at least one internucleotide linkage has a chirally bound phosphorus. In some embodiments, the present disclosureThere is provided a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein at least one internucleotide linkage has the structure of formula I. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein each internucleotide linkage has the structure of formula I. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein at least one internucleotide linkage has the structure of formula I-C. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein each internucleotide linkage has the structure of formula I-C. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein at least one of the phosphoroamidites isIn some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein each internucleotide linkage is
In some embodiments, the modified internucleotide linkage is an internucleotide linkage that is not negatively charged. In some embodiments, the modified internucleotide linkage is a neutral internucleotide linkage. In some embodiments, provided oligonucleotides comprise one or more internucleotide linkages without a negative charge. In some embodiments, the non-negatively charged internucleotide linkage is a positively charged internucleotide linkage. In some embodiments, the non-negatively charged internucleotide linkage is a neutral internucleotide linkage. In some embodiments, the modified internucleotide linkage (e.g., an internucleotide linkage without a negative charge) comprises an optionally substituted triazolyl. In some embodiments, the modified nucleotideAn internucleotide linkage (e.g., an internucleotide linkage without a negative charge) comprises an optionally substituted alkynyl group. In some embodiments, the modified internucleotide linkage comprises a triazole or alkyne moiety. In some embodiments, the triazole moiety (e.g., triazolyl group) is optionally substituted. In some embodiments, the triazole moiety (e.g., triazolyl group) is substituted. In some embodiments, the triazole moiety is unsubstituted. In some embodiments, the modified internucleotide linkage comprises an optionally substituted cyclic guanidine moiety. In some embodiments, the modified internucleotide linkage comprises an optionally substituted cyclic guanidine moiety and has the structure:wherein W is O or S. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, the non-negatively charged internucleotide linkages are stereochemically controlled.
In some embodiments, the internucleotide linkage comprising a triazole moiety (e.g., an optionally substituted triazolyl group) in the provided oligonucleotides (e.g., C9orf72 oligonucleotides) has the following structure:in some embodiments, the internucleotide linkage comprising a triazole moiety has the formula:wherein W is O or S. In some embodiments, the internucleotide linkage comprising an alkyne moiety (e.g., an optionally substituted alkynyl group) has the formula:wherein W is O or S. In some embodiments, the internucleotide linkage comprises a cyclic guanidine moiety. In some embodiments, the internucleotide linkage comprises a cyclic guanidine moiety having the structure:in some embodiments, the neutral internucleotide linkage or the internucleotide linkage comprising a cyclic guanidine moiety is stereochemically controlled.
In some embodiments, the C9off72 oligonucleotide comprises a lipid moiety. In some embodiments, the internucleotide linkage comprises a Tmg groupIn some embodiments, the internucleotide linkage comprises a Tmg group and has(iii) a structure of (i) ("Tmg internucleotide linkage"). In some embodiments, the neutral internucleotide linkage comprises an internucleotide linkage of PNA and PMO and a Tmg internucleotide linkage.
In some embodiments, the non-negatively charged internucleotide linkages have the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, and the like, or a salt form thereof. In some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms. In some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, such heterocyclyl or heteroaryl groups have a 5-membered ring. In some embodiments, such heterocyclyl or heteroaryl groups have 6-membered rings.
In some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms. In some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the non-negatively charged internucleotide linkage comprises a linker having 1-an optionally substituted 5-6 membered heteroaryl group of 4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5-membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the heteroaryl group is directly bonded to the phosphorus linkage. In some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted triazolyl group. In some embodiments, the non-negatively charged internucleotide linkage comprises an unsubstituted triazolyl group, e.g.,in some embodiments, the non-negatively charged internucleotide linkages comprise a substituted triazolyl group, e.g.,
in some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms. In some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5-6 membered heterocyclyl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5-membered heterocyclyl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, at least two heteroatoms are nitrogen. In some embodiments, the heterocyclyl group is directly bonded to the phosphorus linkage. In some embodiments, when a heterocyclyl group is part of a guanidine moiety that is directly bonded to a phosphorus linkage via its ═ N —, the heterocyclyl group is bonded to the phosphorus linkage via a linker (e.g., ═ N —). In some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted internucleotide linkageA group. In some embodiments, the non-negatively charged internucleotide linkages comprise substitutedA group. In some embodiments, the non-negatively charged internucleotide linkage comprisesA group. In some embodiments, each R1Independently is optionally substituted C1-6An alkyl group. In some embodiments, each R1Independently a methyl group.
In some embodiments, the modified internucleotide linkage (e.g., an internucleotide linkage without a negative charge) comprises a triazole or alkyne moiety, each of which is optionally substituted. In some embodiments, the modified internucleotide linkage comprises a triazole moiety. In some embodiments, the modified internucleotide linkage comprises an unsubstituted triazole moiety. In some embodiments, the modified internucleotide linkage comprises a substituted triazole moiety. In some embodiments, the modified internucleotide linkage comprises an alkyl moiety. In some embodiments, the modified internucleotide linkage comprises an optionally substituted alkynyl group. In some embodiments, the modified internucleotide linkage comprises an unsubstituted alkynyl group. In some embodiments, the modified internucleotide linkage comprises a substituted alkynyl group. In some embodiments, the alkynyl group is directly bonded to the phosphorus linkage.
In some embodiments, the oligonucleotides comprise different types of internucleotide phospholinkages. In some embodiments, the chirally controlled oligonucleotide comprises at least one natural phosphate linkage and at least one modified (non-natural) internucleotide linkage. In some embodiments, the oligonucleotide comprises at least one natural phosphate linkage and at least one phosphorothioate. In some embodiments, the oligonucleotide comprises at least one internucleotide linkage that is not negatively charged. In some embodiments, the oligonucleotide comprises at least one dayA phosphate linkage and at least one internucleotide linkage having no negative charge. In some embodiments, the oligonucleotide comprises at least one phosphorothioate internucleotide linkage and at least one non-negatively charged internucleotide linkage. In some embodiments, the oligonucleotide comprises at least one phosphorothioate internucleotide linkage, at least one native phosphate linkage, and at least one non-negatively charged internucleotide linkage. In some embodiments, the oligonucleotide comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, non-negatively charged internucleotide linkages. In some embodiments, the non-negatively charged internucleotide linkages are non-negative in that less than 50%, 40%, 30%, 20%, 10%, 5% or 1% of the internucleotide linkages are present in the form of a negatively charged salt in aqueous solution at a given pH. In some embodiments, the pH is about pH 7.4. In some embodiments, the pH is about 4-9. In some embodiments, the percentage is less than 10%. In some embodiments, the percentage is less than 5%. In some embodiments, the percentage is less than 1%. In some embodiments, the internucleotide linkage is an internucleotide linkage that is not negatively charged, as the neutral form of the internucleotide linkage does not have a pKa in water of no more than about 1, 2, 3, 4, 5, 6, or 7. In some embodiments, none have a pKa of 7 or less. In some embodiments, none have a pKa of 6 or less. In some embodiments, none have a pKa of 5 or less. In some embodiments, none have a pKa of 4 or less. In some embodiments, none have a pKa of 3 or less. In some embodiments, none have a pKa of 2 or less. In some embodiments, none have a pKa of 1 or less. In some embodiments, the pKa of the neutral form of the internucleotide linkage may be represented as having the structure CH3-internucleotide linkage-CH3pKa of the neutral form of the compound of (a). For example, the pKa of the neutral form of the internucleotide linkage having the structure of formula I may be determined from the structuresThe pKa of the neutral form of the compound of (a),can be expressed aspKa of (a). In some embodiments, the non-negatively charged internucleotide linkage is a neutral internucleotide linkage. In some embodiments, the non-negatively charged internucleotide linkage is a positively charged internucleotide linkage. In some embodiments, the non-negatively charged internucleotide linkage comprises a guanidine moiety. In some embodiments, the non-negatively charged internucleotide linkages comprise a heteroaryl base moiety. In some embodiments, the non-negatively charged internucleotide linkage comprises a triazole moiety. In some embodiments, the non-negatively charged internucleotide linkage comprises an alkynyl moiety.
In some embodiments, the non-negatively charged internucleotide linkages have the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof (non-negative charge). In some embodiments, the internucleotide linkage (e.g., an internucleotide linkage without a negative charge) has the structure of formula I-n-1 or a salt form thereof:
in some embodiments, X is a covalent bond and-X-Cy-R1is-Cy-R1. In some embodiments, -Cy-is an optionally substituted divalent group selected from a 5-20 membered heteroaryl ring having 1-10 heteroatoms and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms. In some embodiments, -Cy-is an optionally substituted divalent 5-20 membered heteroaryl ring having 1-10 heteroatoms. In some embodiments, -Cy-R1Is an optionally substituted 5-20 membered heteroaryl ring having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, -Cy-R1Is an optionally substituted 5-membered heteroaryl ring having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments，-Cy-R1Is an optionally substituted 6-membered heteroaryl ring having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, -Cy-R1Is an optionally substituted triazolyl.
In some embodiments, the internucleotide linkage (e.g., an internucleotide linkage without a negative charge) has the structure of formula I-n-2 or a salt form thereof:
in some embodiments, R1Is R'. In some embodiments, L is a covalent bond. In some embodiments, the internucleotide linkage (e.g., an internucleotide linkage without a negative charge) has the structure of formula I-n-3 or a salt form thereof:
in some embodiments, two R' on different nitrogen atoms are taken together to form a ring as described. In some embodiments, the loop formed is 5-membered. In some embodiments, the loop formed is 6-membered. In some embodiments, the ring formed is substituted. In some embodiments, two R' groups that do not together form a ring are each independently R. In some embodiments, two R' groups that do not together form a ring are each independently hydrogen or optionally substituted C1-6An aliphatic group. In some embodiments, two R' groups that do not together form a ring are each independently hydrogen or optionally substituted C1-6An alkyl group. In some embodiments, two R' groups that do not together form a ring are the same. In some embodiments, two R' groups that do not together form a ring are different. In some embodiments, both are-CH3。
In some embodiments, the internucleotide linkage (e.g., an internucleotide linkage without a negative charge) has the structure of formula II or a salt form thereof:
or a salt form thereof, wherein:
PLis P (═ W), P or P → B (R')3；
W is O, N (-L-R)5) S or Se;
x, Y and Z are each independently-O-, -S-, -N (-L-R)5) -, or L;
ring ALIs an optionally substituted 3-to 20-membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms;
each RsIndependently is-H, halogen, -CN, -N3、-NO、-NO2、-L-R′、-L-Si(R)3、-L-OR′、-L-SR′、-L-N(R′)2、-O-L-R′、-O-L-Si(R)3-O-L-OR ', -O-L-SR ' OR-O-L-N (R ')2；
g is 0 to 20;
each L is independently a covalent bond or is selected from C with 1-10 heteroatoms1-30Aliphatic radical and C1-30A divalent optionally substituted straight or branched chain radical of a heteroaliphatic group, wherein one or more methylene units are optionally and independently replaced by: c1-6Alkylene radical, C1-6Alkenylene, -C ≡ C-, divalent C with 1-5 heteroatoms1-C6Heteroaliphatic, -C (R')2-、-Cy-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)O-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-C(O)S-、-C(O)O-、-P(O)(OR′)-、-P(O)(SR′)-、-P(O)(R′)-、-P(O)(NR′)-、-P(S)(OR′)-、-P(S)(SR′)-、-P(S)(R′)-、-P(S)(NR′)-、-P(R′)-、-P(OR′)-、-P(SR′)-、-P(NR′)-、-P(OR′)[B(R′)3]-, -OP (O) (OR ') O-, -OP (O) (SR') O-, -OP (O) (R ') O-, -OP (O) (NR') O-, -OP (OR ') O-, -OP (SR') O-, -OP (NR ') O-, -OP (R') O-, OR-OP (OR ') [ B (R')3]O-, and one or more CH or carbon atoms are optionally and independently CyLReplacement;
each-Cy-is independently an optionally substituted divalent group selected from: c3-20Cycloaliphatic Ring, C6-20An aromatic ring, a 5-to 20-membered heteroaromatic ring having 1-10 heteroatoms, and a 3-to 20-membered heterocyclic ring having 1-10 heteroatoms;
each CyLIndependently is an optionally substituted trivalent or tetravalent group selected from: c3-20Cycloaliphatic Ring, C6-20An aromatic ring, a 5-to 20-membered heteroaromatic ring having 1-10 heteroatoms, and a 3-to 20-membered heterocyclic ring having 1-10 heteroatoms;
each R' is independently-R, -C (O) OR, OR-S (O)2R；
Each R is independently-H, or an optionally substituted group selected from: c1-30Aliphatic, C having 1-10 heteroatoms1-30Heteroaliphatic, C6-30Aryl radical, C6-30Arylaliphatic, C having 1-10 heteroatoms6-30Aryl heteroaliphatic, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or
Two R groups optionally and independently form a covalent bond together, or
Two or more R groups on the same atom optionally and independently form, together with the atom, an optionally substituted 3-30 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms other than the atom, or
Two or more R groups on two or more atoms optionally and independently form, together with their intervening atoms, an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having 0-10 heteroatoms in addition to the intervening atoms.
In some embodiments, the internucleotide linkage (e.g., the non-negatively charged internucleotide linkage of formula II) has the structure of formula II-a-1 or a salt form thereof:
or a salt form thereof.
In some embodiments, the internucleotide linkage (e.g., the non-negatively charged internucleotide linkage of formula II) has the structure of formula II-a-2 or a salt form thereof:
or a salt form thereof.
In some embodiments, ALBonded to-N ═ or L via a carbon atom. In some embodiments, the internucleotide linkage (e.g., a non-negatively charged internucleotide linkage of formula II or formula II-a-1, formula II-a-2) has the structure of formula II-b-1 or a salt form thereof:
in some embodiments, the structure of formula II-a-1 or II-a-2 may be referred to as a structure of formula II-a. In some embodiments, the structure of formula II-b-1 or II-b-2 may be referred to as a structure of formula II-b. In some embodiments, the structure of formula II-c-1 or II-c-2 may be referred to as a structure of formula II-c. In some embodiments, the structure of formula II-d-1 or II-d-2 may be referred to as a structure of formula II-d.
In some embodiments, ALBonded to-N ═ or L via a carbon atom. In some embodiments, the internucleotide linkage (e.g., a non-negatively charged internucleotide linkage of formula II or formula II-a-1, formula II-a-2) has the structure of formula II-b-2 or a salt form thereof:
in some embodiments, ring aLIs an optionally substituted 3-to 20-membered monocyclic ring having (in addition to the two nitrogen atoms of formula II-b) 0-10 heteroatoms. In some embodiments, ring aLIs an optionally substituted 5-membered monocyclic saturated ring.
In some embodiments, the internucleotide linkage (e.g., the non-negatively charged internucleotide linkage of formula II, II-a, or II-b) has the structure of formula II-c-1 or a salt form thereof:
in some embodiments, the internucleotide linkage (e.g., a non-negatively charged internucleotide linkage of formula II, II-a, or II-b) has the structure of formula II-c-2 or a salt form thereof:
in some embodiments, the internucleotide linkage (e.g., the non-negatively charged internucleotide linkage of formula II, II-a, II-b, or II-c) has the structure of formula II-d-1 or a salt form thereof:
in some embodiments, the internucleotide linkage (e.g., the non-negatively charged internucleotide linkage of formula II, II-a, II-b, or II-c) has the structure of formula II-d-2 or a salt form thereof:
in some embodiments, each R' is independently optionally substituted C1-6An aliphatic group. In some embodiments, each R' is independently optionally substituted C1-6An alkyl group. In some embodiments, each R' is independently-CH3. In some embodiments, each Rsis-H.
In some embodiments, the internucleotide linkage without a negative charge hasThe structure of (1). In some embodiments, the internucleotide linkage without a negative charge hasThe structure of (1). In some embodiments, do notThe negatively charged internucleotide linkage hasThe structure of (1). In some embodiments, the internucleotide linkage without a negative charge hasThe structure of (1). In some embodiments, the internucleotide linkage without a negative charge hasThe structure of (1). In some embodiments, the internucleotide linkage without a negative charge hasThe structure of (1). In some embodiments, the internucleotide linkage without a negative charge hasThe structure of (1). In some embodiments, the internucleotide linkage without a negative charge hasThe structure of (1). In some embodiments, the internucleotide linkage without a negative charge hasThe structure of (1). In some embodiments, the internucleotide linkage without a negative charge hasThe structure of (1). In some embodiments, the internucleotide linkage without a negative charge hasThe structure of (1). In some embodiments, the internucleotide linkage without a negative charge hasThe structure of (1). In some embodiments, W is O. In some embodiments, W is S.
In some embodiments, each LP independently has the structure of formula I, formula I-a, formula I-b, formula I-c, formula I-n-1, formula I-n-2, formula I-n-3, formula II-a-1, formula II-a-2, formula II-b-1, formula II-b-2, formula II-c-1, formula II-c-2, formula II-d-1, formula II-d-2, or a salt form thereof.
In some embodiments, the present disclosure provides oligonucleotides comprising one or more internucleotide linkages without a negative charge. In some embodiments, the non-negatively charged internucleotide linkage is a neutral internucleotide linkage. In some embodiments, the present disclosure provides oligonucleotides comprising one or more neutral internucleotide linkages. In some embodiments, the non-negatively charged internucleotide linkages have the structure of formula I-n-1, I-n-2, I-n-3, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.
In some embodiments, the non-negatively charged internucleotide linkage comprises a triazole moiety. In some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted triazolyl group. In some embodiments, the internucleotide linkage without a negative charge hasThe structure of (1). In some embodiments, the internucleotide linkage without a negative charge hasThe structure of (1). In some embodiments, the non-negatively charged internucleotide linkage comprises a substituted triazolyl group. In some embodiments, the non-negatively charged internucleotide linkage hasWherein W is O or S. In some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted alkynyl group. In some embodiments, the non-negatively charged internucleotide linkage has the followingWherein W is O or S.
In some embodiments, the present disclosure provides oligonucleotides comprising internucleotide linkages (e.g., non-negatively charged internucleotide linkages) comprising a cyclic guanidine moiety. In some embodiments, the internucleotide linkage comprises a cyclic guanidine and has the structure:in some embodiments, the cyclic guanidine-containing internucleotide linkage (e.g., an internucleotide linkage without a negative charge) is stereochemically controlled.
In some embodiments, the non-negatively charged internucleotide linkage or the neutral internucleotide linkage is or comprises a structure selected from the group consisting of:wherein W is O or S. In some embodiments, the non-negatively charged internucleotide linkage is a chirally controlled internucleotide linkage. In some embodiments, the neutral internucleotide linkage is a chirally controlled internucleotide linkage. In some embodiments, the nucleic acid or oligonucleotide comprising a modified internucleotide linkage comprising a cyclic guanidine moiety is an siRNA, a double stranded siRNA, a single stranded siRNA, a notch, a skip, a block, an antisense oligonucleotide, an antagomir, a microrna, a pre-microrn, an antimir, a supermir, a ribozyme, a U1 adaptor, an RNA activator, an RNAi agent, a decoy oligonucleotide, a triplex forming oligonucleotide, an aptamer, or an adjuvant.
In some embodiments, the oligonucleotide comprises neutral internucleotide linkages and chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotide comprises a neutral internucleotide linkage and a chirality-controlled internucleotide linkage that is a phosphorothioate in either the Rp or Sp configuration. In some embodiments, the present disclosure provides oligonucleotides comprising one or more non-negatively charged internucleotide linkages and one or more phosphorothioate internucleotide linkages, wherein each phosphorothioate internucleotide linkage in the oligonucleotide is independently a chirally controlled internucleotide linkage. In some embodiments, the present disclosure provides an oligonucleotide comprising one or more neutral internucleotide linkages and one or more phosphorothioate internucleotide linkages, wherein each phosphorothioate internucleotide linkage in the oligonucleotide is independently a chirally controlled internucleotide linkage. In some embodiments, provided oligonucleotides comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more chirally controlled phosphorothioate internucleotide linkages.
Without wishing to be bound by any particular theory, the present disclosure indicates that neutral internucleotide linkages may be more hydrophobic than phosphorothioate internucleotide linkages (PS), which are more hydrophobic than phosphodiester linkages (natural phosphate linkages, PO). Generally, unlike PS or PO, neutral internucleotide linkages carry less charge. Without wishing to be bound by any particular theory, the present disclosure indicates that incorporating one or more neutral internucleotide linkages into an oligonucleotide may increase the ability of the oligonucleotide to be taken up by a cell and/or to escape endosomes. Without wishing to be bound by any particular theory, the present disclosure indicates that the incorporation of one or more neutral internucleotide linkages can be used to modulate the melting temperature between an oligonucleotide and its target nucleic acid.
Without wishing to be bound by any particular theory, the present disclosure indicates that incorporating one or more non-negatively charged internucleotide linkages (e.g., neutral internucleotide linkages) into an oligonucleotide can increase the ability of the oligonucleotide to mediate functions such as exon skipping or gene knockdown. In some embodiments, a knockdown oligonucleotide capable of mediating a level of a nucleic acid or a product encoded thereby comprises one or more non-negatively charged internucleotide linkages. In some embodiments, the oligonucleotide capable of mediating a knockdown in expression of a target gene comprises one or more internucleotide linkages without a negative charge. In some embodiments, the oligonucleotide capable of mediating a knockdown in expression of a target gene comprises one or more neutral internucleotide linkages.
In some embodiments, the non-negatively charged internucleotide linkages are not chirally controlled. In some embodiments, the non-negatively charged internucleotide linkages are chirally controlled. In some embodiments, the internucleotide linkage without a negative charge is chirally controlled and its linkage phosphorus is Rp. In some embodiments, the non-negatively charged internucleotide linkages are chirally controlled and the phosphorus of the linkages is Sp.
In some embodiments, provided oligonucleotides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more internucleotide linkages without negative charge. In some embodiments, provided oligonucleotides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more neutral internucleotide linkages. In some embodiments, the non-negatively charged internucleotide linkages and/or the neutral internucleotide linkages are each optionally and independently chirally controlled. In some embodiments, each non-negatively charged internucleotide linkage in the oligonucleotide is independently a chirally controlled internucleotide linkage. In some embodiments, each neutral internucleotide linkage in the oligonucleotide is independently a chirally controlled internucleotide linkage. In some embodiments, at least one non-negatively charged internucleotide linkage/neutral internucleotide linkage hasWherein W is O or S. In some embodiments, at least one non-negatively charged internucleotide linkage/neutral internucleotide linkage hasThe structure of (1). In some embodiments, at least one non-negatively charged internucleotide linkage/neutral internucleotide linkage hasThe structure of (1). In some embodiments, at least oneThe internucleotide linkage/neutral internucleotide linkage without negative charge hasWherein W is O or S. In some embodiments, at least one non-negatively charged internucleotide linkage/neutral internucleotide linkage hasThe structure of (1). In some embodiments, at least one non-negatively charged internucleotide linkage/neutral internucleotide linkage is identifiedThe structure of (1). In some embodiments, at least one non-negatively charged internucleotide linkage/neutral internucleotide linkage hasWherein W is O or S. In some embodiments, at least one non-negatively charged internucleotide linkage/neutral internucleotide linkage hasThe structure of (1). In some embodiments, at least one non-negatively charged internucleotide linkage/neutral internucleotide linkage hasThe structure of (1). In some embodiments, provided oligonucleotides comprise at least one non-negatively charged internucleotide linkage in which the phosphorated linkage is in the Rp configuration and at least one non-negatively charged internucleotide linkage in which the phosphorated linkage is in the Sp configuration.
In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein at least one of the linkages is Rp, as will be understood by one of ordinary skill in the art, in certain embodiments where the chirally controlled C9orf72 oligonucleotide comprises a base sequence, each T is independently and optionally substituted with U. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein each bonded phosphorus is Rp. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein at least one of the linkages is Sp in some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein each linkage is Sp in some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein the oligonucleotide is a block. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein the oligonucleotide is a stereoblock. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein the oligonucleotide is a P-modified block. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein the oligonucleotide is a linker block. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein the oligonucleotides are alternans. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein the oligonucleotide is a stereo-alternating. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein the oligonucleotide is a P modified alternating. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein the oligonucleotide is a linkage alternating. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein the oligonucleotide is a homopolymer. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein the oligonucleotide is a stereomonomer. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein the oligonucleotide is a P-modified homopolymer. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein the oligonucleotide is a linked homopolymer. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein the oligonucleotide is a notch. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein the oligonucleotide is a skip entity.
In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein each cytosine is optionally and independently replaced with a 5-methylcytosine. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein at least one cytosine is optionally and independently replaced with a 5-methylcytosine. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein, wherein each cytosine is optionally and independently replaced with a 5-methylcytosine.
In some embodiments, the chirally controlled C9orf72 oligonucleotide is designed such that one or more nucleotides comprise a phosphorus modification susceptible to "self release" (auto elease) under certain conditions. That is, under certain conditions, specific phosphorus modifications are designed such that they self-cleave from an oligonucleotide to provide, for example, a phosphateDiesters, such as those found in naturally occurring DNA and RNA. In some embodiments, such phosphorus modifications-O-L-R1Wherein L and R1Each independently described in the present disclosure. In some embodiments, the self-releasing group comprises a morpholino group. In some embodiments, the self-releasing group is characterized by the ability to deliver an agent to the internucleotide phosphorus linker that helps to further modify the phosphorus atom, such as desulfurization. In some embodiments, the agent is water, and the further modification is hydrolysis to form phosphodiesters as found in naturally occurring DNA and RNA.
In some embodiments, the chirally controlled C9orf72 oligonucleotide is designed such that the resulting drug property is improved via one or more specific modifications at the phosphorus. It is well documented in the art that certain oligonucleotides are rapidly degraded by nucleases and exhibit poor cellular uptake through cytoplasmic membranes (Poijarvi-Virta et al, curr. Med. chem. [ medical and chemical new ] (2006), 13 (28); 3441-65; Wagner et al, Med. Res. Rev. [ medical research review ] (2000), 20 (6): 417-51; Peyrottes et al, Mini Rev. Med.chem. [ medical and chemical brief ] (review 2004), 4 (4): 395-408; Gosselin et al, (1996), 43 (1): 196-208; Bologna et al, (2002), Antisense & Nucleic Acid Drug Development [ Antisense and Nucleic Acid Drug Development ] 12: 33-41). For example, Vives et al (Nucleic Acids Research (1999), 27 (20): 4071-76) found that the pro-tert-butyl SATE oligonucleotide (pro-oligonucleotide) exhibited significantly increased cell penetration compared to the parent oligonucleotide.
Base sequence of C9orf72 oligonucleotide
In some embodiments, provided C9orf72 oligonucleotides are capable of directing a decrease in expression, level, and/or activity of C9orf72 gene or gene product thereof. In some embodiments, the C9orf72 target gene comprises repeat amplification. In some embodiments, provided C9orf72 oligonucleotides can comprise any of the base sequences described herein, or portions thereof, wherein a portion is a stretch of at least 15 contiguous bases or a stretch of at least 15 contiguous bases with 1 to 5 mismatches.
In some embodiments, the base sequence of the C9orf72 oligonucleotide is of sufficient length and identity to the C9orf72 transcript target to mediate target-specific knockdown. In some embodiments, the C9orf72 oligonucleotide is complementary to a portion of the transcript target sequence.
In some embodiments, the base sequence of the C9orf72 oligonucleotide is complementary to the base sequence of the C9orf72 target transcript. As used herein, "target transcript sequence," "target gene," and the like refer to a contiguous portion of the nucleotide sequence of an mRNA molecule (including mRNA that is the RNA processing product of the original transcript) formed during transcription of the C9orf72 gene.
The terms "complementary," "fully complementary," and "substantially complementary" herein may be used in terms of base matching between a C9orf72 oligonucleotide and a C9orf72 target sequence, as will be understood from their context of use. In some embodiments, the base sequence of the C9orf72 oligonucleotide is complementary to the base sequence of the C9orf72 target sequence when, at maximum alignment, each base of the oligonucleotide is capable of base pairing with a sequential base on the target strand. As a non-limiting example, if the target sequence has a base sequence such as 5'-GCAUAGCGAGCGAGGGAAAAC-3', an oligonucleotide having a base sequence of 5 'GUUUUCCCUCGCUCGCUAUGC-3' is complementary or fully complementary to the target sequence. Of course, it should be noted that the substitution of T with U or vice versa does not change the amount of complementarity.
As used herein, a polynucleotide that is "substantially complementary" to a C9orf72 target sequence is largely or substantially complementary, but is not 100% complementary. In some embodiments, a substantially complementary sequence (e.g., a C9orf72 oligonucleotide) has 1, 2, 3, 4, or 5 mismatches with a sequence that is 100% complementary to a target sequence.
The disclosure provides, among other things, in table 1A, various oligonucleotides, each having a defined base sequence. In some embodiments, the present disclosure encompasses any oligonucleotide having a base sequence that is, comprises, or is part of the base sequence of any of the oligonucleotides disclosed herein. In some embodiments, the present disclosure encompasses any oligonucleotide having a base sequence that is, comprises, or comprises a portion of the base sequence of any of the oligonucleotides disclosed herein having any chemical modification, stereochemistry, form, structural feature (e.g., any structural or modification pattern or portion thereof), and/or any other modification described herein (e.g., conjugation to another moiety (e.g., a targeting moiety, a carbohydrate moiety, etc.); and/or multimerization). In some embodiments, a "portion" (e.g., a portion of a base sequence or modification pattern) is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 long. In some embodiments, a "portion" of a base sequence is at least 5nt long. In some embodiments, a "portion" of a base sequence is at least 10nt long. In some embodiments, a "portion" of a base sequence is at least 15nt long. In some embodiments, a "portion" of a base sequence is at least 20nt long.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCCACACCTGCTCTTGCTAG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCGGGCAGCAGGGACGGCTG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCGGTTGCGGTGCCTGCGCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCGGTTGTTTCCCTCCTTGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTTGTTCACCCTCAGCGAGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ACCCCCATCTCATCCCGCAT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGACCCGCTGGGAGCGCTGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TGCCGCCTCCTCACTCACCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCTCTCTTTCCTAGCGGGAC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TCCCCATTCCAGTTTCCATC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGCCTCTCAGTACCCGAGGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGATGCCGCCTCCTCACTCA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGCAGCAGGGACGGCTGACA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TACCCGCGCCTCTTCCCGGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ACAGGCTGCGGTTGTTTCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTCTTCCCGGCAGCCGAACC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGGAGGTCCTGCACTTTCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCTGGGTGTCGGGCTTTCGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTTCCTTGCTTTCCCGCCCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CGCTTCTACCCGCGCCTCTT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTTCTACCCGCGCCTCTTCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCAGGCGGTGGCGAGTGGGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCCGCCTCCTCACTCACCCA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ACATCCCCTCACAGGCTCTT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCCTCCTTGTTTTCTTCTGG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTGGCTCTCCAGAAGGCTGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: AGGCTGTCAGCTCGGATCTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CGCCTCCTCACTCACCCACT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTCTTTCCTAGCGGGACACC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CACCCACTCGCCACCGCCTG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TACAGGCTGCGGTTGTTTCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CGCACCTCTCTTTCCTAGCG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TGGCGAGTGGGTGAGTGAGGAGGCGGCATC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCCACCCGCCAGGATGCCGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GATGCACCTGACATCCCCTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCTTGCTACAGGCTGCGGTT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTCACTCACCCACTCGCCAC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTCCTCACTCACCCACTCGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGGAAGGCCGGAGGGTGGGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGGCAGCAGGGACGGCTGAC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCGCAGGCGGTGGCGAGTGGGTGAGTGAGG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGCTGCGGTTGTTTCCCTCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TCTCAGTACCCGAGGCTCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCTTGGTGTGTCAGCCGTCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTGTTCTGTCTTTGGAGCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CGCATAGAATCCAGTACCAT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: AGCGCGCGACTCCTGAGTTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: AGGCTGCGGTTGTTTCCCTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTCAGTACCCGAGGCTCCCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTTCCCGGCAGCCGAACCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CACCCGCCAGGATGCCGCCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ACCTCTCTTTCCTAGCGGGA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCTACAGGCTGCGGTTGTTT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCGCGACTCCTGAGTTCCAG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TCCCGGCAGCCGAACCCCAA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCCAGATCCCCATCCCTTGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTCACCCACTCGCCACCGCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: AGCAACCGGGCAGCAGGGAC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCTGCGGTTGTTTCCCTCCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: AACCGGGCAGCAGGGACGGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGTTGTTTCCCTCCTTGTTT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTTGGTGTGTCAGCCGTCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCTGGAGATGGCGGTGGGCA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCCGCGCCTCTTCCCGGCAG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTTGCTAGACCCCGCCCCCA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGCTCTCCAGAAGGCTGTCA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TCCTCACTCACCCACTCGCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CAGGATGCCGCCTCCTCACT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCTGGTTGCTTCACAGCTCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CGGGCAGCAGGGACGGCTGA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTGCTGCGATCCCCATTCCA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCCGCCAGGATGCCGCCTCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GATCCCCATCCCTTGTCCCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCCTTACTCTAGGACCAAGA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ACCGGGCAGCAGGGACGGCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TGCCAGGCTGGTTATGACTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCATCCTGGCGGGTGGCTGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCTCTTCCCGGCAGCCGAAC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCCCAAACAGCCACCCGCCA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTTGCGGTGCCTGCGCCCGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCCAAACAGCCACCCGCCAG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ACCCGCCAGGATGCCGCCTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TCTTCCCGGCAGCCGAACCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCTCCGGCCTTCCCCCAGGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCATCCGGGCCCCGGGCTTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CACCCCCATCTCATCCCGCA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CAGAGCTTGCTACAGGCTGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCGCTTCTACCCGCGCCTCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTGCAGGCGTCTCCACACCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ACTCACCCACTCGCCACCGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCAGGCGTCTCCACACCCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCTGAGTTCCAGAGCTTGCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GAGAGCCCCCGCTTCTACCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TCCTGAGTTCCAGAGCTTGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: AGCTTGCTACAGGCTGCGGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCAAACAGCCACCCGCCAGG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCGCAGGCGGTGGCGAGTGGGTGAGTGAGGAGGCGGCATC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTGCGGTTGTTTCCCTCCTT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: AGCCGTCCCTGCTGCCCGGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCCTCCTCACTCACCCACTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TGCTACAGGCTGCGGTTGTT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCAGGGACGGCTGACACACC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: AGGATGCCGCCTCCTCACTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGTCTTTTCTTGTTCACCCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCTGCTCTTGCTAGACCCCG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTTCACCCTCAGCGAGTACT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCTAGCGGGACACCGTAGGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCCCTCAGTACCCGAGCTGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTACCCGCGCCTCTTCCCGG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTCTTTTCTTGTTCACCCTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGGTGTCGGGCTTTCGCCTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CGGTTGTTTCCCTCCTTGTT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCACCCTCCGGCCTTCCCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCCTCTCAGTACCCGAGGCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCTCACTCACCCACTCGCCA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GAGCTTGCTACAGGCTGCGG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTGACATCCCCTCACAGGCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCTGTTTGACGCACCTCTCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TGGAATGGGGATCGCAGCAC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCGCCTCCTCACTCACCCAC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCTCTGCCAAGGCCTGCCAC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCGACTTGCATTGCTGCCCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TCACCCTCAGCGAGTACTGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCGCGCCTCTTCCCGGCAGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CGCCTCTTCCCGGCAGCCGA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TTGCTACAGGCTGCGGTTGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCCGGGAAGAGGCGCGGGTAG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCAACCGGGCAGCAGGGACG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TCTTGCTAGACCCCGCCCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCTAGACCCCGCCCCCAAAA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCTGCGATCCCCATTCCAGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCCACTCGCCACCGCCTGCG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTGGCAGGCCTTGGCAGAGG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ACGCACCTCTCTTTCCTAGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: AGGGCCACCCCTCCTGGGAA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCAGGATGCCGCCTCCTCAC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ACCCGCGCCTCTTCCCGGCA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ACCCGAGCTGTCTCCTTCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCCTCTTCCCGGCAGCCGAA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ACTCCTGAGTTCCAGAGCTT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ATTGCCTGCATCCGGGCCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TTCTACCCGCGCCTCTTCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTACCCGAGGCTCCCTTTTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCTGCTGCCCGGTTGCTTCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCGCGCGACTCCTGAGTTCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTCGGTGTGCTCCCCATTCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ACCCACTCGCCACCGCCTGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GACATCCCCTCACAGGCTCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GAGCTGCCCAGGACCACTTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCGGCATCCTGGCGGGTGGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTCCGTGTGCTCATTGGGTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TGGAATGGGGATCGCAGCACA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGCGGAGGCGCAGGCGGTGG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCCAGGATGCCGCCTCCTCA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCACTCGCCACCGCCTGCGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TGCCTGCATCCGGGCCCCGG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TTGCTAGACCCCGCCCCCAA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CAGGCTGCGGTTGTTTCCCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCCGTCCCTGCTGCCCGGTT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TTTCCCCACACCACTGAGCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TTCCAGAGCTTGCTACAGGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCCGGCAGCCGAACCCCAAA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TGTGCTGCGATCCCCATTCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GAGGCCAGATCCCCATCCCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTCGCTGTTTGACGCACCTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTCTTGCTAGACCCCGCCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCGCCAGGATGCCGCCTCCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCTCCTCACTCACCCACTCG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ATCCCCTCACAGGCTCTTGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCTCTTGCTAGACCCCGCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGTCCCTGCCGGCGAGGAGA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCTTCCCTGAAGGTTCCTCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TCCCCTCACAGGCTCTTGTG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TGCTCTTGCTAGACCCCGCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TTCCCGGCAGCCGAACCCCA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTCCCTGCTGCCCGGTTGCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCTTCTACCCGCGCCTCTTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTTTCCTAGCGGGACACCGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTGCAGGACCTCCCTCCTGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTGCTCCCCATTCTGTGGGA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TCCAGAGCTTGCTACAGGCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCCCTCACAGGCTCTTGTGA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TGCTAGACCCCGCCCCCAAA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TCACCCACTCGCCACCGCCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CTGCTCTTGCTAGACCCCGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TGCGGTTGTTTCCCTCCTTG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTTGTTTCCCTCCTTGTTTT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGCGTCTCCACACCCCCATC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGGCTCTCCTCAGAGCTCGA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ACCCTCCGGCCTTCCCCCAG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCGCAGCCTGTAGCAAGCTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: AACCCACACCTGCTCTTGCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: AGTGGTCCTGGGCAGCTCCT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TCCTTGCTTTCCCGCCCTCA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GAGCTCTGAGGAGAGCCCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCACCTCTCTTTCCTAGCGG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CGCCAGGATGCCGCCTCCTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: AACAGCCACCCGCCAGGATG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CAGGGTGGCATCTGCTTCAC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CACTCACCCACTCGCCACCG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCACCCGCCAGGATGCCGCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ACCCACACCTGCTCTTGCTA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ATGCCGCCTCCTCACTCACC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CACCTCTCTTTCCTAGCGGG are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GATCCCCATTCCAGTTTCCA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GTCAGCCGTCCCTGCTGCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: TCACTCACCCACTCGCCACC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGCTCCCTTTTCTCGAGCCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCAGGACCTCCCTCCTGTTT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCTTTCCCGCCCTCAGTACC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GATGCCGCCTCCTCACTCAC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: ACCCCAAACAGCCACCCGCC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCTCCTTGTTTTCTTCTGGT are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GGCCTTGGCAGAGGTGGTGA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GCTCTGAGGAGAGCCCCCGC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: GACAGGGTGGCATCTGCTTC are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CGCGCGACTCCTGAGTTCCA are provided.
In some embodiments, the oligonucleotide targets C9orf72 and its base sequence is, comprises, or comprises a portion of: CCACACCTGCTCTTGCTAGA are provided.
In some embodiments, the portion of the base sequence is a stretch of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more contiguous (contiguous) bases.
In some embodiments, the disclosure discloses C9orf72 oligonucleotides of the sequences recited herein. In some embodiments, the disclosure discloses C9orf72 oligonucleotides of the sequences listed herein, wherein the oligonucleotides are capable of directing a decrease in the expression, level, and/or activity of the C9orf72 gene or gene product thereof. In some embodiments, the C9orf72 oligonucleotide of the recited sequence comprises any structure described herein. In various sequences, U may be replaced by T or vice versa, or the sequence may comprise a mixture of U and T. In some embodiments, the C9orf72 oligonucleotide is up to about 49, 45, 40, 30, 35, 25, 23 total nucleotides in length. In some embodiments, a portion is a sequence segment of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 total nucleotides with 0 to 3 mismatches. In some embodiments, a portion is a sequence segment of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 total nucleotides with 0 to 3 mismatches, where sequence segments with 0 mismatches are complementary and sequence segments with 1 or more mismatches are non-limiting examples of substantial complementarity. In some embodiments where the sequence described above begins with a U at the 5' end, the U may be deleted and/or replaced with another base. In some embodiments, the disclosure encompasses any oligonucleotide having a base sequence that is or comprises a portion of: a base sequence of any of the oligonucleotides disclosed herein, the base sequence having a form or a portion of a form disclosed herein.
In some embodiments, the C9orf72 oligonucleotide may comprise any of the base sequences described herein. In some embodiments, the C9orf72 oligonucleotide may comprise any of the base sequences described herein, or portions thereof. In some embodiments, a C9orf72 oligonucleotide can comprise any of the base sequences described herein, or a portion thereof, wherein a portion is a stretch of 15 contiguous bases or a stretch of 15 contiguous bases with 1 to 5 mismatches. In some embodiments, a C9orf72 oligonucleotide can comprise a combination of any of the base sequences described herein, or portions thereof, and any other structural element or modification described herein.
Non-limiting examples of C9orf72 oligonucleotides having various base sequences and modifications are disclosed in Table 1A below.
Oligonucleotides
In some embodiments, the provided C9orf72 oligonucleotides can direct a decrease in expression, level, and/or activity of a C9orf72 target gene or gene product thereof. In some embodiments, the C9orf72 target gene comprises a hexanucleotide repeat amplification.
In some embodiments, provided C9orf72 oligonucleotides have a structural element or form described herein or a portion thereof.
In some embodiments, provided C9orf72 oligonucleotides capable of directing a reduction in expression, level, and/or activity of a C9orf72 target gene or gene product thereof have a structural element or form described herein or a portion thereof.
In some embodiments, a provided C9orf72 oligonucleotide capable of directing a reduction in expression, level, and/or activity of a C9orf72 target gene or gene product thereof has the form of any of the oligonucleotides disclosed herein, e.g., in table 1A or the figures, or elsewhere herein, or a structural element or form or portion thereof.
In some embodiments, the common pattern of backbone chiral centers (e.g., a pattern of backbone chiral centers in a C9orf72 oligonucleotide) comprises a pattern OSOSO, OSSSO, osssosos, SOSO, sosso, ssoss, ssossossoss, sssssososss, ssssossosssss, sssssssssss, sssssssssssssssssssssss, sssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssss, or RRR, wherein S represents a phosphorothioate in the Sp configuration and O represents a phosphodiester in the Rp configuration.
In some embodiments, provided C9orf72 oligonucleotides comprise at least two pairs of alternating phosphodiester and phosphorothioate internucleotide linkages. In some embodiments, provided C9orf72 oligonucleotides comprise at least 3 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages. In some embodiments, provided C9orf72 oligonucleotides comprise at least 4 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages. In some embodiments, provided C9orf72 oligonucleotides comprise at least 5 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages. In some embodiments, provided C9orf72 oligonucleotides comprise at least 6 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages. In some embodiments, provided C9orf72 oligonucleotides comprise at least 7 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages. In some embodiments, provided C9orf72 oligonucleotides comprise at least 8 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages. In some embodiments, provided C9orf72 oligonucleotides comprise at least 9 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages. In some embodiments, provided C9orf72 oligonucleotides comprise at least 10 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages. In some embodiments, provided C9orf72 oligonucleotides comprise at least two pairs of alternating phosphodiester and phosphorothioate internucleotide linkages; and further comprising a block comprising 5 or more consecutive phosphorothioate internucleotide linkages, wherein at least one phosphorothioate linkage is chirally controlled. In some embodiments, provided C9orf72 oligonucleotides comprise at least 3 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages; and further comprising a block comprising 5 or more consecutive phosphorothioate internucleotide linkages, wherein at least one phosphorothioate linkage is chirally controlled. In some embodiments, provided C9orf72 oligonucleotides comprise at least 4 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages; and further comprising a block comprising 5 or more consecutive phosphorothioate internucleotide linkages, wherein at least one phosphorothioate linkage is chirally controlled. In some embodiments, provided C9orf72 oligonucleotides comprise at least 5 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages; and further comprising a block comprising 5 or more consecutive phosphorothioate internucleotide linkages, wherein at least one phosphorothioate linkage is chirally controlled. In some embodiments, provided C9orf72 oligonucleotides comprise at least 6 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages; and further comprising a block comprising 5 or more consecutive phosphorothioate internucleotide linkages, wherein at least one phosphorothioate linkage is chirally controlled. In some embodiments, provided C9orf72 oligonucleotides comprise at least 7 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages; and further comprising a block comprising 5 or more consecutive phosphorothioate internucleotide linkages, wherein at least one phosphorothioate linkage is chirally controlled. In some embodiments, provided C9orf72 oligonucleotides comprise at least 8 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages; and further comprising a block comprising 5 or more consecutive phosphorothioate internucleotide linkages, wherein at least one phosphorothioate linkage is chirally controlled. In some embodiments, provided C9orf72 oligonucleotides comprise at least 9 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages; and further comprising a block comprising 5 or more consecutive phosphorothioate internucleotide linkages, wherein at least one phosphorothioate linkage is chirally controlled. In some embodiments, provided C9orf72 oligonucleotides comprise at least 10 pairs of alternating phosphodiester and phosphorothioate internucleotide linkages; and further comprising a block comprising 5 or more consecutive phosphorothioate internucleotide linkages, wherein at least one phosphorothioate linkage is chirally controlled. In some embodiments, provided C9orf72 oligonucleotides comprise at least two pairs of alternating phosphodiester and phosphorothioate internucleotide linkages; and further comprising a block comprising 3, 4, 5, 6, 7, or more consecutive phosphorothioate internucleotide linkages, wherein at least one phosphorothioate linkage is chirally controlled. In some embodiments, provided C9orf72 oligonucleotides comprise at least two pairs of alternating phosphodiester and phosphorothioate internucleotide linkages; and further comprising a block comprising 5 or more consecutive phosphodiester internucleotide linkages, wherein at least one phosphorothioate linkage is chirally controlled. In some embodiments, provided C9orf72 oligonucleotides comprise one or more natural phosphate linkages and one or more modified internucleotide linkages. The provided oligonucleotides can comprise various numbers of natural phosphate linkages. In some embodiments, provided C9orf72 oligonucleotides are capable of directing a decrease in expression, level, and/or activity of a C9orf72 target gene or gene product thereof. In some embodiments, the C9orf72 target gene comprises repeat amplification. In some embodiments, 5% or more of the internucleotide linkages in the provided C9orf72 oligonucleotides are natural phosphate linkages. In some embodiments, 10% or more of the internucleotide linkages in the provided C9orf72 oligonucleotides are natural phosphate linkages. In some embodiments, 15% or more of the internucleotide linkages in the provided C9orf72 oligonucleotides are natural phosphate linkages. In some embodiments, 20% or more of the internucleotide linkages in the provided C9orf72 oligonucleotides are natural phosphate linkages. In some embodiments, 25% or more of the internucleotide linkages in the provided C9orf72 oligonucleotides are natural phosphate linkages. In some embodiments, 30% or more of the internucleotide linkages in the provided C9orf72 oligonucleotides are natural phosphate linkages. In some embodiments, 35% or more of the internucleotide linkages in the provided C9orf72 oligonucleotides are natural phosphate linkages. In some embodiments, 40% or more of the internucleotide linkages in the provided C9orf72 oligonucleotides are natural phosphate linkages.
In some embodiments, provided C9orf72 oligonucleotides can bind to a transcript and improve C9orf72 knockdown of the transcript. In some embodiments, the provided C9orf72 oligonucleotides improve C9orf72 knockdown with improved efficiency over similar oligonucleotides under one or more suitable conditions.
In some embodiments, under one or more suitable conditions, the improvement in C9orf72 knockdown obtained is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% greater than that of a similar oligonucleotide, or is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more times greater than that of a similar oligonucleotide.
In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% by administering the C9orf72 oligonucleotide. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% due to C9orf72 oligonucleotide-directed knock down. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% in total due to C9orf72 oligonucleotide-directed RNase H mediated knock-down of C9orf 72. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% as a result of in vitro administration of the C9orf72 oligonucleotide. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% due to in vitro C9orf72 oligonucleotide-directed knockdown. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% due to the C9orf72 oligonucleotide-directed RNase H-mediated knock down of C9orf72 in vitro. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% as a result of administering the C9orf72 oligonucleotide in one or more cells in vitro. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% due to C9orf72 oligonucleotide-directed knock down in one or more cells in vitro. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70% or 80% due to C9orf72 oligonucleotide-directed RNase H-mediated C9orf72 knockdown in one or more cells in vitro. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% as a result of administering the C9orf72 oligonucleotide at a concentration of 1uM or less in one or more cells in vitro. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% due to 1uM or less concentration of C9orf72 oligonucleotide-directed knockdown in one or more cells in vitro. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% due to RNase H-mediated C9orf72 knockdown in one or more cells in vitro at a concentration of 1uM or less of C9orf72 oligonucleotide. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% as a result of administering the C9orf72 oligonucleotide at a concentration of 10uM or less in one or more cells in vitro. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% due to C9orf72 oligonucleotide-directed knockdown at a concentration of 10uM or less in one or more cells in vitro. In some embodiments, the expression or level of the C9orf72 target gene or gene product thereof is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% due to RNase H-mediated C9orf72 knockdown at a concentration of 10uM or less in one or more cells in vitro. In some embodiments, a concentration of 1nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification by at least 10% in cells in vitro relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 5nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification by at least 10% in cells in vitro relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 10nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification in cells in vitro by at least 10% relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 1nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification in cells in vitro by at least 20% relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 5nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification in cells in vitro by at least 20% relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 10nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification in cells in vitro by at least 20% relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 1nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification in cells in vitro by at least 30% relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 5nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification in cells in vitro by at least 30% relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 10nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification in cells in vitro by at least 30% relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 1nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification by at least 40% in cells in vitro relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 5nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification by at least 40% in cells in vitro relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 10nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification by at least 40% in cells in vitro relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 1nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification by at least 50% in cells in vitro relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 5nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification by at least 50% in cells in vitro relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 10nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification by at least 50% in cells in vitro relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 1nm or less of the C9orf72 oligonucleotide is capable of mediating an in vitro cell reduction in expression, level, and/or activity of a C9orf72 transcript containing repeat amplification by at least 75% relative to a C9orf72 transcript without repeat amplification. In some embodiments, a concentration of 5nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification in cells in vitro of at least 75% relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 10nm or less of the C9orf72 oligonucleotide is capable of mediating a reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification in cells in vitro of at least 75% relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 1nm or less of the C9orf72 oligonucleotide is capable of mediating an at least 90% reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification in cells in vitro relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 5nm or less of the C9orf72 oligonucleotide is capable of mediating an at least 90% reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification in cells in vitro relative to C9orf72 transcripts not containing repeat amplification. In some embodiments, a concentration of 10nm or less of the C9orf72 oligonucleotide is capable of mediating an at least 90% reduction in expression, level, and/or activity of C9orf72 transcripts containing repeat amplification in cells in vitro relative to C9orf72 transcripts not containing repeat amplification.
In some embodiments, IC50 is an inhibitory concentration that reduces the expression or level of a C9orf72 target gene or gene product thereof by 50% in one or more cells in vitro. In some embodiments, the C9orf72 oligonucleotide has an IC50 of no more than about 10nM in one or more cells in vitro. In some embodiments, the C9orf72 oligonucleotide has an IC50 of no more than about 5nM in one or more cells in vitro. In some embodiments, the C9orf72 oligonucleotide has an IC50 of no more than about 2nM in one or more cells in vitro. In some embodiments, the C9orf72 oligonucleotide has an IC50 of no more than about 1nM in one or more cells in vitro. In some embodiments, the C9orf72 oligonucleotide has an IC50 of no more than about 0.5nM in one or more cells in vitro. In some embodiments, the C9orf72 oligonucleotide has an IC50 of no more than about 0.1nM in one or more cells in vitro. In some embodiments, the C9orf72 oligonucleotide has an IC50 of no more than about 0.01nM in one or more cells in vitro. In some embodiments, the C9orf72 oligonucleotide has an IC50 of no more than about 0.001nM in one or more cells in vitro.
In some embodiments, provided C9orf72 oligonucleotides comprise any of the stereochemical patterns described herein. In some embodiments, provided C9orf72 oligonucleotides comprise any of the stereochemical patterns described herein, and are capable of directing RNase H-mediated C9orf72 knockdown. In some embodiments, provided C9orf72 oligonucleotides comprise any of the stereochemical patterns described herein, and are capable of directing RNase H-mediated C9orf72 knockdown.
In some embodiments, provided C9orf72 oligonucleotides comprise any modification or modification pattern described herein. In some embodiments, provided C9orf72 oligonucleotides comprise any of the modification patterns described herein, and are capable of directing RNase H-mediated C9orf72 knockdown. In some embodiments, the modification or modification pattern is a modification or modification pattern at the 2' position of the saccharide. In some embodiments, the modification OR modification pattern is at the 2 'position of the sugar, including but not limited to 2' -deoxy, 2 '-F, 2' -OMe, 2 '-MOE, and 2' -OR1, wherein R1 is optionally substituted C1-6 alkyl.
In some embodiments, the disclosure demonstrates that Sp internucleotide linkages at the 5 'and 3' ends can improve oligonucleotide stability, among other things. In some embodiments, the present disclosure demonstrates that native phosphate linkages and/or Rp internucleotide linkages, among others, can improve removal of oligonucleotides from a system. As will be appreciated by one of ordinary skill in the art, various analyses known in the art can be employed to assess the characteristics in accordance with the present disclosure.
In some embodiments, provided C9orf72 oligonucleotides capable of directing knock down of C9orf72 comprise one or more modified sugar moieties. In some embodiments, the modified sugar moiety comprises a 2' -modification. In some embodiments, the modified sugar moiety comprises a 2' -modification. In some embodiments, the 2 '-modification is 2' -OR1. In some embodiments, the 2 '-modification is 2' -OMe. In some embodiments, the 2 '-modification is 2' -MOE. In some embodiments, the 2' -modification is a LNA sugar modification. In some embodiments, the 2 '-modification is 2'-F. In some embodiments, each sugar modification is independently a 2' -modification. In some embodiments, each sugar modification is independently 2' -OR1Or 2' -F. In some embodiments, each sugar modification is independently 2' -OR1Or 2' -F, wherein R1Is optionally substituted C1-6An alkyl group. In some embodiments, each sugar modification is independently 2' -OR1Or 2 '-F, at least one of which is 2' -F. In some embodiments, each sugar modification is independently 2' -OR1Or 2' -F, wherein R1Is optionally substituted C1-6Alkyl, and at least one of them is 2' -OR1. In some embodiments, each sugar modification is independently 2' -OR1OR 2 ' -F, at least one of which is 2 ' -F and at least one of which is 2 ' -OR1. In some embodiments, each sugar modification is independently 2' -OR1Or 2' -F, wherein R1Is optionally substituted C1-6Alkyl groups, and at least one of which is 2 '-F and at least one is 2' -OR1。
In some embodiments, provided C9orf72 oligonucleotides capable of directing knock down of C9orf72 comprise one or more 2' -fs. In some embodiments, provided C9orf72 oligonucleotides capable of directing knock down of C9orf72 comprise at least one 2' -OMe. In some embodiments, provided C9orf72 oligonucleotides capable of directing knock down of C9orf72 comprise at least two or more contiguous 2' -fs. In some embodiments, provided C9orf72 oligonucleotides capable of directing knock down of C9orf72 comprise at least two or more contiguous 2' -OMe.
In some embodiments, provided C9orf72 oligonucleotides capable of directing C9orf72 knock down comprise alternating 2 '-F modified sugar moieties and 2' -OR1A modified sugar moiety. In some embodiments, provided C9orf72 oligonucleotides capable of directing C9orf72 knockdown comprise alternating 2 '-F modified sugar moieties and 2' -OMe modified sugar moieties, e.g., [ (2 '-F) (2' -OMe)]x、[(2′-OMe)(2′-F)]x, and the like, wherein x is 1 to 50. In some embodiments, provided C9orf72 oligonucleotides capable of directing C9orf72 knockdown comprise at least two pairs of alternating 2 '-F and 2' -OMe modifications. In some embodiments, provided C9orf72 oligonucleotides comprise alternating phosphodiester and phosphorothioate internucleotide linkages, e.g., [ (PO) (PS)]x、[(PS)(PO)]x, and the like, wherein x is 1 to 50.
In some embodiments, the present disclosure provides a C9orf72 oligonucleotide composition comprising a first plurality of oligonucleotides, wherein:
the first plurality of oligonucleotides have the same base sequence; and is
The first plurality of oligonucleotides comprises one or more modified sugar moieties, or comprises one or more natural phosphate linkages and one or more modified internucleotide linkages.
In some embodiments, provided C9orf72 oligonucleotides comprise one or more 2' -fs. In some embodiments, in the provided C9orf72 oligonucleotides, the nucleoside comprising the 2' modification is followed by a modified internucleotide linkage. In some embodiments, in the provided C9orf72 oligonucleotides, the nucleoside comprising the 2' modification is preceded by a modified internucleotide linkage. In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate. In some embodiments, the chiral internucleotide linkage is Sp. In some embodiments, in the provided C9orf72 oligonucleotides, the nucleoside comprising the 2' modification is followed by an Sp chiral internucleotide linkage. In some embodiments, provided C9orf72 oligonucleotides wherein the 2' -F containing nucleoside is followed by an Sp chiral internucleotide linkage. In some embodiments, in the provided C9orf72 oligonucleotides, the nucleoside comprising the 2' modification is preceded by an Sp chiral internucleotide linkage. In some embodiments, a C9orf72 oligonucleotide is provided in which the nucleoside comprising a 2' -F is preceded by an Sp chiral internucleotide linkage. In some embodiments, the chiral internucleotide linkage is Rp. In some embodiments, in the provided C9orf72 oligonucleotides, the nucleoside comprising a 2' modification is followed by an Rp chiral internucleotide linkage. In some embodiments, in the provided C9orf72 oligonucleotides, the nucleoside comprising a 2' -F is followed by an Rp chiral internucleotide linkage. In some embodiments, in the provided C9orf72 oligonucleotides, the nucleoside comprising a 2' modification is preceded by an Rp chiral internucleotide linkage. In some embodiments, in the provided C9orf72 oligonucleotides, the nucleoside comprising a 2' -F is preceded by an Rp chiral internucleotide linkage. In some embodiments, provided C9orf72 oligonucleotides are capable of directing a decrease in expression, level, and/or activity of a C9orf72 target gene or gene product thereof. In some embodiments, the C9orf72 target gene comprises repeat amplification. In some embodiments, the first plurality of C9orf72 oligonucleotides comprise one or more natural phosphate linkages and one or more modified internucleotide linkages.
In some embodiments, provided compositions alter transcript C9orf72 knockdown such that target and/or biological function inhibition is not desired. In some embodiments, in such cases, provided compositions can also induce cleavage of the transcript after hybridization.
In some embodiments, the provided chirality controlled C9orf72 oligonucleotide compositions are unexpectedly effective compared to reference conditions. In some embodiments, a desired biological effect (e.g., as measured by an increased level of a desired mRNA, protein, etc., a decreased level of an undesired mRNA, protein, etc.) may be enhanced by more than 5, 10, 15, 20, 25, 30, 40, 50, or 100-fold. In some embodiments, the alteration is measured by an increase in the level of the desired mRNA compared to a reference condition.
In some embodiments, provided C9orf72 oligonucleotides contain increased levels of one or more isotopes. In some embodiments, provided C9orf72 oligonucleotides are labeled, e.g., with one or more isotopes of one or more elements (e.g., hydrogen, carbon, nitrogen, etc.). In some embodiments, a provided C9orf72 oligonucleotide (e.g., a first plurality of C9orf72 oligonucleotides) in a provided composition comprises base modifications, sugar modifications, and/or internucleotide linkage modifications, wherein the oligonucleotide contains an enriched level of deuterium. In some embodiments, provided C9orf72 oligonucleotides are deuterium labeled (with deuterium) at one or more positions-2H replacement-1H) In that respect In some embodiments, one or more of the C9orf72 oligonucleotides or any moiety conjugated to the oligonucleotide (e.g., targeting moiety, etc.)1H channel2H-substitution of such oligonucleotides can be used in any of the compositions or methods described herein.
In some embodiments, the present disclosure provides a C9orf72 oligonucleotide composition comprising a first plurality of oligonucleotides that:
1) has a common base sequence that is complementary to the C9orf72 target sequence in the transcript; and is
2) Comprising one or more modified sugar moieties and modified internucleotide linkages.
In some embodiments, the disclosure provides a C9orf72 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing C9orf72 knockdown, wherein the C9orf72 oligonucleotide type is defined by:
1) a base sequence;
2) a skeletal linkage mode;
3) pattern of backbone chiral centers; and
4) the mode of the phosphorus modification of the skeleton,
the composition is chirally controlled in that the composition is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence,
the oligonucleotide composition is characterized in that: when contacted with a transcript in a C9orf72 knockdown system, the C9orf72 knockdown of the transcript, C9orf72 knockdown of low conductance, is improved relative to the knockdown observed under reference conditions selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.
In some embodiments, the present disclosure provides a C9orf72 oligonucleotide composition comprising a first plurality of oligonucleotides, wherein: the first plurality of oligonucleotides have the same base sequence; the first plurality of oligonucleotides comprises the following structural elements: (a)2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleoside units comprising a sugar moiety modified with 2 '-F, 2' -OMe, 2 '-deoxy and/or 2' -MOE; (b)2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more modified internucleotide linkages; (c)2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more chirally controlled modified internucleotide linkages; and (d)2, 3, 4, 5, 6, 7, 8, 9, 10 or more natural phosphate linkages. In some embodiments, the first plurality of oligonucleotides comprises structural elements (a), (b), and (c). In some embodiments, the first plurality of oligonucleotides comprises structural elements (b), (c), and (d). In some embodiments, the first plurality of oligonucleotides comprises structural elements (a), (b), and (d). In some embodiments, the first plurality of oligonucleotides comprises structural elements (a), (c), and (d). In some embodiments, the first plurality of oligonucleotides comprises structural elements (a) and (b). In some embodiments, the first plurality of oligonucleotides comprises structural elements (a) and (c). In some embodiments, the first plurality of oligonucleotides comprises structural elements (a) and (d). In some embodiments, the first plurality of oligonucleotides comprises structural elements (b) and (c). In some embodiments, the first plurality of oligonucleotides comprises structural elements (b) and (d). In some embodiments, the first plurality of oligonucleotides comprises structural elements (c) and (d).
In some embodiments, the modified internucleotide linkage has the structure of formula I. In some embodiments, the modified internucleotide linkage has the structure of formula I-a.
As demonstrated in the present disclosure, in some embodiments, provided C9orf72 oligonucleotide compositions are characterized by improved C9orf72 knockdown of low conductance C9orf72 knockdown of a transcript when contacted with the transcript in a C9orf72 knockdown system relative to the knockdown observed under reference conditions selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof. In some embodiments, the C9orf72 knockdown is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 fold or more. In some embodiments, as exemplified in the present disclosure, the level of the plurality of oligonucleotides (e.g., the first plurality of oligonucleotides) in the provided compositions is predetermined.
In some embodiments, the common base sequence and length may be referred to as a common base sequence. In some embodiments, C9orf72 oligonucleotides having a common base sequence may have the same pattern of nucleoside modifications, e.g., sugar modifications, base modifications, and the like. In some embodiments, the nucleoside modification pattern can be represented by a combination of positions and modifications. In some embodiments, the nucleoside modification pattern can be represented by a combination of position and modification. In some embodiments, the backbone linkage pattern comprises the position and type of each internucleotide linkage (e.g., phosphate, phosphorothioate, substituted phosphorothioate, etc.). The backbone chiral center pattern of the C9orf72 oligonucleotide can be specified by a combination of 5 'to 3' bonded phosphorus stereochemistry (Rp/Sp). As exemplified above, the position of the achiral linkage can be obtained, for example, from a pattern of backbone linkages.
In some embodiments, the disclosure provides a C9orf72 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing C9orf72 knockdown, wherein the oligonucleotides are of a specific oligonucleotide type, characterized by:
1) a common base sequence and length;
2) a common backbone linkage pattern; and
3) a common pattern of backbone chiral centers;
the compositions are chirally controlled in that they are enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length.
As understood by those of ordinary skill in the art, a stereorandom or racemic formulation of an oligonucleotide is prepared by non-stereoselective and/or low stereoselective coupling of nucleotide monomers, typically without the use of any chiral auxiliary agents, chiral modifying reagents, and/or chiral catalysts. In some embodiments, in substantially racemic (or chirally uncontrolled) oligonucleotide formulations, all or most of the coupling steps are not chirally controlled, as the coupling steps are not specifically performed to provide enhanced stereoselectivity. An exemplary substantially racemic formulation of the oligonucleotide is synthesized from commonly used phosphoramidite oligonucleotides (methods well known in the art) by sulfurizing phosphite triester with dithiotetraethylthiuram or (TETD) or 3H-1, 2-benzodithiol-3-one 1, 1-dioxide (BDTD). In some embodiments, a substantially racemic formulation of an oligonucleotide provides a substantially racemic oligonucleotide composition (or chiral uncontrolled oligonucleotide composition). In some embodiments, at least one coupling of nucleotide monomers has a diastereoselectivity of less than about 60: 40, 70: 30, 80: 20, 85: 15, 90: 10, 91: 9, 92: 8, 97: 3, 98: 2, or 99: 1.
As understood by one of ordinary skill in the art, in some embodiments, the coupled or linked diastereoselectivity can be assessed by the diastereoselectivity of dimer formation under identical or comparable conditions, where the dimers have identical 5 '-and 3' -nucleosides and internucleotide linkages.
In some embodiments, C9orf72 oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, C9orf72 oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, C9orf72 oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have the same structure.
In some embodiments, C9orf72 oligonucleotides of a certain C9orf72 oligonucleotide type have a common backbone phosphorus modification pattern and a common sugar modification pattern. In some embodiments, C9orf72 oligonucleotides of a certain C9orf72 oligonucleotide type have a common backbone phosphorus modification pattern and a common base modification pattern. In some embodiments, C9orf72 oligonucleotides of a certain C9orf72 oligonucleotide type have a common backbone phosphorus modification pattern and a common nucleoside modification pattern. In some embodiments, the C9orf72 oligonucleotides of a certain C9orf72 oligonucleotide type are identical.
In some embodiments, a C9orf72 oligonucleotide is a substantially pure preparation of a certain C9orf72 oligonucleotide type, and oligonucleotides in the composition that are not the oligonucleotide type are in an impurity form during the preparation of the oligonucleotide type (in some cases after certain purification steps).
In some embodiments, at least about 20% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 25% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 30% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 35% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 40% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 45% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 50% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 55% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 60% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 65% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 70% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 75% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 80% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 85% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 90% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 92% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 94% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 95% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, greater than about 99% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, the purity of a C9orf72 oligonucleotide of C9orf72 oligonucleotides can be expressed as a percentage of oligonucleotides in a composition having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.
In some embodiments, C9orf72 oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modification. In some embodiments, C9orf72 oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, C9orf72 oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of sugar modifications. In some embodiments, C9orf72 oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, C9orf72 oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, C9orf72 oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers are the same.
As indicated above and understood in the art, in some embodiments, the base sequence of a C9orf72 oligonucleotide can refer to the identity and/or modification state of nucleoside residues (e.g., sugar and/or base components, relative to standard naturally occurring nucleotides such as adenine, cytosine, guanosine, thymine and uracil) in the oligonucleotide and/or can refer to the hybridization characteristics of the residues (i.e., the ability to hybridize to a particular complementary residue).
In some embodiments, the purity of the C9orf72 oligonucleotide can be controlled by the stereoselectivity of each coupling step in its preparation. In some embodiments, the coupling step has a stereoselectivity (e.g., diastereoselectivity) of 60% (60% of the new internucleotide linkages formed by the coupling step have the expected stereochemistry).
In some embodiments, provided compositions comprise oligonucleotides comprising one or more residues modified at a sugar moiety. In some embodiments, provided compositions comprise oligonucleotides comprising one or more residues modified at the 2 'position of the sugar moiety (referred to herein as a "2' modification"). Examples of such modifications are described above and herein and include, but are not limited to, 2 '-OMe, 2' -MOE, 2 '-LNA, 2' -F, FRNA, FANA, S-cEt, and the like. In some embodiments, provided compositions comprise oligonucleotides comprising one or more 2' modified residues. For example, in some embodiments, provided C9orf72 oligonucleotides contain one or more residues that are 2 '-O-methoxyethyl (2' -MOE) -modified residues. In some embodiments, provided compositions comprise oligonucleotides that do not contain any 2' modifications. In some embodiments, provided compositions are oligonucleotides that do not contain any 2' -MOE residues. That is, in some embodiments, provided C9orf72 oligonucleotides are not MOE modified. Other example sugar modifications are described in the present disclosure.
In some embodiments, the sugar moiety without the 2' -modification is a sugar moiety found in a natural DNA nucleoside.
One of ordinary skill in the art understands that the provided compositions and methods can target various regions of the C9orf72 target transcript. In some embodiments, the base sequence of a provided C9orf72 oligonucleotide comprises an intron sequence. In some embodiments, the base sequence of a provided C9orf72 oligonucleotide comprises an exon sequence. In some embodiments, the base sequence of a provided C9orf72 oligonucleotide comprises an intron and an exon sequence.
As understood by one of ordinary skill in the art, the provided C9orf72 oligonucleotides and compositions are particularly targeted to a large number of nucleic acid polymers. For example, in some embodiments, provided C9orf72 oligonucleotides and compositions can target transcripts of nucleic acid sequences in which a common base sequence of the oligonucleotides (e.g., a base sequence of a certain C9orf72 oligonucleotide type) comprises or is complementary to a sequence of the transcript.
In some embodiments, the provided C9orf72 oligonucleotides and compositions, as described in the present disclosure, can provide new cleavage patterns, higher cleavage rates, higher degrees of cleavage, higher cleavage selectivities, and the like. In some embodiments, provided compositions can selectively inhibit (e.g., cleave) a transcript of a C9orf72 target nucleic acid sequence having one or more analogous sequences within an individual or population, each of the target sequence and its analogous sequences containing a particular nucleotide signature sequence element that defines the target sequence relative to the analogous sequence.
In some embodiments, a similar sequence has greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a C9orf72 target sequence. In some embodiments, the C9orf72 target sequence is a pathogenic copy of a nucleic acid sequence comprising one or more mutations, and the analogous sequence is a copy that does not cause disease (wild-type). In some embodiments, the C9orf72 target sequence comprises a mutation, wherein the analogous sequence is the corresponding wild-type sequence. In some embodiments, the C9orf72 target sequence is a mutant allele, and the analogous sequence is a wild-type allele. In some embodiments, the C9orf72 target sequence is located in an intron comprising a hexanucleotide repeat amplification. In some embodiments, a region of the C9orf72 target sequence that is complementary to a common base sequence of the provided C9orf72 oligonucleotide compositions differs from the corresponding region of the analogous sequence at less than 5, less than 4, less than 3, less than 2, or only 1 base pair.
In some embodiments, the common base sequence comprises or is complementary to a sequence element that is characteristic of the sequence. In some embodiments, the common base sequence comprises a sequence complementary to the characteristic sequence element. In some embodiments, the common base sequence is a sequence complementary to the characteristic sequence element. In some embodiments, the common base sequence comprises or is 100% complementary to the characteristic sequence element. In some embodiments, the common base sequence comprises a sequence that is 100% complementary to the characteristic sequence element. In some embodiments, the common base sequence is a sequence that is 100% complementary to the characteristic sequence element.
The present disclosure recognizes, among other things, that base sequences may have an effect on oligonucleotide properties. In some embodiments, when an oligonucleotide having a base sequence is used to inhibit a C9orf72 target, e.g., by a pathway involving rnase H, the base sequence may have an effect on the cleavage pattern of the C9orf72 target: for example, oligonucleotides with different sequences that are structurally similar (all phosphorothioate linkages, all stereo-random) may have different cleavage patterns.
As understood by one of ordinary skill in the art, the provided C9orf72 oligonucleotide compositions and methods have a variety of uses known to those of ordinary skill in the art. Methods for evaluating the provided compositions and their properties and uses are also well known and practiced by those of ordinary skill in the art. Exemplary characteristics, uses and/or methods include, but are not limited to, those described in WO/2014/012081 and WO/2015/107425.
In some embodiments, the chiral internucleotide linkage has the structure of formula I. In some embodiments, the chiral internucleotide linkage is a phosphorothioate. In some embodiments, each chiral internucleotide linkage in the individual oligonucleotides of the provided compositions independently has the structure of formula I. In some embodiments, each chiral internucleotide linkage in a single oligonucleotide of a provided composition is a phosphorothioate.
In some embodiments, the C9orf72 oligonucleotides of the present disclosure comprise one or more modified sugar moieties. In some embodiments, the C9orf72 oligonucleotides of the present disclosure comprise one or more modified base moieties. Various modifications can be introduced into the sugars and/or moieties as known to those of ordinary skill in the art and described in the present disclosure. For example, in some embodiments, the modifications are those described in US 9006198, WO 2014/012081, and WO/2015/107425, the respective sugar and base modifications of which are incorporated herein by reference.
In some embodiments, the sugar modification is a 2' -modification. Common 2 'modifications include, but are not limited to, 2' -OR1Wherein R is1Is not hydrogen. In some embodiments, the modification is 2' -OR, wherein R is optionally substituted aliphatic. In some embodiments, the modification is 2' -OMe. In some embodiments, the modification is 2' -O-MOE. In some embodiments, the present disclosure demonstrates that inclusion and/or location of particular chirally pure internucleotide linkages can provide stability improvements comparable to or better than those achieved by use of modified backbone linkages, bases, and/or sugars. In some embodiments, provided single oligonucleotides of provided compositions do not have modifications on the sugar. In some embodiments, a provided single oligonucleotide of a provided composition does not have a modification at the 2 'position of the sugar (i.e., the two groups at the 2' position are-H/-H or-H/-OH). In some embodiments, provided single oligonucleotides of provided compositions do not have any 2' -MOE modifications.
In some embodiments, the 2 '-modification is-O-L-or-L-, which links the 2' -carbon of the sugar moiety to another carbon of the sugar moiety. In some embodiments, the 2 ' -modification is-O-L-or-L-, which links the 2 ' -carbon of the sugar moiety to the 4 ' -carbon of the sugar moiety. In some embodiments, the 2' -modification is S-cEt. In some embodiments, the modified sugar moiety is an LNA moiety.
In some embodiments, the locked nucleic acid or LNA nucleoside or LNA nucleotide is or comprises a nucleic acid monomer having a bridge connecting two carbon atoms between the 4 ' and 2 ' positions of the nucleoside sugar unit, thereby forming a bicyclic sugar examples of such bicyclic sugars include, but are not limited to α -L-methyleneoxy (4 ' -CH)2-O-2 ') LNA, β -D-methyleneoxy (4' -CH)2-O-2 ') LNA, ethyleneoxy (4' - (CH)2)2-O-2') LNA, aminoOxy (4' -CH)2-O-N (R) -2 ') LNA and oxyamino (4' -CH)2-N (R) -O-2') LNA. In some embodiments, R is R1Or R2。
In some embodiments, LNA compounds include, but are not limited to, compounds having at least one bridge between the 4 'and 2' positions of the sugar, wherein each bridge independently comprises 1 or 2 to 4 linking groups independently selected from: - [ C (R)1)(R2)]n-、-C(R1)＝C(R2)-、-C(R1)＝N-、-C(＝NR1)-、-C(＝O)-、-C(＝S)-、-O-、-Si(R1)2-、-S(＝O)x-and-N (R)1) -; wherein: x is 0, 1 or 2; n is 1, 2, 3 or 4; each R1And R2Independently is H, a protecting group, hydroxy, C1-C12Alkyl, substituted C1-C12Alkyl radical, C2-C12Alkenyl, substituted C2-C12Alkenyl radical, C2-C12Alkynyl, substituted C2-C12Alkynyl, C5-C20Aryl, substituted C5-C20Aryl, heterocyclyl, substituted heterocyclyl, heteroaryl, substituted heteroaryl, C5-C7Alicyclic radical, substituted C5-C7Alicyclic radical, halogen, OJ1、NJ1J2、SJ1、N3、COOJ1Acyl (C ═ O) -H), substituted acyl, CN, sulfonyl (S ═ O)2-J1) Or sulfo (S (═ O) -J1) (ii) a And each J1And J2Independently is H, C1-C12Alkyl, substituted C1-C12Alkyl radical, C2-C12Alkenyl, substituted C2-C12Alkenyl radical, C2-C12Alkynyl, substituted C2-C12Alkynyl, C5-C20Aryl, substituted C5-C20Aryl, acyl (C (═ O) -H), substituted acyl, heterocyclic, substituted heterocyclic, C1-C12Aminoalkyl, substituted C1-C12Aminoalkyl groups, or protecting groups. Non-limiting examples of 4 '-2' bridging groups encompassed within the definition of LNA include, but are not limited to, one of the following formulae: - [ C (R)1)(R2)]n-、-[C(R1)(R2)]n-O-、-C(R1R2)-N(R1) -O-or C (R)1R2)-O-N(R1) -. Furthermore, other bridging groups encompassed within the definition of LNA are 4' -CH2-2′、4′-(CH2)2-2′、4′-(CH2)3-2′、4′-CH2-O-2′、4′-(CH2)2-O-2′、4′-CH2-O-N(R1) -2 'and 4' -CH2-N(R1) -O-2' -bridging group, wherein each R1And R2Independently is H, a protecting group or C1-C12An alkyl group. Also included within the definition of LNA is where the 2 ' -hydroxyl group of the ribosyl sugar ring is attached to the 4 ' carbon atom of the sugar ring, thereby forming a methyleneoxy group (4 ' -CH)2-O-2') to form an LNA of the bicyclic sugar moiety. The bridging group can also be a methylene group (-CH) linking the 2 'oxygen atom to the 4' carbon atom2-) group, the term methyleneoxy (4' -CH) being used2-O-2') LNA. In some embodiments, where the bicyclic sugar moiety has an ethylene bridging group in this position, the term ethyleneoxy (4' -CH) is used2CH2-O-2 ') LNA. α -L-Methyleneoxy (4' -CH)2-O-2 ') is methyleneoxy (4' -CH)2-O-2') isomers of LNA, which are also encompassed within the definition of LNA as used herein.
In some embodiments, the 2' -modification is-F. In some embodiments, the 2' -modification is FANA. In some embodiments, the 2' -modification is FRNA.
In some embodiments, the sugar modification is a 5 ' -modification, such as R-5 ' -Me, S-5 ' -Me, and the like.
In some embodiments, the sugar modification alters the size of the sugar ring. In some embodiments, the sugar modification is a sugar moiety in FHNA.
In some embodiments, the sugar modification replaces the sugar moiety with another cyclic or acyclic moiety. Examples of such moieties are widely known in the art, including but not limited to those moieties used in morpholino (optionally with its phosphorodiamidite linkage), diol nucleic acids, and the like.
In some embodiments, the C9orf72 oligonucleotide is selected from the group consisting of the C9orf72 oligonucleotides disclosed herein and any C9orf72 oligonucleotide in any form described herein. One of ordinary skill in the art reading this disclosure will appreciate that this disclosure does not specifically exclude the possibility that any of the oligonucleotides labeled as C9orf72 oligonucleotides described herein may also or alternatively function via another mechanism (e.g., as antisense oligonucleotides; mediate knockdown via the RNase H mechanism; sterically hinder translation; or any other biochemical mechanism).
In some embodiments, the antisense oligonucleotide (ASO) is or comprises a C9orf72 oligonucleotide selected from the group consisting of: any of the C9orf72 oligonucleotides disclosed herein, and any of the oligonucleotides in any form described herein. One of ordinary skill in the art reading this disclosure will appreciate that this disclosure does not specifically exclude the possibility that any of the oligonucleotides labeled as antisense oligonucleotides (ASOs) described herein may also or alternatively function via another mechanism (e.g., as C9orf72 knockdown using RISC); the invention also indicates that the various oligonucleotides can act via different mechanisms (using RNaseH, spatially blocking translation or other post-transcriptional processes, altering the conformation of the C9orf72 target nucleic acid, etc.).
Chirally controlled oligonucleotides and chirally controlled oligonucleotide compositions
In some embodiments, provided C9orf72 oligonucleotides are capable of directing a decrease in expression, level, and/or activity of a C9orf72 target gene or gene product thereof. In some embodiments, the C9orf72 target gene comprises repeat amplification. In some embodiments, the C9orf72 target gene comprises a hexanucleotide repeat amplification.
The present disclosure provides chirally controlled C9orf72 oligonucleotides and chirally controlled C9orf72 oligonucleotide compositions having high crude product purity and high diastereomeric purity. In some embodiments, the disclosure provides chirally controlled C9orf72 oligonucleotides and chirally controlled C9orf72 oligonucleotide compositions with high crude product purity. In some embodiments, the disclosure provides chirally controlled C9orf72 oligonucleotides and chirally controlled C9orf72 oligonucleotide compositions with high diastereomeric purity.
In some embodiments, a C9orf72 oligonucleotide is a substantially pure preparation of a certain C9orf72 oligonucleotide type, and oligonucleotides in the composition that are not the oligonucleotide type are in an impurity form during the preparation of the oligonucleotide type (in some cases after certain purification steps).
In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different stereochemistry and/or different P modifications relative to each other. In certain embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide, wherein at least two individual internucleotide linkages within the oligonucleotide have different P modifications relative to each other. In certain embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different P modifications relative to each other, and wherein the chirally controlled C9orf72 oligonucleotide comprises at least one phosphodiester internucleotide linkage. In certain embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different P modifications relative to each other, and wherein the chirally controlled C9orf72 oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least one phosphorothioate diester internucleotide linkage. In certain embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different P modifications relative to each other, and wherein the chirally controlled C9orf72 oligonucleotide comprises at least one phosphorothioate triester internucleotide linkage. In certain embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different P modifications relative to each other, and wherein the chirally controlled C9orf72 oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least one phosphorothioate triester internucleotide linkage.
Internucleotide linkage
In some embodiments, provided C9orf72 oligonucleotides are capable of directing a decrease in expression, level, and/or activity of a C9orf72 target gene or gene product thereof. In some embodiments, the C9orf72 target gene comprises repeat amplification. In some embodiments, provided C9orf72 oligonucleotides comprise any internucleotide linkage described herein or known in the art.
In some embodiments, the C9orf72 oligonucleotide may comprise any internucleotide linkage described herein or known in the art.
Non-limiting examples of internucleotide linkages or unmodified internucleotide linkages are phosphodiesters; non-limiting examples of modified internucleotide linkages include those in which one or more of the oxygens of the phosphodiester are replaced by sulfur (as in phosphorothioate), H, an alkyl, or another moiety or element (as non-limiting examples) that is not an oxygen. A non-limiting example of an internucleotide linkage is a moiety that does not contain phosphorus but is used to link two sugars. A non-limiting example of an internucleotide linkage is a moiety that does not contain a phosphorus but serves to link two sugars in the backbone of a C9orf72 oligonucleotide. Disclosed herein are additional non-limiting examples of nucleotides, modified nucleotides, nucleotide analogs, internucleotide linkages, modified internucleotide linkages, bases, modified bases, and base analogs, sugars, modified sugars, and sugar analogs, as well as nucleosides, modified nucleosides, and nucleoside analogs.
In certain embodiments, the internucleotide linkage has the structure of formula I:
wherein the variables are as defined and described below. In some embodiments, the linkage of formula I is chiral. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide comprising one or more modified internucleotide linkages of formula I. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising one or more modified internucleotide linkages of formula I, and wherein individual internucleotide linkages of formula I within the oligonucleotide have different P modifications relative to each other. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising one or more modified internucleotide linkages of formula I, and wherein individual internucleotide linkages of formula I within the oligonucleotide have different-X-L-R relative to each other1. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising one or more modified internucleotide linkages of formula I, and wherein the individual internucleotide linkages of formula I within the oligonucleotide have a different X relative to each other. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising one or more modified internucleotide linkages of formula I, and wherein individual internucleotide linkages of formula I within the oligonucleotide have different-L-R relative to each other1。
In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different stereochemistry and/or different P modifications relative to each other. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different stereochemistry relative to each other, and wherein at least a portion of the structure of the chirally controlled C9orf72 oligonucleotide is characterized by a repeating pattern of alternating stereochemistry.
In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide, wherein at least two of the individual internucleotide linkages within the oligonucleotide have a difference relative to each otherP modification of (2), which differs therefrom by-XLR1The moieties having different X atoms, and/or-XLR thereof1The moieties having different L groups, and/or-XLR thereof1Moieties having different R1Atom(s) in which XLR1Is equal to X-L-R1And X, L and R1As defined in formula I as disclosed herein.
In some embodiments, L is a covalent bond or an optionally substituted linear or branched C1-C10Alkylene, wherein one or more methylene units of L are optionally and independently replaced by: optionally substituted C1-C6Alkylene radical, C1-C6Alkenylene, -C ≡ C-, -C (R')2-、-Cy-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)-、-N(R′)C(O)O-、-OC(O)N(R′)-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-N(R′)S(O)2-, -SC (O) -, -C (O) S-, -OC (O) -, or-C (O) O-;
R1is halogen, R or optionally substituted C1-C50Aliphatic, wherein one or more methylene units are optionally and independently replaced by: optionally substituted C1-C6Alkylene radical, C1-C6Alkenylene, -C ≡ C-, -C (R')2-、-Cy-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)-、-N(R′)C(O)O-、-OC(O)N(R′)-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-N(R′)S(O)2-, -SC (O) -, -C (O) S-, -OC (O) -, or-C (O) O-;
each R' is independently-R, -C (O) R, -CO2R, or-SO2R, or
Two R' on the same nitrogen, taken together with the intervening atoms, form an optionally substituted heterocyclic or heteroaryl ring, or
Two R' on the same carbon, together with the intervening atoms, form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring;
-Cy-is an optionally substituted divalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, and heterocyclylene;
each R is independently hydrogen or an optionally substituted group selected from: c1-C6Aliphatic, phenyl, carbocyclyl, aryl, heteroaryl and heterocyclyl; and is
Each one of which isIndependently represents a linkage to a nucleoside.
In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises one or more modified internucleotide phosphorus linkages. Examples of such modified internucleotide phosphorus linkages are further described herein.
In some embodiments, the chirally controlled C9orf72 oligonucleotides comprise different internucleotide phosphorus linkages. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least one modified internucleotide linkage. Examples of such modified internucleotide phosphorus linkages are further described herein.
In some embodiments, the phosphorothioate triester linkage comprises a chiral auxiliary, for example to control the stereoselectivity of the reaction. In some embodiments, the phosphorothioate triester linkage does not comprise a chiral auxiliary. In some embodiments, phosphorothioate triester linkages are intentionally maintained until administration to a subject, and/or phosphorothioate triester linkages are intentionally maintained during administration to a subject.
In some embodiments, the chirally controlled C9orf72 oligonucleotide is attached to a solid support. In some embodiments, the chirally controlled C9orf72 oligonucleotide is cleaved from the solid support.
In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least two consecutive modified internucleotide linkages. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least two consecutive phosphorothioate triester internucleotide linkages.
In some embodiments, the disclosure provides compositions comprising or consisting of a plurality of the provided C9orf72 oligonucleotides (e.g., chirally controlled C9orf72 oligonucleotide compositions). In some embodiments, all of the provided C9orf72 oligonucleotides are of the same type, i.e., they all have the same base sequence, backbone linkage pattern (i.e., internucleotide linkage type pattern, e.g., phosphate, phosphorothioate, etc.), backbone chiral center pattern (i.e., bonded phosphorus stereochemistry (Rp/Sp) pattern), and backbone phosphorus modification pattern (e.g., -XLR in formula I disclosed herein)1"mode of group"). In some embodiments, all oligonucleotides of the same type are the same. However, in many embodiments, provided compositions comprise a plurality of oligonucleotide types (typically in predetermined relative amounts).
In some embodiments, the C9orf72 oligonucleotide may comprise any internucleotide linkage described herein or known in the art. In some embodiments, a C9orf72 oligonucleotide may comprise a combination of any internucleotide linkage described herein or known in the art and any other structural element or modification described herein including, but not limited to, a base sequence or portion thereof, a sugar, a base (nucleobase); stereochemistry or modes thereof; additional chemical moieties including, but not limited to, targeting moieties, carbohydrate moieties, and the like; additional chemical moieties including, but not limited to, targeting moieties, and the like; in the form thereof or any structural element; and/or any other structural element or modification described herein; and in some embodiments, the disclosure relates to multimers of any such oligonucleotides.
In some embodiments, the disclosure provides C9orf72 oligonucleotides comprising one or more modified internucleotide linkages independently having the structure of formula I disclosed herein.
In some embodiments of formula I, TLDP in (1) is P*. In some embodiments, P is an asymmetric phosphorus atom and is Rp or Sp. In some embodiments, P is Rp. At itIn other embodiments, P is Sp. In some embodiments, the C9orf72 oligonucleotide comprises one or more internucleotide linkages of formula I, wherein each P is independently Rp or Sp. In some embodiments, the C9orf72 oligonucleotide comprises one or more internucleotide linkages of formula I wherein each P is*Is Rp. In some embodiments, the C9orf72 oligonucleotide comprises one or more internucleotide linkages of formula I wherein each P is*Is Sp. In some embodiments, the C9orf72 oligonucleotide comprises at least one internucleotide linkage of formula I, wherein P is Rp. In some embodiments, the C9orf72 oligonucleotide comprises at least one internucleotide linkage of formula I, wherein P*Is Sp. In some embodiments, the C9orf72 oligonucleotide comprises at least one internucleotide linkage of formula I wherein P is Rp and at least one wherein P is*Is an internucleotide linkage of formula I of Sp.
In some embodiments of formula I, W is O, S or Se. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, W is Se. In some embodiments, the C9orf72 oligonucleotide comprises at least one internucleotide linkage of formula I, wherein W is O. In some embodiments, the C9orf72 oligonucleotide comprises at least one internucleotide linkage of formula I, wherein W is S. In some embodiments, the C9orf72 oligonucleotide comprises at least one internucleotide linkage of formula I, wherein W is Se.
In some embodiments of formula I, the C9orf72 oligonucleotide comprises at least one internucleotide linkage of formula I, wherein W is O. In some embodiments, the C9orf72 oligonucleotide comprises at least one internucleotide linkage of formula I, wherein W is S.
In some embodiments, each R is independently hydrogen or selected from C1-C6Optionally substituted groups of aliphatic, phenyl, carbocyclyl, aryl, heteroaryl and heterocyclyl.
In some embodiments, R is hydrogen. In some embodiments, R is selected from C1-C6Optionally substituted groups of aliphatic, phenyl, carbocyclyl, aryl, heteroaryl and heterocyclyl.
In some embodiments, R is optionally substituted C1-C6Aliphatic seriesAnd (4) a base. In some embodiments, R is optionally substituted C1-C6An alkyl group. In some embodiments, R is an optionally substituted straight or branched chain hexyl. In some embodiments, R is an optionally substituted straight or branched chain pentyl group. In some embodiments, R is an optionally substituted straight or branched chain butyl. In some embodiments, R is an optionally substituted straight or branched chain propyl group. In some embodiments, R is an optionally substituted ethyl. In some embodiments, R is optionally substituted methyl.
In some embodiments, R is optionally substituted phenyl. In some embodiments, R is substituted phenyl. In some embodiments, R is phenyl.
In some embodiments, R is optionally substituted carbocyclyl. In some embodiments, R is optionally substituted C3-C10A carbocyclic group. In some embodiments, R is an optionally substituted monocyclic carbocyclyl. In some embodiments, R is optionally substituted cycloheptyl. In some embodiments, R is optionally substituted cyclohexyl. In some embodiments, R is an optionally substituted cyclopentyl. In some embodiments, R is an optionally substituted cyclobutyl. In some embodiments, R is optionally substituted cyclopropyl. In some embodiments, R is an optionally substituted bicyclic carbocyclyl.
In some embodiments, R is optionally substituted aryl. In some embodiments, R is an optionally substituted bicyclic aryl ring.
In some embodiments, R is optionally substituted heteroaryl. In some embodiments, R is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, and oxygen. In some embodiments, R is a substituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an unsubstituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, and oxygen.
In some embodiments, R is an optionally substituted 5-membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, and oxygen. In some embodiments, R is an optionally substituted 6-membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, R is an optionally substituted 5-membered monocyclic heteroaryl ring having 1 heteroatom selected from nitrogen, oxygen, and sulfur. In some embodiments, R is selected from pyrrolyl, furanyl, and thienyl.
In some embodiments, R is an optionally substituted 5-membered heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-membered heteroaryl ring having 1 nitrogen atom and another heteroatom selected from sulfur and oxygen. Exemplary R groups include optionally substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, or isoxazolyl.
In some embodiments, R is a 6-membered heteroaryl ring having 1-3 nitrogen atoms. In other embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1-2 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 2 nitrogen atoms. In certain embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1 nitrogen. Exemplary R groups include optionally substituted pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl.
In certain embodiments, R is an optionally substituted 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 1 heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted indolyl. In some embodiments, R is an optionally substituted azabicyclo [3.2.1] octyl. In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted azaindolyl. In some embodiments, R is optionally substituted benzimidazolyl. In some embodiments, R is optionally substituted benzothiazolyl. In some embodiments, R is optionally substituted benzoxazolyl. In some embodiments, R is optionally substituted indazolyl. In certain embodiments, R is an optionally substituted 5, 6-fused heteroaryl ring having 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In certain embodiments, R is an optionally substituted 6, 6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6, 6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 6, 6-fused heteroaryl ring having 1 heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted quinolinyl. In some embodiments, R is optionally substituted isoquinolinyl. According to one aspect, R is an optionally substituted 6, 6-fused heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is quinazoline or quinoxaline.
In some embodiments, R is optionally substituted heterocyclyl. In some embodiments, R is an optionally substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is a substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an unsubstituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, R is optionally substituted heterocyclyl. In some embodiments, R is an optionally substituted 6-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having 2 oxygen atoms.
In certain embodiments, R is a 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is oxiranyl, oxetanyl, tetrahydrofuryl, tetrahydropyranyl, oxepanyl, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, thiepanyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl, thiazolidinyl, dithiolane, dioxanyl, morpholinyl, oxathietanyl, piperazinyl, thiomorpholinyl, dithianyl, dioxacycloheptyl, oxazepanyl, oxathiepinyl, dithycloheptyl, diazepinyl, dihydrofuranonyl, tetrahydropyranonyl, oxepinyl, pyrrolidinonyl, piperidinonyl, azepinyl, dihydrothiophenyl, dihydrothiophenonyl, oxacycloheptanyl, oxacycloheptanonyl, pyrrolidinonyl, piperidinonyl, azacycloheptanonyl, dihydrothiophenonyl, dihydrothiophenyl, oxacycloheptanyl, and mixtures thereof, Tetrahydrothiopyronyl, thiepinonyl, oxazolidinonyl, oxazepinonyl, dioxapinonyl, oxathiepinonyl, oxathiacetonyl, thiazolidinonyl, thiazinonenyl, thiazepinonyl, imidazolidinonyl, tetrahydropyrimidinone, diazepanyl, imidazolidinedionyl, oxazolidinedione, thiazolidinedione, dioxolanedionyl, oxathiazepinonyl, piperazinedionyl, morpholinodione, thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, tetrahydrothiophenyl, or tetrahydrothiopyranyl. In some embodiments, R is an optionally substituted 5-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
In some embodiments, the structure of formula I is a structure of formula I as described in WO 2017/210647. In some embodiments, the internucleotide linkage of formula I has the structure of formula I-a:
wherein each variable is independently described in the present disclosure, as in formula I.
In some embodiments, the internucleotide linkage of formula I has the structure of formula I-b:
wherein each variable is independently described in the present disclosure, as in formula I.
In some embodiments, the internucleotide linkage of formula I is a phosphorothioate triester linkage having the structure of formula I-c:
wherein:
p is an asymmetric phosphorus atom and is Rp or Sp;
l is a covalent bond or an optionally substituted straight or branched chain C1-C10Alkylene, wherein one or more methylene units of L are optionally and independently replaced by: optionally substituted C1-C6Alkylene radical, C1-C6Alkenylene, -C ≡ C-, -C (R')2-、-Cy-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)-、-N(R′)C(O)O-、-OC(O)N(R′)-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-N(R′)S(O)2-, -SC (O) -, -C (O) S-, -OC (O) -, or-C (O) O-;
R1is halogen, R or optionally substituted C1-C50Aliphatic, wherein one or more methylene units are optionally and independentlyIs replaced by: optionally substituted C1-C6Alkylene radical, C1-C6Alkenylene, -C ≡ C-, -C (R')2-、-Cy-、-O-、-S-、-S-S-、-N(R′)-、-C(O)-、-C(S)-、-C(NR′)-、-C(O)N(R′)-、-N(R′)C(O)N(R′)-、-N(R′)C(O)-、-N(R′)C(O)O-、-OC(O)N(R′)-、-S(O)-、-S(O)2-、-S(O)2N(R′)-、-N(R′)S(O)2-, -SC (O) -, -C (O) S-, -OC (O) -, or-C (O) O-;
each R' is independently-R, -C (O) R, -CO2R, or-SO2R, or
Two R' on the same nitrogen, taken together with the intervening atoms, form an optionally substituted heterocyclic or heteroaryl ring, or
Two R' on the same carbon, together with the intervening atoms, form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring;
-Cy-is an optionally substituted divalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, and heterocyclylene;
each R is independently hydrogen or an optionally substituted group selected from: c1-C6Aliphatic, phenyl, carbocyclyl, aryl, heteroaryl and heterocyclyl;
each one of which isIndependently represents a linkage to a nucleoside; and is
When L is a covalent bond, R1Is not-H.
In some embodiments, the internucleotide linkage of the structure of formula I isOr internucleotide linkages as shown in the art (e.g. WO 2017/210647).
In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising one or more phosphodiester linkages, and one or more modified internucleotide linkages having formula I-a, formula I-b, or formula I-C.
In some embodiments, the modified internucleotide linkage has the structure I. In some embodiments, the modified internucleotide linkage has the I-a structure. In some embodiments, the modified internucleotide linkage has the structure I-b. In some embodiments, the modified internucleotide linkage has the structure I-c.
In some embodiments, the modified internucleotide linkage is a phosphorothioate. Examples of internucleotide linkages having the structure of formula I are widely known in the art and include, but are not limited to, those described in US 20110294124, US 20120316224, US20140194610, US20150211006, US 20150197540, WO 2015107425, PCT/US 2016/043542, and PCT/US 2016/043598 (each of which is incorporated herein by reference). In some embodiments, the modified internucleotide linkage is a vinylphosphonate. Whittaker et al 2008Tetrahedron Letters [ Tetrahedron Letters ] 49: 6984-6987.
Non-limiting examples of internucleotide linkages also include those described in the art, including but not limited to those described in any of the following: gryaznov, s.; chen, j. -k.j.am.chem.soc. [ journal of the american chemical society ]1994, 116, 3143; jones et al j.org.chem. [ journal of organic chemistry ]1993, 58, 2983; koshkin et al 1998Tetrahedron 54: 3607-; lauritsen et al 2002chem. 530- > 531; lauritsen et al 2003 bio.med.chem.lett. [ biomedical and chemical communication ] 13: 253-256; memsaeker et al, angelw.chem., int.ed.engl. [ international edition of applied chemistry english ]1994, 33, 226; petersen et al 2003TRENDS Biotech [ Biotech TRENDS ] 21: 74-81; schultz et al 1996 Nucleic acids sRes. [ Nucleic acids research ] 24: 2966; ts' o et al ann.n.y.acad.sci. [ journal of new york academy of sciences ]1988, 507, 220; and Vasseur et al j.am.chem.soc. [ journal of the american chemical society ]1992, 114, 4006.
In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising at least one phosphodiester internucleotide linkage and at least one phosphorothioate triester linkage having the structure of formula I-C. In some embodiments, the chirally controlled C9orf72 oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least two phosphorothioate triester linkages. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising at least one phosphodiester internucleotide linkage and at least two phosphorothioate triester linkages having the structure of formula I-C. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising at least one phosphodiester internucleotide linkage and at least three phosphorothioate triester linkages having the structure of formula I-C. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising at least one phosphodiester internucleotide linkage and at least four phosphorothioate triester linkages having the structure of formula I-C. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising at least one phosphodiester internucleotide linkage and at least five phosphorothioate triester linkages having the structure of formula I-C.
In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein one or more U is replaced with a T or vice versa. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein the sequence has more than 50% identity to the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein the sequence has more than 60% identity to the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein the sequence has more than 70% identity to the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein the sequence has more than 80% identity to the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein the sequence has more than 90% identity to the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein the sequence has greater than 95% identity to the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide comprising the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide having the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the disclosure provides a chirality controlled C9orf72 oligonucleotide comprising a sequence present in any of the oligonucleotides disclosed herein, wherein the oligonucleotide has a backbone linkage pattern, a backbone chiral center pattern, and/or a backbone phosphorus modification pattern described herein.
In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising a sequence (or portion of at least 10 consecutive bases thereof) present in any of the oligonucleotides disclosed herein, wherein at least one internucleotide linkage has a chirally bound phosphorus. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide having a sequence present in any of the oligonucleotides disclosed herein, wherein at least one internucleotide linkage has the structure of formula I. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising a sequence (or portion of at least 10 consecutive bases thereof) present in any of the oligonucleotides disclosed herein, wherein each internucleotide linkage has the structure of formula I. In some implementationsIn an example, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising a sequence (or portion of at least 10 consecutive bases thereof) present in any of the oligonucleotides disclosed herein, wherein at least one internucleotide linkage has the structure of formula I-C. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising a sequence (or portion of at least 10 consecutive bases thereof) present in any of the oligonucleotides disclosed herein, wherein each internucleotide linkage has the structure of formula I-C. In some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising a sequence (or portion of at least 10 consecutive bases thereof) present in any of the oligonucleotides disclosed herein, wherein at least one internucleotide linkage isIn some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising a sequence (or portion of at least 10 consecutive bases thereof) present in any of the oligonucleotides disclosed herein, wherein each internucleotide linkage isIn some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising a sequence (or portion of at least 10 consecutive bases thereof) present in any of the oligonucleotides disclosed herein, wherein at least one internucleotide linkage isIn some embodiments, the disclosure provides a chirally controlled C9orf72 oligonucleotide comprising a sequence (or portion of at least 10 consecutive bases thereof) present in any of the oligonucleotides disclosed herein, wherein each internucleotide linkage is
In some embodiments, the modification of the phospho-link is characterized by its ability to convert to phosphodiester by one or more esterases, nucleases and/or cytochrome P450 enzymes, such as those phosphodiester found in naturally occurring DNA and RNA, including but not limited to CYP1A1, CYP1A2, CYP1B1 (family: CYP1), CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1(CYP2), CYP3A 2, CYP2A 2, CYP2F 2, CYP 6F, CYP2, CYP.
In some embodiments, the modification at the phosphorus results in a P-modified moiety characterized in that it acts as a prodrug, e.g., the P-modified moiety facilitates delivery of the C9orf72 oligonucleotide to a desired location prior to removal. For example, in some embodiments, the P-modifying moiety results from pegylation at the phosphorus linkage. One skilled in the relevant art will appreciate that a variety of PEG chain lengths are available, and the choice of chain length will depend in part on the results sought to be achieved by pegylation. For example, in some embodiments, to reduce RES uptake and extend the in vivo circulating life of C9orf72 oligonucleotides, pegylation is performed.
In some embodiments, the molecular weight of the pegylation agent for use according to the present disclosure is about 300g/mol to about 100,000 g/mol. In some embodiments, the molecular weight of the pegylation agent is about 300g/mol to about 10,000 g/mol. In some embodiments, the molecular weight of the pegylation agent is about 300g/mol to about 5,000 g/mol. In some embodiments, the molecular weight of the pegylation agent is about 500 g/mol. In some embodiments, the molecular weight of the pegylation agent is about 1000 g/mol. In some embodiments, the molecular weight of the pegylation agent is about 3000 g/mol. In some embodiments, the molecular weight of the pegylation agent is about 5000 g/mol.
In certain embodiments, the pegylation agent is PEG 500. In certain embodiments, the pegylation agent is PEG 1000. In certain embodiments, the pegylation agent is PEG 3000. In certain embodiments, the pegylation agent is PEG 5000.
In some embodiments, the P-modified moiety is characterized in that it acts as an agent that promotes cell entry and/or endosomal escape, such as a membrane-disruptive lipid or peptide.
In some embodiments, the P-modified moiety is characterized in that it acts as a targeting agent. In some embodiments, the P-modifying moiety is or comprises a targeting agent. As used herein, the phrase "targeting agent" is an entity that associates with a load of interest (e.g., with a C9orf72 oligonucleotide or oligonucleotide composition) and also interacts with the relevant C9orf72 target site such that the load of interest, when associated with the targeting agent, targets the relevant target site to a much greater extent than would be observed under otherwise similar conditions when the load of interest is not associated with the targeting agent. The targeting agent can be or comprise any of a variety of chemical moieties including, for example, small molecule moieties, nucleic acids, polypeptides, carbohydrates, and the like. Targeting agents are further described by Adarsh et al, "Organelle Specific Targeted Drug Delivery-A Review [ Organelle Specific Targeted Drug Delivery-Review ]", International Journal of Research in Pharmaceutical and BiomedicalSciences [ Journal of International Drug and biomedical sciences ], 2011, page 895.
Examples of such targeting agents include, but are not limited to, proteins (e.g., transferrin), C9orf72 oligopeptides (e.g., cyclic and acyclic RGDd-containing oligopeptides), antibodies (monoclonal and polyclonal antibodies, e.g., IgG, IgA, IgM, IgD, IgE antibodies), sugars/carbohydrates (e.g., mono-and/or oligosaccharides (mannose, mannose-6-phosphate, galactose, and the like)), vitamins (e.g., folate), or other small biological molecules. In some embodiments, the targeting moiety is a steroid molecule (e.g., a bile acid including cholic acid, deoxycholic acid, dehydrocholic acid; cortisone; digoxin (digoxigenin); testosterone; cholesterol; cationic steroids such as cortisone with a trimethylaminomethylhydrazide group attached at the 3-position of the cortisone ring via a double bond, etc.). In some embodiments, the targeting moiety is a lipophilic molecule (e.g., alicyclic hydrocarbons, saturated and unsaturated fatty acids, waxes, terpenes, and polycyclohydrocarbons such as diamondoids (adamantine) and buckminsterfullerene (buckminsterfullerene)).
In some embodiments, the P-modifying moiety is a peptide having- -X-L-R1Wherein X, L and R1Each as defined in formula I as disclosed herein.
In some embodiments, the P-modified moiety is characterized in that it facilitates cell-specific delivery.
In some embodiments, the P-modified moiety is characterized in that it belongs to one or more of the above categories. For example, in some embodiments, the P-modifying moiety acts as a PK enhancer and targeting ligand. In some embodiments, the P-modifying moiety acts as a prodrug and an endosome escape. One skilled in the relevant art will recognize that many other such combinations are possible and are encompassed by the present disclosure.
In some embodiments, the carbocyclyl, aryl, heteroaryl, or heterocyclyl, or divalent or multivalent groups thereof, is C3-C30Carbocyclyl, aryl, heteroaryl or heterocyclyl or divalent and/or polyvalent groups thereof.
Base (nucleobase)
In some embodiments, provided C9orf72 oligonucleotides are capable of directing a decrease in expression, level, and/or activity of a C9orf72 target gene or gene product thereof. In some embodiments, the C9orf72 target gene comprises repeat amplification. In some embodiments, provided C9orf72 oligonucleotides comprise any nucleobase described herein or known in the art.
In some embodiments, the nucleobase present in the provided C9orf72 oligonucleotides is a natural nucleobase or a modified nucleobase derived from a natural nucleobase. Examples include, but are not limited to, uracil, thymine, adenine, cytosine, and guanine, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2, 6-diaminopurine, azacytosine, pyrimidine analogs (e.g., pseudoisocytosine and pseudouracil), and other modified nucleobases (e.g., 8-substituted purines, xanthines, or hypoxanthines, the latter two being natural degradation products) whose respective amino groups are protected by an acyl protecting group. Exemplary modified nucleobases are disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048; nucleic acids research, 1994, 22, 2183-; and Revankar and Rao, Comprehensive Natural products Chemistry [ Natural products Integrated Chemistry ], Vol.7, 313. In some embodiments, the modified nucleobase is a substituted uracil, thymine, adenine, cytosine, or guanine. In some embodiments, the modified nucleobase is a functional surrogate for uracil, thymine, adenine, cytosine, or guanine, e.g., in terms of hydrogen bonding and/or base pairing. In some embodiments, the nucleobase is an optionally substituted uracil, thymine, adenine, cytosine, 5-methylcytosine or guanine. In some embodiments, the nucleobase is uracil, thymine, adenine, cytosine, 5-methylcytosine or guanine.
In some embodiments, the modified base is optionally substituted adenine, cytosine, guanine, thymine, or uracil. In some embodiments, the modified nucleobase is an adenine, cytosine, guanine, thymine, or uracil that is independently modified by one or more modifications by:
(1) the nucleobases are modified with one or more optionally substituted groups independently selected from: acyl, halogen, amino, azido, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heteroaryl, carboxyl, hydroxyl, biotin, avidin, streptavidin, substituted silyl, and combinations thereof;
(2) one or more atoms of the nucleobase are independently replaced by a different atom selected from carbon, nitrogen and sulfur;
(3) one or more double bonds in the nucleobase are independently hydrogenated; or
(4) One or more aryl or heteroaryl rings are independently inserted into the nucleobase.
In some embodiments, the modified nucleobases are modified nucleobases displayed in the art (e.g., WO 2017/210647). Modified nucleobases also include size extended nucleobases in which one or more aryl rings, such as benzene rings, have been added. Covers the Glen Research catalog (Glen Research, stirling, virginia); krueger at et al, acc, chem, res. [ chemical research description ], 2007, 40, 141-; kool, ET, Acc. chem. Res. [ chemical research Specification ], 2002, 35, 936-943; benner s.a. et al, nat.rev.genet. [ review of natural genetics ], 2005, 6, 553-; romesberg, f.e., et al, curr. opin. chem.biol. [ new chemical and biological notes ], 2003, 7, 723-; the nucleobase substitutions described in Hirao, i., curr, opin, chem, biol. [ new chemical and biological, see ], 2006, 10, 622-. In some embodiments, the size expanded nucleobase is a size expanded nucleobase as displayed in the art (e.g., WO 2017/210647). Herein, modified nucleobases also encompass structures that are not considered nucleobases but are considered other moieties, such as, but not limited to, corrin or porphyrin-derived rings. Porphyrin-derived base substitutions have been described in Morales-Rojas, H and Kool, ET, org. Lett. [ organic letters ], 2002, 4, 4377-. In some embodiments, the porphyrin-derived ring is a porphyrin-derived ring as displayed in the art (e.g., WO 2017/219647). In some embodiments, the modified nucleobase is a modified nucleobase displayed in the art (e.g., WO 2017/219647). In some embodiments, the modified nucleobase is fluorescent. Examples of such fluorogenic modified nucleobases include phenanthrene, pyrene, stilbene (stillbene), isoxanthine, isoflavopterin, terphenyl, trithiophene, benzotrithiophene, coumarin, dioxotetrahydropyridine, tethering stilbene (tethered stilbene), benzouracil and naphthouracil as demonstrated in the art (e.g., WO 2017/210647). In some embodiments, the nucleobase or modified nucleobase is selected from: c5-propyne T, C5-propyne C, C5-thiazole, phenoxazine, 2-thio-thymine, 5-triazolylphenyl-thymine, diaminopurine and N2-aminopropylguanine.
In some embodiments, the modified nucleobase is selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl-or alkynyl-substituted pyrimidines, alkyl-substituted purines, and N-2, N-6, and 0-6 substituted purines. In certain embodiments, the modified nucleobase is selected from: 2-aminopropyladenine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C.ident.C-CH)3) Uracil, 5-propynylcytosine, 6-azauracil, 6-azacytosine, 6-azathymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halopurine, 8-aminopurine, 8-thiopurine, 8-thioalkylpurine, 8-hydroxypurine, 8-azapurine and other 8-substituted purines, 5-halo, especially 5-bromo, 5-trifluoromethyl, 5-halouracil and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl-4-N-benzoylcytosine, 5-methyl-4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-extended bases, and fluorinated bases. Additional modified nucleobases include tricyclic pyrimidines such as 1, 3-diazophenoxazin-2-one, 1, 3-diazophenthiazin-2-one, and 9- (2-aminoethoxy) -1, 3-diazophenoxazin-2-one (G-clamp). Modified nucleobases may also include those nucleobases in which a purine or pyrimidine base is replaced by another heterocyclic ring, e.g., 7-deaza-adenine, 7-deaza-guanosine, 2-aminoPyridine, and 2-pyridone. Additional nucleobases include those nucleobases disclosed in U.S. Pat. No. 3,687,808; the sense Encyclopedia of Polymer Science And d Engineering]Kroschwitz, j.i. editions, john willi father and son, 1990, 858-; englisch et al, Angewandte Chemie, International Edition]1991, 30, 613; sanghvi, Y.S., Chapter 15, Antisense Research and Applications [ Antisense Research and Applications]Crooke, S.T. and Lebleu, B. editions, CRC Press]1993, 273-288; and chapter 6 and 15, Antisense Drug Technology]Edit crook s.t., CRC Press]2008, 163-166 and 442-443.
Exemplary U.S. patents that teach the preparation of certain modified nucleobases mentioned above, as well as other modified nucleobases, include, but are not limited to, US 2003/0158403, u.s.3,687,808, 4,845,205; 5,130, 302; 5,134,066, respectively; 5,175,273, respectively; 5,367,066, respectively; 5,432,272; 5,434,257, respectively; 5,457,187, respectively; 5,459,255; 5,484,908, respectively; 5,502,177, respectively; 5,525,711, respectively; 5,552,540, respectively; 5,587,469, respectively; 5,594,121, respectively; 5,596,091, respectively; 5,614,617, respectively; 5,645,985, respectively; 5,681,941, respectively; 5,750,692, respectively; 5,763,588, respectively; 5,830,653, respectively; and 6,005,096.
In some embodiments, the modified nucleobase is unsubstituted. In some embodiments, the modified nucleobase is substituted. In some embodiments, the modified nucleobase is substituted such that it contains, for example, a heteroatom, alkyl group, or linking moiety attached to a fluorescent moiety, biotin or avidin moiety, or other protein or peptide. In some embodiments, the modified nucleobase is a "universal base" that is not a nucleobase in the most classical sense, but that functions similarly to a nucleobase. A representative example of such a universal base is 3-nitropyrrole.
In some embodiments, other nucleosides can also be used in the methods disclosed herein and include nucleosides incorporating a modified nucleobase or a nucleobase covalently bound to a modified sugar. Nuclei incorporating modified nucleobasesSome examples of glycosides include 4-acetylcytidine, 5- (carboxyhydroxymethyl) uridine, 2 ' -O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2 ' -O-methylpseudouridine, β, D-galactosylQ nucleoside (beta, D-galactosylguanosine), 2 ' -O-methylguanosine, N6-isopentenyl adenosine; 1-methyladenosine; 1-methylpseudouridine; 1-methylguanosine; l-methylinosine; 2, 2-dimethylguanosine; 2-methyladenosine; 2-methylguanosine; n is a radical of7-methylguanosine; 3-methyl-cytidine; 5-methylcytidine; 5-hydroxymethylcytidine; 5-formylcytosine; 5-carboxycytosine; n is a radical of6-methyladenosine, 7-methylguanosine, 5-methylaminoethyluridine, 5-methoxyaminomethyl-2-thiouridine, β, D-mannosyl Q nucleoside, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N-methyluridine6-isopentenyladenosine, N- ((9- β, D-ribofuranosyl-2-methylthiopurin-6-yl) carbamoyl) threonine, N- ((9- β, D-ribofuranosyl-purin-6-yl) -N-methylcarbamoyl) threonine, uridine-5-oxoacetic acid methyl ester, uridine-5-oxoacetic acid (v), pseudouridine, Q nucleoside, 2-thiocytidine, 5-methyl-2-thiouridine, 4-thiouridine, 5-methyluridine, 2 '-O-methyl-5-methyluridine, and 2' -O-methyluridine.
In some embodiments, nucleosides include 6-modified bicyclic nucleosides having (R) or (S) chirality at the 6' position and include analogs described in U.S. patent No. 7,399,845. In other embodiments, nucleosides include 5 "-modified bicyclic nucleosides having (R) or (S) chirality at the 5' position and include analogs described in U.S. patent application publication No. 20070287831.
In some embodiments, the nucleobase or modified nucleobase comprises one or more biomolecule binding moieties, such as, for example, antibodies, antibody fragments, biotin, avidin, streptavidin, receptor ligands, or chelating moieties. In other embodiments, the nucleobase or modified nucleobase is 5-bromouracil, 5-iodouracil, or 2, 6-diaminopurine. In some embodiments, the nucleobase or modified nucleobase is modified by substitution with a fluorescent moiety or a biomolecule-binding moiety. In some embodiments, the nucleobase or modified nucleobase substituents are fluorescent moieties. In some embodiments, the substituent on the nucleobase or modified nucleobase is biotin or avidin.
Representative U.S. patents that teach the preparation of certain of the above-mentioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-mentioned U.S. patent nos. 3,687,808, and U.S. patent nos. 4,845,205, 5,130,30, 5,134,066; 5,175,273, respectively; 5,367,066, respectively; 5,432,272; 5,457,187, respectively; 5,457,191, respectively; 5,459,255; 5,484,908, respectively; 5,502,177, respectively; 5,525,711, respectively; 5,552,540, respectively; 5,587,469, respectively; 5,594,121, 5,596,091, 5,614,617; 5,681,941, respectively; 5,750,692, respectively; 6,015,886, respectively; 6,147,200, respectively; 6,166,197, respectively; 6,222,025, respectively; 6,235,887, respectively; 6,380,368, respectively; 6,528,640, respectively; 6,639,062, respectively; 6,617,438, respectively; 7,045,610, respectively; 7,427,672, respectively; and 7,495,088, the modified nucleobases, sugars and internucleotide linkages in each of said documents are incorporated by reference.
In some embodiments, the base is optionally substituted A, T, C, G or U, wherein one or more-NH groups2Independently and optionally substituted by-C (-L-R)1)3Alternatively, one or more of-NH-are independently and optionally substituted with-C (-L-R)1)2-substitution, one or more ═ N-independently and optionally by-C (-L-R)1) -substitution, one or more ═ CH-independently and optionally substituted by ═ N-, and one or more ═ O independently and optionally substituted by ═ S, ═ N (-L-R)1) Or ═ C (-L-R)1)2Alternatively, wherein two or more-L-R1Optionally together with intervening atoms, form a 3-30 membered bicyclic or polycyclic ring having 0-10 heteroatom ring atoms. In some embodiments, the modified base is optionally substituted A, T, C, G or U, wherein one or more-NH groups2Independently and optionally substituted by-C (-L-R)1)3Alternatively, one or more of-NH-are independently and optionally substituted with-C (-L-R)1)2-substitution, one or more ═ N-independently and optionally by-C (-L-R)1) -a substitution, one or more-CH-is independently and optionally substituted with-N-, and one or more-O is independently and optionally substituted withS、＝N(-L-R1) Or ═ C (-L-R)1)2Alternatively, wherein two or more-L-R1Optionally together with intervening atoms, form a 3-30 membered bicyclic or polycyclic ring having 0-10 heteroatom ring atoms, wherein the modified base is other than natural A, T, C, G and U. In some embodiments, the base is optionally substituted A, T, C, G or U. In some embodiments, the modified base is substituted A, T, C, G or U, wherein the modified base is different from native A, T, C, G and U.
In some embodiments, the nucleoside is any nucleoside described in any one of: gryaznov, S; chen, j. -k.j.am.chem.soc. [ journal of the american chemical society ]1994, 116, 3143; hendrix et al 1997 chem.Eur.J. [ European journal of chemistry ] 3: 110; hyrup et al 1996 Bioorg.Med.chem. [ Bio-organic chemistry and medical chemistry ] 4: 5; jepsen et al 2004Oligo [ oligonucleotide ] 14: 130-146; j.org.chem. [ journal of organic chemistry ]1993, 58, 2983; koizumi et al 2003 nuc. acids Res. [ nucleic acid research ] 12: 3267-3273; koshkin et al 1998Tetrahedron 54: 3607-; kumar et al 1998 bio.med.chem.let. [ bio-organic chemistry and medicinal chemistry communication ] 8: 2219-2222; lauritsen et al 2002chem. 530- > 531; lauritsen et al 2003 bio.med.chem.lett. [ bio-organic chemistry and medicinal chemistry communication ] 13: 253-256; mesmaeker et al, Angew.chem., int.Ed.Engl. [ International edition of applied chemical English ]1994, 33, 226; morita et al, 2001 nucl. 241-242; morita et al 2002bio, med, chem, lett [ bio-organic chemistry and medicinal chemistry communication ] 12: 73-76; morita et al 2003 Bio.Med.chem.Lett. [ Bioorganic chemistry and medicinal chemistry communication ] 2211-2226; nielsen et al 1997 chem.soc.rev. [ review of chemical society ] 73; nielsen et al 1997j.chem.soc.perkins trans. [ journal of the chemical society bokings edition ] 1: 3423-; obika et al 1997Tetrahedron Lett. [ Tetrahedron communication ]38 (50): 8735-8; obika et al 1998Tetrahedron Lett. [ Tetrahedron communication ] 39: 5401-5404; pallan et al, 2012 chem, comm. [ chemical communication ] 48: 8195-; petersen et al 2003TRENDS Biotech [ Biotech TRENDS ] 21: 74-81; rajwanshi et al 1999 chem.commu. [ chemical communication ] 1395-; schultz et al 1996 Nucleic Acids Res. [ Nucleic acid research ] 24: 2966; seth et al, 2009 j.med.chem. [ journal of medical chemistry ] 52: 10-13; seth et al 2010j.med.chem. [ journal of medical chemistry ] 53: 8309-8318; seth et al 2010j. org.chem. [ journal of organic chemistry ] 75: 1569-1581; seth et al, 2012 bio.med.chem.lett. [ bio-organic chemistry and medicinal chemistry communication ] 22: 296-; seth et al 2012mol. ther-nuc. acids. [ molecular therapy and nucleic acids ]1, e 47; seth, Punit P; siwkowski, Andrew; allerson, Charles R; vasquez, Guillermo; lee, Sam; prakash, Thazha P; kinberger, Garth; migawa, Michael T; gaus, Hans; bhat, balkrishn; and the like. From Nucleic acids symposium Series (2008), 52(1), 553-; singh et al 1998chem. Comm. [ chemical communication ] 1247-; singh et al, 1998J. org.chem. [ J. org. chem. ] 63: 10035-39; singh et al, 1998J. org.chem. [ J. org. chem. ] 63: 6078-6079; sorensen 2003chem. [ chem ] 2130-; ts' o et al, ann.n.y.acad.sci. [ journal of new york academy of sciences ]1988, 507, 220; van amerschot et al, 1995 angel w.chem.int.ed.engl. [ international edition of applied chemical english ] 34: 1338; vasseur et al j.am.chem.soc. [ journal of the american chemical society ]1992, 114, 4006; WO 20070900071; WO 20070900071; or WO 2016/079181.
Exemplary nucleobases are also described in US 20110294124, US 20120316224, US20140194610, US20150211006, US 20150197540, WO 2015107425, PCT/US 2016/043542 and PCT/US 2016/043598, each of which is incorporated herein by reference.
In some embodiments, the C9orf72 oligonucleotide comprises Feldman et al 2017 j.am.chem.soc. [ journal of american chemical society ] 139: 11427-11433; feldman et al 2017 proc. natl.acad.sci.usa [ journal of the american academy of sciences ] 114: E6478-E6479; hwang et al 2009 nucleic acids Res [ nucleic acid studies ] 37: 4757-4763; hwang et al 2008 j.am.chem.soc. [ journal of american chemical society ] 130: 14872 vs 14882; lavergne et al 2012 chem. Eur.J. [ European journal of chemistry ] 18: 1231-; lavergne et al 2013 J.am.chem.Soc. [ journal of American chemical society ] 135: 5408-5419; ledbetter et al 2018 j.am.chem.soc. [ journal of american chemical society ] 140: 758-; malyshiev et al 2009 j.am.chem.soc. [ journal of american chemical society ] 131: 14620-14621; seo et al 2009 ChemBiochem [ chemical and biochemical ] 10: 2394 nucleobases, synthetic or modified nucleobases, nucleosides or nucleotides or modified nucleosides or modified nucleotides described in 2400, including but not limited to d3FB, d2Py analogue, d2Py, d3MPy, d4MPy, d5MPy, d34DMPy, d35DMPy, d45DMPy, d5FM, d5PrM, d5SICS, dFEMO, dMMO2, dNaM, dNM01, dTPT 3; nucleotides having a2 ' -azido sugar, a2 ' -chloro sugar, a2 ' -amino sugar, or arabinose; isoquinolone nucleotides, naphthyl nucleotides, and azaindole nucleotides; and modified and derivatives and functionalized forms thereof, including but not limited to those in which the sugar comprises a 2' modification and/or other modification, and dMMO2 derivatives having m-chloro, -bromo, -iodo, -methyl, or-propynyl substituents.
Candy
In some embodiments, provided C9orf72 oligonucleotides are capable of directing a decrease in expression, level, and/or activity of a C9orf72 target gene or gene product thereof. In some embodiments, the C9orf72 target gene comprises repeat amplification. In some embodiments, provided C9orf72 oligonucleotides comprise any sugar described herein or known in the art.
In some embodiments, provided C9orf72 oligonucleotides capable of directing knock down of C9orf72 comprise one or more modified sugar moieties in addition to a native sugar moiety.
The most common naturally occurring nucleotides comprise ribose linked to the nucleobases adenosine (a), cytosine (C), guanine (G) and thymine (T) or uracil (U). Also encompassed are modified nucleotides, wherein a phosphate group or a bonded phosphorus in the nucleotide can be attached to each position of the sugar or modified sugar. As non-limiting examples, a phosphate group or a phosphorus linkage may be attached to the 2 ", 3", 4 ", or 5" hydroxyl moiety of the sugar or modified sugar. Also encompassed in this case are nucleotides as described herein and having a modified nucleobase. In some embodiments, nucleotides or modified nucleotides comprising an unprotected-OH moiety are used in accordance with the methods of the present disclosure.
In some embodiments, the C9orf72 oligonucleotide may comprise any base (nucleobase), modified base, or base analog described herein or known in the art. In some embodiments, a C9orf72 oligonucleotide may comprise any base described herein or known in the art in combination with any other structural element or modification described herein, including but not limited to a base sequence or portion thereof, a sugar; an internucleotide linkage; stereochemistry or modes thereof; additional chemical moieties including, but not limited to, targeting moieties, and the like; a modification pattern of sugar, base, or internucleotide linkages; in the form thereof or any structural element; and/or any other structural element or modification described herein; and in some embodiments, the disclosure relates to multimers of any such oligonucleotides.
In some embodiments, the C9orf72 oligonucleotide may comprise any sugar.
In some embodiments, the saccharide has the following structure:
the modified sugar may be incorporated into the provided C9orf72 oligonucleotides. In some embodiments, the modified sugar contains one or more substituents at the 2 "position, including one of the following: -F; -CF3、-CN、-N3、-NO、-NO2-OR ', -SR ', OR-N (R ')2Wherein each R' is independently described in the present disclosure; -O- (C)1-C10Alkyl), -S- (C)1-C10Alkyl), -NH- (C)1-C10Alkyl), or-N (C)1-C10Alkyl radical)2；-O-(C2-C10Alkenyl), -S- (C)2-C10Alkenyl), -NH- (C)2-C10Alkenyl), or-N (C)2-C10Alkenyl)2；-O-(C2-C10Alkynyl), -S- (C)2-C10Alkynyl), -NH- (C)2-C10Alkynyl), or-N (C)2-C10Alkynyl)2(ii) a or-O- - (C)1-C10Alkylene) -O- - (C)1-C10Alkyl), -O- (C)1-C10Alkylene) -NH- (C)1-C10Alkyl) or-O- (C)1-C10Alkylene) -NH (C)1-C10Alkyl radical)2、-NH-(C1-C10Alkylene) -O- (C)1-C10Alkyl), or-N (C)1-C10Alkyl group) - (C1-C10Alkylene) -O- (C)1-C10Alkyl), wherein alkyl, alkylene, alkenyl, and alkynyl groups may be substituted or unsubstituted. Examples of substituents include, and are not limited to, -O (CH)2)nOCH3and-O (CH)2)nNH2(where n is from 1 to about 10), MOE, DMAOE, DMAEOE. Also encompassed herein are WO 2001/088198; and Martin et al, Helv, Chim, acta [ Helveti chemical journal of]1995, 78, 486-. In some embodiments, the modified saccharide comprises one or more groups selected from: substituted silyl groups, RNA cleaving groups, reporter groups, fluorescent labels, intercalators, groups for improving the pharmacokinetic properties of nucleic acids, groups for improving the pharmacodynamic properties of nucleic acids, or other substituents with similar properties. In some embodiments, the modification is at one or more of the 2 ', 3 ', 4 ', 5 ' or 6 ' positions of the sugar or modified sugar (including the 3 ' position of the sugar on the 3 ' -terminal nucleotide or the 5 ' position of the 5 ' -terminal nucleotide).
In some embodiments, the 2 '-modification is 2' -F.
In some embodiments, the 2' -OH of the ribose is replaced with a substituent comprising one of: -H, -F; -CF3、-CN、-N3、-NO、-NO2-OR ', -SR ', OR-N (R ')2Wherein each R' is independently described in the present disclosure; -O- (C)1-C10Alkyl), -S- (C)1-C10Alkyl), -NH- (C)1-C10Alkyl), or-N (C)1-C10Alkyl radical)2；-O-(C2-C10Alkenyl), -S- (C)2-C10Alkenyl), -NH- (C)2-C10Alkenyl), or-N (C)2-C10Alkenyl)2；-O-(C2-C10Alkynyl), -S- (C)2-C10Alkynyl), -NH- (C)2-C10Alkynyl), or-N (C)2-C10Alkynyl)2(ii) a or-O- - (C)1-C10Alkylene) -O- - (C)1-C10Alkyl), -O- (C)1-C10Alkylene) -NH- (C)1-C10Alkyl) or-O- (C)1-C10Alkylene) -NH (C)1-C10Alkyl radical)2、-NH-(C1-C10Alkylene) -O- (C)1-C10Alkyl), or-N (C)1-C10Alkyl group) - (C1-C10Alkylene) -O- (C)1-C10Alkyl), wherein alkyl, alkylene, alkenyl, and alkynyl groups may be substituted or unsubstituted. In some embodiments, the 2' -OH is replaced with-H (deoxyribose). In some embodiments, the 2' -OH is replaced with — F. In some embodiments, the 2 '-OH is replaced with — OR'. In some embodiments, the 2' -OH is replaced with-OMe. In some embodiments, 2' -OH is-OCH2CH2And (4) OMe replacement.
The modified sugar also includes Locked Nucleic Acids (LNA). In some embodiments, two substituents on a sugar carbon atom together form a divalent moiety. In some embodiments, the two substituents are on two different sugar carbon atoms. In some embodiments, the divalent moiety formed has the structure-L-as defined herein. In some embodiments, -L-is-O-CH2-2-is optionally substituted. In some embodiments, -L-is-O-CH2-. In some embodiments, -L-is-O-CH (Et) -. In some embodiments, -L-is between C2 and C4 of the sugar moiety. In some embodiments, the locked nucleic acid has the structure shown below. Shows a locked nucleic acid having the structure, wherein B represents a nucleobase or a modified nucleobase as described herein, and wherein for example R2sAnd R4sIs R which forms a ring together with its intervening atoms. In some casesIn embodiments, the modified nucleoside has the following structure:
wherein B is a base.
In some embodiments, the modified sugar is ENA, such as in, for example, Seth et al, J Am Chem Soc. [ journal of american chemical society ]10 months and 27 days 2010; 132(42): 14942 those described in 14950. In some embodiments, the modified sugar is any of those found in XNA (xenogenic nucleic acid), such as arabinose, anhydrohexitol, threose, 2' fluoroarabinose, or cyclohexene.
The modified sugar includes a cyclobutyl or cyclopentyl moiety in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. patent nos.: 4,981,957, 5,118,800; 5,319,080, respectively; and 5,359,044. Some modified sugars contemplated include sugars in which an oxygen atom within the ribose ring is replaced with nitrogen, sulfur, selenium, or carbon. In some embodiments, the modified sugar is a modified ribose sugar in which an oxygen atom within the ribose ring is replaced with a nitrogen, and wherein the nitrogen is optionally substituted with an alkyl group (e.g., methyl, ethyl, isopropyl, etc.).
Non-limiting examples of modified sugars include glycerol, which forms Glycerol Nucleic Acid (GNA). An example of GNA is shown below and described in Zhang, R et al, j.am.chem.soc. [ journal of american chemical society ], 2008, 130, 5846-; zhang L, et al, j.am.chem.soc. [ journal of the american chemical society ], 2005, 127, 4174-. In some embodiments, the nucleoside has the structure:
wherein B is a base.
Flexible formyl glycerol-based mixed acetal aminal nucleic acids (FNA) are described in Joyce GF et al, PNAS [ journal of the national academy of sciences USA ], 1987, 84, 4398-. In some embodiments, the nucleoside has the structure:
wherein B is a base.
Additional non-limiting examples of modified sugars and/or modified nucleosides and/or modified nucleotides include hexopyranosyl sugars (6 'to 4'), pentopyranosyl sugars (4 'to 2'), pentopyranosyl sugars (4 'to 3'), 5 '-deoxy-5' -C-malonyl sugars, squarylium diamide (squarylium diamide), and tetrafuranosyl (3 'to 2') sugars. In some embodiments, the modified nucleoside comprises a hexopyranosyl (6 'to 4') sugar and has the structure of any one of the following formulae:
wherein XsCorresponding to the P modification group "-XLR described herein1", wherein XLR1Is equal to X-L-R1And X, L and R1As defined in formula I disclosed herein, and B is a base.
In some embodiments, the modified nucleotide comprises a pentopyranosyl (4 'to 2') sugar and has the structure of any one of the following formulae:
wherein XsCorresponding to the P-modifying group "-XLR described herein1", wherein XLR1Equivalent to X-L-R1And X, L and R1As defined by formula I as disclosed herein, and B is a base.
In some embodiments, the modified nucleotide comprises a pentopyranosyl (4 'to 3') sugar and has any one of the following formulae:
wherein XsCorresponding to the P-modifying group "-XLR described herein1", wherein XLR1Equivalent to X-L-R1And X, L and R1As defined by formula I as disclosed herein, and B is a base.
In some embodiments, the modified nucleotide comprises a tetrafuranosyl (3 'to 2') sugar and has any one of the following formulae:
wherein XsCorresponding to the P-modifying group "-XLR described herein1", wherein XLR1Equivalent to X-L-R1And X, L and R1As defined by formula I as disclosed herein, and B is a base.
In some embodiments, the modified nucleotide comprises a modified sugar and has any one of the following formulae:
wherein XsCorresponding to the P-modifying group "-XLR described herein1", wherein XLR1Equivalent to X-L-R1And X, L and R1As defined by formula I as disclosed herein, and B is a base.
In some embodiments, one or more hydroxyl groups in the sugar moiety are optionally and independently substituted with halogen, R '-N (R')2-OR ', OR-SR ', wherein each R ' is independently described in the disclosure.
In some embodiments, the modified nucleotides are shown below, wherein Xs corresponds to the P modification group "-XLR" described herein1", wherein XLR1Is equal to X-L-R1And X, L and R1As defined in formula I disclosed herein, B is a base, and X1Is selected from-S-, -Se-, -CH2-, -NMe-, -NEt-and-NiPr-
Modified sugars can be prepared by methods known in the art, including but not limited to: eschenmoser, Science [ Science ] (1999), 284: 2118; m. bohringer et al, helv, chim, acta [ journal of herville chemical (1992), 75: 1416-; m.egli et al, j.am.chem.soc. [ journal of the american chemical society ] (2006), 128 (33): 10847-56; eschenmoser in Chemical Synthesis: gnosis toPrognosis [ chemical synthesis: glandular disease prognosis ], c.chatgilialoglu and v.snikus, editors (kluweracademy, netherlands, 1996), p.293; K. schoning et al, Science [ Science ] (2000), 290: 1347-1351; eschenmoser et al, Helv. Chim. acta [ Helverett chemical journal ] (1992), 75: 218; j.huntziker et al, helv.chim.acta [ journal of herville chemical ] (1993), 76: 259; g.otting et al, helv.chim.acta [ journal of herville chemical ] (1993), 76: 2701; k.groebke et al, helv.chim.acta [ journal of herville chemical ] (1998), 81: 375; and a. eschenmoser, Science [ Science ] (1999), 284: 2118. modifications to the 2' modification can be found in Verma, s. et al annu.rev.biochem [ review of biochemistry ].1998, 67, 99-134 and all references therein. Specific modifications to ribose can be found in the following references: 2' -fluoro (Kawasaki et al, J.Med.chem. [ J.Med.chem. ] [ J.Med.Chem. ] [ 1993, 36, 831-. In some embodiments, the modified sugar is any of those described in PCT publication No. WO 2012/030683 (incorporated herein by reference) and/or depicted herein. In some embodiments, the modified sugar is any modified sugar described in any one of: gryaznov, S; chen, j. -k.j.am.chem.soc. [ journal of the american chemical society ]1994, 116, 3143; hendrix et al 1997 chem.Eur.J. [ European journal of chemistry ] 3: 110; hyrup et al 1996 bioorg.Med.chem. [ bio-organic chemistry and medicinal chemistry ] 4: 5; jepsen et al 2004Oligo [ oligonucleotide ] 14: 130-146; j.org.chem. [ journal of organic chemistry ]1993, 58, 2983; koizumi et al 2003 nuc. acids Res. [ nucleic acid research ] 12: 3267-3273; koshkin et al 1998Tetrahedron 54: 3607-; kumar et al 1998 bio.med.chem.let. [ bio-organic chemistry and medicinal chemistry communication ] 8: 2219-2222; lauritsen et al 2002chem. 530- > 531; lauritsen et al 2003 bio.med.chem.lett. [ bio-organic chemistry and medicinal chemistry communication ] 13: 253-256; mesmaeker et al, Angew. chem., int.Ed. Engl. [ International edition of applied chemistry English ]1994, 33, 226; morita et al, 2001 nucl. acids res. sup. [ nucleic acid research supplement ] 1: 241-242; morita et al 2002bio, med, chem, lett [ bio-organic chemistry and medicinal chemistry communication ] 12: 73-76; morita et al 2003 Bio.Med.chem.Lett. [ Bioorganic chemistry and medicinal chemistry communication ] 2211-2226; nielsen et al 1997 chem.soc.rev. [ review of chemical society ] 73; nielsen et al 1997j.chem.soc.perkins trans. [ journal of the chemical society, bockin's edition ] 1: 3423-; obika et al 1997Tetrahedron Lett. [ Tetrahedron communication ]38 (50): 8735-8; obika et al 1998Tetrahedron Lett. [ Tetrahedron communication ] 39: 5401-5404; pallan et al, 2012 chem, comm. [ chemical communication ] 48: 8195-; petersen et al 2003TRENDS Biotech [ Biotech TRENDS ] 21: 74-81; rajwanshi et al 1999 chem.commu. [ chemical communication ] 1395-; schultz et al 1996 Nucleic Acids Res. [ Nucleic acid research ] 24: 2966; seth et al, 2009 j.med.chem. [ journal of medicinal chemistry ] 52: 10-13; seth et al 2010j.med.chem. [ journal of medicinal chemistry ] 53: 8309-8318; seth et al 2010j. org.chem. [ journal of organic chemistry ] 75: 1569-1581; seth et al, 2012 bio.med.chem.lett. [ bio-organic chemistry and medicinal chemistry communication ] 22: 296-; seth et al 2012mol. ther-nuc. acids. [ molecular therapy and nucleic acids ]1, e 47; seth, Punit P; siwkowski, Andrew; allerson, Charles R; vasquez, Guillermo; lee, Sam; prakash, ThazhaP; kinberger, Garth; migawa, Michael T; gaus, Hans; bhat, balkrishn; from nucleic acids Symposium Series (2008), 52(1), 553-; singh et al 1998chem. Comm. [ chemical communication ] 1247-; singh et al, 1998J. org.chem. [ J. org. chem. ] 63: 10035-39; singh et al, 1998J. org.chem. [ J. org. chem. ] 63: 6078-6079; sorensen 2003chem. [ chemical communication ] 2130-; ts' o et al, ann.n.y.acad.sci. [ journal of new york academy of sciences ]1988, 507, 220; van amerschot et al, 1995 angel w.chem.int.ed.engl. [ international edition of applied chemistry ] 34: 1338; vasseur et al j.am.chem.soc. [ journal of the american chemical society ]1992, 114, 4006; WO 20070900071; WO 20070900071; or WO 2016/079181.
In some embodiments, the modified sugar moiety is an optionally substituted pentose or hexose moiety. In some embodiments, the modified sugar moiety is an optionally substituted pentose moiety. In some embodiments, the modified sugar moiety is an optionally substituted hexose moiety. In some embodiments, the modified sugar moiety is an optionally substituted ribose or hexitol moiety. In some embodiments, the modified sugar moiety is an optionally substituted ribose moiety. In some embodiments, the modified sugar moiety is an optionally substituted hexitol moiety.
In some embodiments, exemplary modified nucleotides are selected from:
2 ' -fluoro-N3 ' -P5 ' -phosphoramidate
In some embodiments, the nucleotide has a structure selected from any one of:
in some embodiments, the modified nucleoside has a structure selected from the group consisting of:
β -D-oxy-LNA
Wherein R is1And R2independently-H, -F, -OMe, -MOE or substituted or unsubstituted C1-6An alkyl group;2 '-O, 3' -C-linked bicyclic rings
β -D-sulfanyl-LNA
β -D-amino-LNA
Wherein Re is substituted or unsubstituted C1-6Alkyl or H
xylose-LNA [ c]α-L-LNA
ENAβ-D-ENAmethylphosphonate-LNA(R，S)-cEt(R，S)-cMOE(R，S)-5′-Me-LNAS-Me cLNAmethylene-cLNA
3′-Me-α-L-LNAR-6′-Me-α-L-LNA
S-5′-Me-α-L-LNAR-5′-Me-α-L-LNA。
Additional chemically modified sugars are described in WO 2008/101157, WO 2007/134181, WO 2016/167780 and published U.S. patent application US 2005-0130923.
In some embodiments, the nucleotide and adjacent nucleoside have the following structures:
amide linked LNA
Examples of nucleosides having modified sugar moieties include, without limitation, nucleosides comprising 5 '-vinyl, 5'-methyl (R or S), 4 ' -S, 2 ' -F, 2 ' -OCH3、2′-OCH2CH3、2′-OCH2CH2F and 2' -O (CH)2)20CH3A nucleoside of substituents. The substituent at the 2' position may also be selected from allyl, amino, azido, thio, O-allyl, O-C1-C10Alkyl radical, OCF3、OCH2F、O(CH2)2SCH3、O(CH2)2-O-N(Rm)(Rn)、O-CH2-C(＝O)-N(Rm)(Rn) And O-CH2-C(＝O)-N(R1)-(CH2)2-N(Rm)(Rn) Wherein each R1、RmAnd RnIndependently is H or substituted or unsubstituted C1-C10An alkyl group.
In some embodiments, bicyclic nucleosides include any modified nucleoside comprising a bicyclic sugar moiety. Examples of Bicyclic Nucleic Acids (BNAs) include, but are not limited to, nucleosides comprising a bridge between a4 'ribose ring atom and a 2' ribose ring atom. In some embodiments, antisense compounds provided herein comprise one or more BNA nucleosides, wherein the bridge comprises one of the following formulae: 4' - (CH)2)-O-2′(LNA)；4′-(CH2)-S-2′；4，-(CH2)2-O-2′(ENA)；4′-CH(CH3) -O-2 'and 4' -CH (CH)2OCH3) -O-2' (and analogs thereof; see us patent 7,399,845); 4' -C (CH)3)(CH3) -O-2' (and analogs thereof; see PCT/US 2008/068922 published as WO/2009/006478); 4' -CH2-N(OCH3) -2' (and analogs thereof; see PCT/US 2008/064591 published as WO/2008/150729); 4' -CH2-O-N(CH3) -2' (see published U.S. patent application US 2004-0171570); 4' -CH2-N (R) -O-2', wherein R is H, C1-C12Alkyl or protecting groups (see U.S. patent 7,427,672); 4' -CH2-C(H)(CH3) -2' (see Chattopadhyaya et al, J.org.chem. [ J.Org.Organischen. chem. ] [ J.Organic chemistry]2009, 74, 118-); and 4, -CH2-C(＝CH2) -2' (and thereof)(ii) an analog; see PCT/US 2008/066154 published as WO 2008/154401).
Other bicyclic nucleosides have been reported in the literature (see, e.g., Srivastava et al, J.Am. chem. Soc. [ journal of the American chemical society ], 2007, 129(26)8362-, chem. [ journal of organic chemistry ], 1998, 63, 10035-; U.S. patent nos.: 7,399,845, respectively; 7,053,207, respectively; 7,034,133; 6,794,499, respectively; 6,770,748; 6,670,461; 6,525,191, respectively; 6,268,490; U.S. patent publication nos.: US 2008-0039618; US 2007-0287831; US 2004-0171570; U.S. patent application serial No.: 12/129,154, respectively; 61/099,844, respectively; 61/097,787, respectively; 61/086,231, respectively; 61/056,564, respectively; 61/026,998, respectively; 61/026,995, respectively; 60/989,574, respectively; international application WO 2007/134181; WO 2005/021570; WO 2004/106356; and PCT international application No.: PCT/US 2008/068922; PCT/US 2008/066154; and PCT/US 2008/064591).
In some embodiments, bicyclic nucleosides (see PCT International application PCT/DK98/00393, published as WO 99/14226) can be prepared having one or more stereochemical sugar configurations, including, for example, α -L-ribofuranose and β -D-ribofuranose(ii) (2 'carbon atom to 4' carbon atom connecting the sugar ring). In some embodiments, the bicyclic sugar moiety of a BNA nucleoside includes, but is not limited to, a compound having at least one bridge between the 4 'carbon atom and the 2' carbon atom of the pentofuranosyl sugar moiety, including, but not limited to, bridges comprising 1 or 1 to 4 linking groups independently selected from: - [ C (R)a)(Rb)]n-、-C(Ra)＝C(Rb)-、-C(Ra)＝N-、-C(＝NRa)-、-C(＝O)-、-C(＝S)-、-O-、-Si(Ra)2-, -S (═ O) x-and-N (R)a) -; wherein: x is 0, 1 or 2; n is 1, 2, 3or 4; each RaAnd RbIndependently is H, a protecting group, a hydroxy group C1-C12Alkyl, substituted C1-C12Alkyl radical, C2-C12Alkenyl, substituted C2-C12Alkenyl radical, C2-C12Alkynyl, substituted C2-C12Alkynyl, C5-C20Aryl, substituted C5-C20Aryl, heterocyclyl, substituted heterocyclyl, heteroaryl, substituted heteroaryl, C5-C7Alicyclic radical, substituted C5-C7Alicyclic radical, halogen, OJ1、NJ1J2、SJ1、N3、COOJ1Acyl (C ═ O) -H), substituted acyl, CN, sulfonyl (S ═ O)2-J1) Or sulfo (S (═ O) -J1) (ii) a And each J1And J2Independently is H, C1-C12Alkyl, substituted C1-C12Alkyl radical, C2-C12Alkenyl, substituted C2-C12Alkenyl radical, C2-C12Alkynyl, substituted C2-C12Alkynyl, C5-C20Aryl, substituted C5-C20Aryl, acyl (C (═ O) -H), substituted acyl, heterocyclyl, substituted heterocyclyl, C1-C12Aminoalkyl, substituted C1-C12Aminoalkyl groups or protecting groups.
In some embodiments, the bridging of the bicyclic sugar moiety is- [ C (R)a)(Rb)]n-、-[C(Ra)(Rb)]n-O-、-C(RaRb) -N (R) -O-or-C (R)aRb) -O-N (R) -. In some embodiments, the bridging group is 4' -CH2-2′、4′-(CH2)2-2′、4′-(CH2)3-2′、4′-CH2-O-2′、4′-(CH2)2-O-2′、4′-CH2-O-N (R) -2 'and 4' -CH2-N (R) -O-2', wherein each R is independently H, a protecting group or C1-C12An alkyl group.
In some embodiments, bicyclic nucleosides are further defined by isomeric configurations. For example, containing 4' - (CH)2) The nucleoside of the-O-2 'bridging group may be in the α -L configuration or the β -D configuration α -L-methyleneoxy (4' -CH)2O-2') BNA has been incorporated into antisense oligonucleotides exhibiting antisense activity (Frieden et al, Nucleic Acids Research [ Nucleic Acids Research ]]，2003，21，6365-6372)。
In some embodiments, bicyclic nucleosides include those having a4 ' to 2 ' bridging group, including but not limited to a-L-4 ' - (CH)2)-O-2′、P-D-4′-CH2-O-2′、4′-(CH2)2-O-2′、4′-CH2-O-N(R)-2′、4′-CH2-N(R)-O-2′、4′-CH(CH3)-O-2′、4′-CH2-S-2′、4′-CH2-N(R)-2′、4′-CH2-CH(CH3) -2 'and 4' - (CH)2)3-2', wherein R is H, a protecting group or C1-C12An alkyl group.
Various bicyclic nucleoside analogs have also been prepared having, for example, 4' -CH2-O-2 'and 4' -CH2The 4 ' to 2 ' bridging group of-S-2 ' (Kumar et al, bioorg.Med.chem.Lett. [ Bioorganic chemistry and medicinal chemistry communication ]],1998,8, 2219-2222). The preparation of oligodeoxyribonucleotide duplexes comprising bicyclic nucleosides, which serve as substrates for nucleic acid polymerases, is also described (Wengel et al, WO 99/14226). Further, it is described in the artSynthesis of 2' -amino-BNA, a novel configurationally constrained high affinity oligonucleotide analog (Singh et al, J.org.chem. [ J.Org.Chem. [ J.Ore.Or. ], J.Ore.Ore.],1998, 63, 10035-10039). In addition, 2 '-amino-BNA and 2' -methylamino-BNA have been prepared and their duplex thermal stability to complementary RNA and DNA strands has been previously reported.
Has already described a compound having 4' - (CH)2)3-2 'bridging group and alkenyl-like bridging group 4' -CH ═ CH-CH22' -carbocyclic bicyclic nucleosides (Frier et al, Nucleic Acids Research [ Nucleic Acids Research ]]1997, 25(22), 4429-4443 and Albaek et al, J.org.chem. [ J.Org.Chem. [ J.Ore.Organic chemistry ]],2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides and their oligomerization and biochemical studies are also described (Srivastava et al, j.am.chem.soc. [ journal of the american chemical society]2007，129(26)，8362-8379)。
In some embodiments, bicyclic nucleosides include, but are not limited to, α -L-methyleneoxy (4' -CH)2-O-2 ') BNA, β -D-methyleneoxy (4' -CH)2-O-2 ') BNA, ethyleneoxy (4' - (CH)2)2-O-2 ') BNA, aminooxy (4' -CH)2-O-N (R) -2 ') BNA, oxyamino (4' -CH)2-N (R) -O-2 ') BNA, methyl (methyleneoxy) (4' -CH (CH)3) -O-2 ') BNA (also known as constrained ethyl or cEt), methylene-thio (4' -CH)2-S-2 ') BNA, methylene-amino (4' -CH)2-N (R) -2 ') BNA, methyl carbocycle (4' -CH)2-CH(CH3) -2 ') BNA, propylenylcyclo (4' - (CH)2)3-2') BNA and vinylBNA.
In some embodiments, the modified tetrahydropyran nucleoside or modified THP nucleoside is a nucleoside having a six-membered tetrahydropyran "sugar" substituted for the pentofuranosyl residue in a normal nucleoside, and may be referred to as a sugar substitute. Modified THP nucleosides include, but are not limited to, those known in the art as Hexitol Nucleic Acids (HNA), Anitol Nucleic Acids (ANA), Mannitol Nucleic Acids (MNA) (see Leumann, bioorg.med.chem. [ bio-organic chemistry and medicinal chemistry ], 2002, 10, 841-854), or fluoro HNA (F-HNA) having a tetrahydropyranyl ring system as described below.
In some embodiments, the sugar substitute comprises a ring having more than 5 atoms and more than one heteroatom. For example, nucleosides containing morpholino sugar moieties and their use in oligomeric compounds have been reported (see, e.g., Braasch et al, Biochemistry [ Biochemistry ], 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685, 5,166,315, 5,185,444, and 5,034,506).
Also provided are combinations of modifications, such as, but not limited to, 2 '-F-5' -methyl substituted nucleosides (see PCT International application WO 2008/101157 for other disclosed 5 ', 2' -disubstituted nucleosides); and replacement of the ribosyl epoxy atom with S and further substitution at the 2' position (see published U.S. patent application US 2005-0130923); or alternatively 5 'substitution of a bicyclic nucleic acid (see PCT International application WO 2007/134181, wherein the 4' -CH is2-O-2 'bicyclic nucleoside further substituted in 5' position with 5 '-methyl or 5' -vinyl). The synthesis and preparation of carbocyclic bicyclic nucleosides and their oligomerization and biochemical studies are also described (see, e.g., Srivastava et al, j.am.chem.soc. [ journal of the american chemical society]2007，129(26)，8362-8379)。
In some embodiments, the antisense compound comprises one or more modified cyclohexenyl nucleosides that are nucleosides having a six-membered ring hexenyl group in place of a pentofuranosyl residue in a naturally occurring nucleoside. Modified cyclohexenyl Nucleosides include, but are not limited to, those described in the art (see, e.g., commonly owned published PCT application WO 2010/036696; Robeyns et al, J.Am. chem. Soc. [ J.Am. chem. Soc. ], 2008, 130(6), 1979-1984; Horvath et al, Tetrahedron Letters [ Tetrahedron communication ], 2007, 48, 3621-9323; Nauwelerts et al, J.Am. chem. Soc. [ J.Am. chem. J.Soc. [ J.Am. chem. C.C. ], 2007, 129(30), 9340-9348; Gu et al, Nucleic Acids & Nucleic Acids [ nucleoside, nucleotide and Nucleic Acids ], 2005, 24 (5-2457), 993-998; Nauwelerts et al, Nucleic Acids Research [ 2005, 33, 9-11, 92, crystallization [ gradient F. ], crystal structures [ gradient & propagation [ 12-61 ], tetrahedron 2004, 60(9), 2111-2123; gu et al Oligonucleotides, 2003, 13(6), 479-; wang et al, j. org. chem. [ journal of organic chemistry ], 2003, 68, 4499-; vercure et al, Nucleic Acids Research [ Nucleic Acids Research ], 2001, 29(24), 4941-4947; wang et al, j.org.chem. [ journal of organic chemistry ], 2001, 66, 8478-82; wang et al, Nucleotides & Nucleic Acids [ Nucleosides, Nucleotides and Nucleic Acids ], 2001, 20(4-7), 785-788; wang et al, J.Am.chem. [ J.Chem., USA ], 2000, 122, 8595-; published PCT applications, WO 06/047842; and published PCT application WO 01/049687.
Many other monocyclic, bicyclic and tricyclic systems are known in the art and are suitable for use as sugar substitutes that can be used to modify nucleosides for incorporation into oligomeric compounds as provided herein (see, e.g., review article: Leumann, christianj.&Chem. [ journal of bio-organic chemistry and medicinal chemistry],2002, 10, 841-854). Such ring systems may be subjected to various additional substitutions to further enhance their activity. In some embodiments, the 2 '-modified sugar is a furanosyl sugar modified at the 2' position. In some embodiments, such modifications include substituents selected from the group consisting of: halides, including but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted aminoalkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In some embodiments, the 2' modification is selected from the following substituents, including but not limited to: o [ (CH)2)nO]mCH、O(CH2)nNH2、O(CH2)nCH3、O(CH2)nF、O(CH2)nONH2、OCH2C(＝O)N(H)CH3And O (CH)2)nON[(CH2)nCH3]2Wherein n and m are 1 to about 10. The other 2' -substituents may also be selected from: c1-C12Alkyl, substituted alkyl, alkenyl, alkynyl, alkylaryl, arylalkyl, O-alkylaryl or O-arylalkyl, SH, SCH3、OCN、Cl、Br、CN、F、CF3、OCF3、SOCH3、SO2CH3、ONO2、NO2、N3、NH2Heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, R A cleavage groups, reporter groups, intercalators, groups for improved pharmacokinetic properties, or groups for improved pharmacokinetic properties of antisense compounds, and other substituents with similar properties. In some embodiments, the modified nucleoside includes a 2' -MOE side chain (Baker et al, j.biol.chem. [ journal of biochemistry)],1997, 272, 11944-12000). Such 2 '-MOE substitutions have been described as having improved binding affinity compared to unmodified nucleosides and other modified nucleosides (e.g., 2' -O-methyl, O-propyl, and O-aminopropyl). Oligonucleotides with 2' -MOE substituents have also been shown to be gene expression antisense inhibitors with promising characteristics for in vivo use (MMartin, Helv. Chim. acta [ Hellvidi chemical journal of chemistry)]1995, 78, 486-; altmann et al, Chimia [ chemistry]1996, 50, 168-; altmann et al, biochem]1996, 24, 630-; and Altmann et al, Nucleotides]，1997，16，917-926)。
In some embodiments, a2 ' -modified or 2 ' -substituted nucleoside is a nucleoside comprising a sugar containing a substituent other than H or OH at the 2 ' position. In some embodiments, 2' -modified nucleosides include, but are not limited to: bicyclic nucleosides, wherein the bridge connecting two carbon atoms of the sugar ring connects the 2' carbon of the sugar ring to another carbon; and nucleosides having non-bridging 2' substituents such as allyl, amino, azido, thio, O-allyl, O-C1-C10Alkyl, -OCF3、O-(CH2)2-O-CH3、2′-O(CH2)2SCH3、O-(CH2)2-O-N(Rm)(Rn) Or O-CH2-C(＝O)-N(Rm) (R,) wherein each RmAnd RnIndependently is H or substituted or unsubstituted C1-C10An alkyl group.
Methods for preparing modified sugars are well known to those skilled in the art. Some representative us patents teaching the preparation of such modified sugars include, but are not limited to, the us: 4,981,957, respectively; 5,118,800, respectively; 5,319,080, respectively; 5,359,044, respectively; 5,393,878, respectively; 5,446,137, respectively; 5,466,786, respectively; 5,514,785, respectively; 5,519,134, respectively; 5,567,811, respectively; 5,576,427, respectively; 5,591,722, respectively; 5,597,909, respectively; 5,610,300, respectively; 5,627,053, respectively; 5,639,873, respectively; 5,646,265, respectively; 5,670,633, respectively; 5,700,920, respectively; 5,792,847 and 6,600,032, and international application PCT/US 2005/019219, published as WO 2005/121371.
In some embodiments, R1Is R as defined and described. In some embodiments, R2Is R. In some embodiments, ReIs R. In some embodiments, ReIs H, CH3、Bn、COCF3Non-limiting example internucleotide linkages or sugars are or comprise a component of any of N-methanocarba (N-methanocarba), C3-amide, methylal, thioacetal, MMI, PMO (phosphorodiamidate-linked morpholino), PNA (peptide nucleic acid), LNA, cMEBNA, cEt BNA, α -L-NA or related analogs, HNA, Me-ANA, MOE-ANA, Ara-FHNA, R-6 '-Me-FHNNA, S-6' -Me-FHNA, ENA or C-ANA. in some embodiments, non-limiting example internucleotide linkages or sugars are or comprise a component of any of those described in Allerson et al, cheJ]48: 901-4; BMCL 201121: 1122; BMCL 201121: 588; BMCL 201222: 296; chattopadhyaya et al 2007 J.am.chem.Soc. [ journal of the American chemical society]129: 8362; chem.bio.chem. [ chemical and biochemical]201314: 58; curr. prot. nucleic acids Chem. [ current scheme of nucleic acid chemistry]20111.24.1, respectively; egli et al 2011j.am.chem.soc. [ journal of the american chemical society]133: 16642; hendrix et al 1997 chem.Eur.J. [ European journal of chemistry]3: 110; hyrup et al 19Med, chem, 96 bioorg, bio-organic chemistry and medicinal chemistry]4: 5; imanishi1997 Tet.Lett. [ tetrahedron communication ]]38: 8735; am chem.soc. [ journal of american chemical society]1994, 116, 3143; med, chem. [ journal of medicinal chemistry]200952: 10; chem. [ journal of organic chemistry ]]201075: 1589; jepsen et al 2004Oligo [ oligonucleotides]14: 130-146; jones et al j. org.chem. [ journal of organic chemistry]1993, 58, 2983; jung et al 2014 ACIIEE 53: 9893; kodama et al 2014 AGDS; koizumi 2003 BMC 11: 2211; koizumi et al 2003 Nuc. acids Res. [ nucleic acid research ]]12: 3267-3273; koshkin et al 1998Tetrahedron]54: 3607-; kumar et al 1998Bio o.Med.chem.Let. [ Bioorganic chemistry and medicinal chemistry communication]8: 2219-2222; lauritsen et al 2002chem]5: 530- > 531; lauritsen et al 2003 Bio.Med.chem.Lett. [ Bioorganic chemistry and medicinal chemistry communication]13: 253-256; lima et al 2012 Cell]150: 883-894; mesmaeker et al, Angew. chem., int.Ed. Engl. [ International edition of applied chemistry English ]]1994, 33, 226; migawa et al 2013org.Lett. [ organic chemistry Communication]15: 4316; acids [ molecular therapy and nucleic acids ]]20121: e 47; morita et al 2001 Nucl. acids Res. supp. [ supplement to nucleic acid research ]]1: 241-242; morita et al 2002Bio o.Med.chem.Lett. [ bio-organic chemistry and medicinal chemistry communication]12: 73-76; morita et al 2003 Bio.Med.chem.Lett. [ Bioorganic chemistry and medicinal chemistry communication]2211-2226; murray et al 2012 nucleic acids Res. [ nucleic acid research]40: 6135; nielsen et al 1997 chem.Soc.Rev. [ review of the chemical society]73; nielsen et al 1997J.chem.Soc.Perkins Transl [ J.Chemie Bojinsk edition]1: 3423-; obika et al 1997Tetrahedron Lett. [ Tetrahedron communication ]]38(50): 8735-8; obika et al 1998Tetrahedron Lett. [ Tetrahedron communication ]]39: 5401-5404; obika et al 2008 J.am.chem.Soc. [ journal of American chemical society]130: 4886; obika et al 2011 org]13: 6050; oestergaard et al 2014 JOC 79: 8877; pallan et al 2012 Biochem]51: 7; pallan et al 2012 chem]48: 8195-; petersen et al 2003 TRENDSBIOTech [ trends in biotechnology]21: 74-81; prakash et al 2010J. MeChem. [ journal of medicinal chemistry ]]53: 1636; prakash et al 2015 Nucl acids Res. [ nucleic acid research]43: 2993-3011; prakash et al 2016bioorg.Med.chem.Lett. [ Bioorganic chemistry and medicinal chemistry communication]26: 2817-2820; rajwanshi et al 1999chem]1395-1396; schultz et al 1996 Nucleic Acids Res. [ Nucleic acid research ]]24: 2966; seth et al, 2008 nuclear. acid sym.ser, [ nucleic acid topic discussion society series]52: 553; seth et al 2009 J.Med.chem. [ journal of medicinal chemistry]52: 10-13; seth et al 2010 J.am.chem.Soc. [ journal of the American chemical society]132: 14942, respectively; seth et al 2010j.med.chem. [ journal of medicinal chemistry]53: 8309-8318; seth et al 2010J. org.chem. [ journal of organic chemistry]75: 1569-1581; seth et al 2011 BMCL 21: 4690; seth et al 2012 Bio o.Med.chem.Lett. [ bio-organic chemistry and medicinal chemistry communication]22: 296-; seth et al 2012mol, ther-Nuc acids [ molecular therapies and nucleic acids ]]1, e 47; seth et al, nucleic acids Symposium Series](2008) 52(1), 553-; singh et al 1998chem]1247-1248; singh et al 1998J. org.chem. [ J. org. chem. ]]63: 10035-39; singh et al 1998J. org.chem. [ J. org. chem. ]]63: 6078-6079; comm.2003 comm. [ chemical communication ]]2130-2131; acids Res [ nucleic acid research ]2010 nucleic acids et al]38: 7100; swayze et al 2007 Nucl. acids Res. [ nucleic acid research]35: 687; ts' o et al, Ann.N.Y.Acad.Sci. [ Proc. Natl. Acad. Sci. [ Proc. Nuch. Acad. Sci. ]]1988, 507, 220; van amerschot et al, 1995 angel]34: 1338; vasseur et al, j.am.chem.soc. [ journal of the american chemical society]1992, 114, 4006; WO 20070900071; WO 2016/079181; US6,326,199; US6,066,500; and US6,440,739; their respective base and sugar modifications are incorporated herein by reference.
In some embodiments, the C9orf72 oligonucleotide may comprise any sugar described herein or known in the art. In some embodiments, a C9orf72 oligonucleotide may comprise a combination of any of the sugars described herein or known in the art with any other structural element or modification described herein, including but not limited to a base sequence or portion thereof, a base; an internucleotide linkage; stereochemistry or modes thereof; additional chemical moieties including, but not limited to, targeting moieties, and the like; a modification pattern of sugar, base, or internucleotide linkages; in the form thereof or any structural element; and/or any other structural element or modification described herein; and in some embodiments, the disclosure relates to multimers of any such oligonucleotides.
Biological applications
As described herein, the provided compositions and methods are capable of improving knock-down of RNA, including knock-down of C9orf72RNA transcripts. In some embodiments, the provided compositions and methods provide improved knockdown of C9orf72 transcripts (including but not limited to those comprising repeated amplifications) compared to a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.
In some embodiments, the C9orf72 oligonucleotide is capable of preferentially reducing the expression, level, and/or activity (knockdown) of a mutant or repeat-containing amplified C9orf72 gene or gene product (e.g., a C9orf72 gene or gene product comprising a hexanucleotide repeat amplification) relative to a wild-type or repeat-free amplified C9orf72 gene or gene product (e.g., a C9orf72 gene or gene product without a hexanucleotide repeat amplification).
For example, preferential knockdown of C9orf72 oligonucleotides containing repeated amplifications is illustrated in fig. 4A and 4B. The C9orf72 oligonucleotides WV-3688, WV-6408, WV-7658, WV-7659, WV-8011, and WV-8012 are all capable of preferentially reducing the level of C9orf72RNA transcripts containing repeat amplification relative to the level of C9orf72RNA transcripts without repeat amplification (e.g., total transcripts, most of which are normal transcripts not containing repeat amplification).
WV-3688, WV-6408, WV-7658, WV-7659, WV-8011 and WV-8012 all have the nucleotide sequence CCUCACTCACCCACTCGCCA (WV-3688) or CCTCACTCACCCACTCGCCA (the rest), and have the following sequences: SC 5 sme Sm SC, m5Ceo teom5Ceo aoo C SC, m5Ceo se o SC. Total transcripts included normal (healthy, without repeat amplification) and mutant (pathological, including repeat amplification) V2, V3, and V1. The various transcripts are illustrated in FIG. 1. It was reported that V1 was transcribed at very low levels (about 1% of total C9orf72 transcripts) and did not significantly increase the level of transcripts comprising the hexanucleotide repeat amplification or increase the level of transcripts detected in the V3 transcript assay.
V1, V2 and V3 are naturally occurring pre-mRNA variants of the C9orf72 transcript produced by alternative pre-mRNA splicing. DeJesus-Hemandez et al 2011. In variants 1 and 3, the amplified GGGGCC repeats are located in an intron between two alternatively spliced exons, whereas in variant 2, the repeats are located in the promoter region and thus are not present in the transcript. V1 is the C9orf72 variant 1 transcript, which represents the shortest transcript and encodes the shorter C9orf72 protein (isoform b), see NM — 145005.5. V2 is a C9orf72 variant 2 transcript that differs in the 5 'UTR and 3' coding region and UTR compared to variant 1. The resulting C9orf72 protein (isoform a) was longer compared to isoform 1. Variants 2 and 3 encode the same C9orf72 protein; see NM _ 018325.3. V3 is the C9orf72 variant 3 transcript, which differs in the 5 'UTR and 3' coding region and UTR compared to variant 1. The resulting C9orf72 protein (isoform a) was longer compared to isoform 1; variants 2 and 3 encode the same protein, see NM — 001256054.1. Transcript variants 1 and 3 were predicted to encode a 481 amino acid long protein encoded by exons 2-11 of C9ORF72 (NP-060795.1; isoform a); while variant 2 is predicted to encode the shorter 222 amino acid protein encoded by exons 2-5 (NP-659442.2; isoform b). It should be noted that, according to some reports, the V1, V2, and V3 transcripts were not equally abundant; it was reported that V2 is the major transcript, representing 90% of the total transcripts, V3 representing 9% and V1 representing 1%. Thus, without being bound by any particular theory, the present disclosure suggests that the total transcript reduction mediated by some C9orf72 oligonucleotides includes the presentation of knockdown of transcripts containing repeat amplifications. The data show that many C9orf72 oligonucleotides are thus able to modulate the preferred knock-down of C9orf72 transcripts containing repeat amplification versus C9orf72 transcripts without repeat amplification. For example, WV-6408 implements repeat-related transcripts (V3): 80% of total (mostly normal) C9 mRNA: 35% knockdown. WV-3537 and WV-3174 are also capable of modulating some preferred knockdown of transcripts containing repeat amplifications. In contrast, SEQ ID NOs: 0553 and SEQ ID NO of WO 2016168592: 0057 complementary sequences of C9orf72 oligonucleotides WV-3662 and WV-3536 were unable to modulate the preferred knock-down of C9orf72 transcripts containing repeat amplification relative to C9orf72 transcripts not containing repeat amplification (FIGS. 4A and 4B).
In these experiments, patient-derived ALS neurons (detailed in example 9) were used for screening. The negative control oligonucleotide WV-2376 did not target C9orf 72. The control oligonucleotide WV-3542 is described in Table 1A. In FIGS. 4C and 4D, the oligonucleotides were tested at 1. mu.M and 10. mu.M
Fig. 5 and 6 present example data demonstrating the preferred knockdown in vivo ability of C9orf72 oligonucleotide to modulate C9-BAC mouse spinal cord and cortex containing repeatedly amplified C9orf72 transcripts, respectively. The presented data are those of: WV-6408, WV-8009, WV-8010, WV-8011 and WV-8012. FIGS. 5A and 6A show knockdown of total transcripts (including transcripts with and without repeat amplification). FIGS. 5B and 6B show knock-down of V3 (containing repeat amplified) transcripts. FIGS. 5C and 6C show knock-down of intron/AS transcripts (probes amplified 3' to repeat transcripts using targeting regions, including sense and antisense transcripts of intron regions). Additional experimental details are provided in example 9. Additional information relating to preferred knockdown of C9orf72 transcripts containing repeat amplifications is presented herein.
In some embodiments, the C9orf72 oligonucleotide may preferably knock down or reduce the expression, level, and/or activity of mutated (e.g., containing repeat amplified) V3C9orf72 transcripts relative to total C9orf72 transcripts.
In some embodiments, the C9orf72 oligonucleotide is capable of modulating a decrease in expression, activity, and/or level of a DPR protein translated from the repeat amplification.
In some embodiments, the C9orf72 oligonucleotide is capable of modulating expression, activity, and/or a decrease in the level of a C9orf72 gene product. In some embodiments, the C9orf72 gene product is a protein, such as a dipeptide repeat (DPR) protein. In some embodiments, DPRs can be made by RAN translation of any of the six reading frames containing repeated C9orf72 transcripts. In some embodiments, the dipeptide repeat protein is produced via RNA (repeat-related and ATG-independent translation) of either the sense or antisense strand of the hexanucleotide repeat region. DPR proteins are described, for example, in Zu et al 2011proc. natl. acad. sci. usa [ journal of the american academy of sciences ] 108: 260-265; zu et al proc.natl.acad.sci.us a. [ proceedings of the american academy of sciences ]2013 Dec 17; 110(51): e4968-77; Lopez-Gonzalez et al, 2016, Neuron 92, 1-9; may et al Acta Neuropathol [ neuropathology report ] (2014) 128: 485- & ltSUB & gt 503-; and Freibaum et al 2017 front. mol. neurosci [ molecular neuroscience frontier ]10, article 35; and Westergard et al, 2016, Cell Reports 17, 645-. In some embodiments, the C9orf72 dipeptide repeat is or comprises any one of: poly- (proline-alanine) (poly PA or) or poly- (alanine-proline) or (poly AP); poly- (proline-arginine) (polypr) or poly- (arginine-proline) (poly RP); or poly- (proline-glycine) (polypg) or poly- (glycine-proline) (polygp). It was reported that polyGA is well expressed in C9orf72 brain, followed by polyGP and polyGR, whereas polyPA and polyPR resulting from translation of antisense transcript are rare. It is reported how polyga and another DPR species are transported between cells and DPR uptake affects recipient cells. Zhou et al detected that all hydrophobic DPR species were cell-to-cell transported and showed that polyga increased repeat RNA levels and DPR expression, confirming that DPR transport could trigger a vicious cycle; treatment of cells with anti-GA antibodies reduced intracellular aggregation of DPR. Zhou et al 2017 EMBO mol.med. [ EMBO molecular medicine ]9 (5): 687-702. Chang et al reported that the glycine-alanine dipeptide repeat protein forms toxic amyloid with cell-to-cell transport properties. Chang et al 2016.j.biol.chem. [ journal of biochemistry ] 291: 4903-4911.
In some embodiments, the DPR protein is polygp. As a non-limiting example, the amino acid sequence of a DPR protein is or comprises any of: GAGAGAGAGAGAGAGAGAGAWSGRARGRARGGAAVAVPAPA-AAEAQAVASG, GPGPGPGPGPGPGPGPGPGRGRGGPGGGPGAGLRLRCLRPRRRRRRR-WRVGE, or GRGRGRGRGRGRGRGRGRGVVGAGPGAGPGRGCGCGACARGGGGAGG-GEWVSEEAASWRVAVWGSAAGKRRG (from sense boxes); or PRPRPRPRPR-PRPRPRPRPLARDS, GPGPGPGPGPGPGPGPGP, or PAPAPAPAPAPAPAPAPAPSARLLSS-RACYRLRLFPSLFSSG (from antisense boxes).
As shown in FIG. 10 and detailed in example 13, C9orf72 oligonucleotides WV-6408, WV-8009, WV-8010, WV-8011 and WV-8012 all reduced poly-GP (pGP, dipeptide repeat protein) levels in the hippocampus of C9-BAC mice. In addition, C9orf72 oligonucleotides WV-8549 and WV-8551 also reduced polyGP levels in mouse hippocampus (data not shown).
The C9orf72 gene product also includes foci comprising complexes (e.g., deleted introns) of C9orf72RNA or portions thereof bound by multiple RNA binding proteins. Lesions are described, for example, in Mori et al, 2013 ActaNeuropath [ neuropathology report ] 125: 413 and 423. In some embodiments, the C9orf72 oligonucleotide is capable of modulating the number of cells comprising the lesion, and/or a reduction in the number of lesions per cell.
As non-limiting example data, administration of C9orf72 oligonucleotides WV-7658 and WV-7659 in mice showed a 51.8% and 62.2% reduction in the number of foci per 100 motoneuron nuclei counts in the anterior horn of the spinal cord [ compared to PBS (negative control) ]; the cell number of more than 5 focuses/cells is respectively reduced by 58.3 percent and 70.9 percent; and the number of foci per 100 motor neurons decreased 49.1% and 55.0%, respectively.
Without wishing to be bound by any particular theory, the present disclosure suggests that significant knock-down of V3C9orf72 transcripts and/or decreased expression, activity, and/or levels of DPR proteins and/or decreased numbers of cells comprising lesions and/or numbers of lesions per cell may result in or be associated with significant inhibition of cytopathology, with the underlying biological rationale being that amplification of the hexanucleotide repeat allele results in longer residence times of the pre-spliced C9orf72 transcript and spliced intron, which makes it more susceptible to the introns of the targeting oligonucleotide. Without wishing to be bound by any particular theory, the present disclosure suggests that about 50% knock-down of V3C9orf72 transcript may result in or correlate with about 90% inhibition of cytopathology.
The improvement mediated by the C9orf72 oligonucleotide may be an improvement in any desired biological function, including but not limited to treatment and/or prevention of a C9orf 72-related disorder or a symptom thereof. In some embodiments, the C9orf 72-related disorder is Amyotrophic Lateral Sclerosis (ALS), frontotemporal dementia (FTD), corticobasal degeneration syndrome (CBD), atypical parkinsonism, olivopontocerebellar degeneration (OPCD), Primary Lateral Sclerosis (PLS), progressive amyotrophic lateral sclerosis (PMA), pseudophenotypic Huntington's Disease (HD), Alzheimer's Disease (AD), bipolar disorder, schizophrenia, or other non-movement disorder. In some embodiments, the symptoms of the C9orf 72-related disorder are selected from: agitation, anxiety, blunted mood, altered food preferences, reduced energy and/or motivation, dementia, depression, dyspnea, dysphagia, dyspnea, distraction, muscle fasciation and/or spasm, impaired balance, impaired motor function, inappropriate social behavior, loss of mental capacity, loss of memory, mood swings, muscle twitching, muscle weakness, neglecting personal hygiene, repetitive or compulsive behavior, shortness of breath, slurred speech, gait instability, visual abnormalities, weakness of limbs.
In some embodiments, the symptom of the C9orf 72-related disorder is semantic dementia, impaired speech understanding, or difficulty using the correct or precise language. In some embodiments, the c9orf 72-related disorder or symptom thereof is corticobasal degeneration syndrome (CBD), tremor, lack of coordination, muscle stiffness and/or spasm, Progressive Supranuclear Palsy (PSP), walking and/or balance problems, frequent falls, muscle stiffness in the neck and/or upper body, loss of physiological function, and/or abnormal eye movement.
In some embodiments, the FTD is behavioral modification frontotemporal dementia (bvFTD). In some embodiments, the most significant initial symptoms are reported to be related to personality and behavior in bvFTD. In some embodiments, the c9orf72 oligonucleotide is capable of reducing the degree or rate at which an individual experiences disinhibition, which is exhibited as limits of personal relationships and social life are lost, as assessed according to methods well known in the art.
In some embodiments, the disclosure provides a method of treating a disease by administering a composition comprising a first plurality of oligonucleotides sharing a common base sequence comprising a common base sequence that is complementary to a target sequence in a target C9orf72 transcript,
the improvement comprises using as an oligonucleotide composition a stereocontrolled oligonucleotide composition characterized in that, when it is contacted with a C9orf72 transcript in an oligonucleotide or a knockdown system, RNase H-mediated knockdown of the C9orf72 transcript is improved relative to the knockdown observed under reference conditions selected from the group consisting of absence of said composition, presence of a reference composition, and combinations thereof.
Evaluation and testing of the efficacy of C9orf72 oligonucleotides
A wide variety of techniques and tools, including but not limited to those known in the art, can be used to evaluate and test the C9orf72 oligonucleotides.
In some embodiments, the assessment and testing of the efficacy of the C9orf72 oligonucleotides can be performed by quantifying a change or improvement in the level, activity, expression, allele-specific expression, and/or intracellular distribution of the C9orf72 target nucleic acid or the corresponding gene product following delivery of the C9orf72 oligonucleotide. In some embodiments, delivery may be via a transfection agent or not via a transfection agent (e.g., in vitro (gynstic)).
In some embodiments, the assessment and testing of the efficacy of the C9orf72 oligonucleotide can be performed by quantifying the level, activity, expression, and/or intracellular changes in the C9orf72 gene product (including but not limited to transcripts, DPR, or foci) following the introduction of the C9orf72 oligonucleotide. The C9orf72 gene product includes RNA produced from the C9orf72 gene or locus.
In some embodiments, the present disclosure provides a method of identifying and/or characterizing an oligonucleotide composition, the method comprising the steps of:
providing at least one composition comprising a first plurality of oligonucleotides; and is
Delivery is assessed relative to a reference composition.
In some embodiments, the present disclosure provides a method of identifying and/or characterizing an oligonucleotide composition, the method comprising the steps of:
providing at least one composition comprising a first plurality of oligonucleotides; and is
Cellular uptake was assessed relative to a reference composition.
In some embodiments, the characteristics of the provided oligonucleotide compositions are compared to a reference oligonucleotide composition.
In some embodiments, the reference oligonucleotide composition is a sterically random oligonucleotide composition. In some embodiments, the reference oligonucleotide composition is a sterically random composition of oligonucleotides in which all internucleotide linkages are phosphorothioates. In some embodiments, the reference oligonucleotide composition is a DNA oligonucleotide composition having all phosphate linkages.
In some embodiments, the reference composition is a composition of oligonucleotides having the same base sequence and the same chemical modification. In some embodiments, the reference composition is a composition of oligonucleotides having the same base sequence and the same pattern of chemical modifications. In some embodiments, the reference composition is a chiral uncontrolled (or stereorandom) composition of oligonucleotides having the same base sequence and chemical modifications.
In some embodiments, the reference composition is a composition of oligonucleotides having the same base sequence but different chemical modifications (including, but not limited to, the chemical modifications described herein). In some embodiments, the reference composition is a composition of stereochemically and/or chemically modified oligonucleotides having the same base sequence but different patterns of internucleotide linkages and/or internucleotide linkages.
Various methods are known in the art for detecting the C9orf72 gene product, the expression, level, and/or activity of which can be altered following introduction and administration of the C9orf72 oligonucleotide. As non-limiting examples: c9orf72 transcripts and their knockdown can be quantified using qPCR, C9orf72 protein levels can be determined via western blotting, RNA foci by Fluorescence In Situ Hybridization (FISH), DPR by western blotting, ELISA or mass spectrometry. Commercially available C9orf72 antibodies include the anti-C9 orf72 antibody GT779 (1: 2000; GeneTex, Ill.W. (Irvine), Calif.). In addition, functional assays can be performed on Motor Neurons (MN) expressing wild-type and/or mutant C9orf72 by electrophysiology and NMJ formation.
In some embodiments, the assessment and testing of the efficacy of the C9orf72 oligonucleotide can be performed in vitro in a cell. In some embodiments, the cell is a cell expressing C9orf 72. In some embodiments, the cell is a SH-SY5Y (human neuroblastoma) cell engineered to express C9orf 72. In some embodiments, the cell is a SH-SY5Y cell engineered to express C9orf72, as described in WO 2016/167780. In some embodiments, the cell is a patient-derived cell, a patient-derived fibroblast, an iPSC, or an iPSN. In some embodiments, the cell is an iPSC-derived neuron or a motor neuron. Various cells suitable for testing C9orf72 oligonucleotides include patient-derived fibroblasts, iPSC, and iPSN and are described, for example, in Donelly et al, 2013 Neuron 80, 415-428; sareen et al 2013sci. trans. med. [ scientific transformation medicine ] 5: 208ra 149; swartz et al STEM CELLS TRANSLATIONAL EDICINE [ Stem cell transformation medicine ] 2016; 5: 1 to 12; and Almeida et al 2013 Acta neuropathohol. [ neuropathology report ] 126: 385-. In some embodiments, the cell is a BAC transgenic mouse-derived cell, including (but not limited to) a mouse embryonic fibroblast or cortical primordial neuron. In some embodiments, the assessment and testing involves a population of cells. In some embodiments, the population of cells is a mixed population of iCell neurons (also known as iineurons), iPS cell-derived human cortical neurons, which exhibit natural electrical and biochemical activity, available from cellular dynamics International, madison, wisconsin. Additional cells including spinal motor neurons, midbrain, dopaminergic neurons, glutamatergic neurons, gabaergic neurons, mixed cortical neurons, medium spiny gabaergic neurons, microalbumin-rich cortical gabaergic neurons, and V-layer cortical glutamatergic neurons are available from BrainXell, madison, wisconsin.
In some embodiments, the evaluation of C9orf72 oligonucleotides can be performed in animals. In some embodiments, the animal is a mouse. The C9orf72 mouse model and experimental procedures using the same are described in Hukema et al 2014 acta neuropath comm. [ neuropathology report communication ] 2: 166, a water-soluble polymer; ferguson et al 2016 j.anat. [ J.anat ] 226: 871-; lagier-tournene et al proc.natl.acad.sci.usa. [ proceedings of the american academy of sciences ]2013, 11 months and 19 days; 110(47): e4530-9; konpers et al, ann.neuron. [ neurological yearbook ] 2015; 78: 426-438; kramer et al 2016 Science 353: 708; liu et al, 2016, Neuron 90, 521-534; peters et al, 2015, Neuron 88, 902-; ActaNeuropathic Communications [ neuropathology report communication ] (2016) 4: 70 (c). The C9-BAC mouse model is described herein (see example 9).
In some embodiments, target nucleic acid levels can be quantified by any method known in the art, many of which can be accomplished using commercially available kits and materials, and which are well known and conventional in the art. Such methods include, for example, northern blot analysis, competitive Polymerase Chain Reaction (PCR), or real-time quantitative PCR. RNA analysis may be performed on total cellular RNA or poly (A) + mRNA. Probes and primers were designed to hybridize to C9orf72 nucleic acid. Methods for designing real-time PCR probes and primers are well known in the art.
In some embodiments, the assessment and testing of the efficacy of the C9orf72 oligonucleotide can be performed using luciferase assays. A non-limiting example of this analysis is detailed in example 3 below. In some embodiments, the luciferase assay employs constructs comprising a luciferase gene (or an effective portion thereof) linked to a portion of the sense C9orf72 transcript, such as nt 1-374 or nt 158-900 (both of which comprise a six nucleotide repeat amplification). In some embodiments, nt 1-374 includes exon 1a and an intron between exons 1a and 1 b. In some embodiments, the luciferase assay employs a construct comprising a luciferase gene (or an effective portion thereof) linked to a portion of an antisense C9orf72 transcript, such as nt 900 to 1 (which comprises a six nucleotide repeat amplification). In some embodiments, luciferase assays are performed in transfected COS-7 cells.
In some embodiments, the C9orf72 protein level can be assessed or quantified by any method known in the art, including but not limited to enzyme-linked immunosorbent assay (ELISA), western blot analysis (immunoblot), immunocytochemistry, Fluorescence Activated Cell Sorting (FACS), immunohistochemistry, immunoprecipitation, protein activity analysis (e.g., caspase activity analysis), and quantitative protein analysis. Antibodies suitable for the detection of mouse, rat, monkey, and human C9orf72 are commercially available; additional antibodies to C9orf72 can be generated via methods known in the art.
Assays for detecting levels of oligonucleotides or other nucleic acids are described herein (e.g., in example 14). By way of non-limiting example, this assay can be used to detect C9orf72 oligonucleotides or any other nucleic acid of interest, including nucleic acids or other oligonucleotides that do not target C9orf72 and nucleic acids.
The assessment and testing of the efficacy of the C9orf72 oligonucleotide can be performed by determining the change in the number of repeated RNA foci (or RNA foci) in the cell after delivery of the C9orf72 oligonucleotide. A repetitive RNA lesion is a structure formed when an RNA-sequestering RNA-binding protein comprising a hexanucleotide repeat is formed and is a measure and/or cause of RNA-mediated toxicity. In some embodiments, the RNA lesion may be a sense or antisense RNA lesion. When C9orf72 oligonucleotide is administered to an animal in vivo, the presence and/or number of RNA lesions can be determined or detected in the brain or a portion thereof of the animal (such as, but not limited to, the cerebellum, cerebral cortex, hippocampus, thalamus, medulla, or any other portion of the brain). The number of foci per cell (e.g., up to 5 or greater than 5) or an average thereof and/or the number of cells comprising a foci can be determined after delivery of the C9orf72 oligonucleotide. A decrease in any or all of these numbers indicates the efficacy of the C9orf72 oligonucleotide. RNA lesions can be detected by methods known in the art, including but not limited to Fluorescence In Situ Hybridization (FISH); a non-limiting example of FISH is presented in example 14.
The assessment and testing of the efficacy of the C9orf72 oligonucleotide can be performed in vitro by determining the change in single dose insufficiency in cells following delivery of the C9orf72 oligonucleotide. For example, a haploid dose deficit results when the hexanucleotide repeat RNA acts as a negative effector on C9orf72 transcription and/or expression of the C9orf72 gene, thereby reducing the total amount of C9orf72 transcripts or gene products. A single dose underreduction indicates the efficacy of the C9orf72 oligonucleotide.
In some embodiments, the C9orf72 oligonucleotide does not significantly reduce the expression, activity, and/or level of C9orf72 protein. In some embodiments, the C9orf72 oligonucleotide reduces the expression, activity, and/or level of C9orf72 repeat amplification or a gene product thereof, but does not significantly reduce the expression, activity, and/or level of C9orf72 protein.
In some embodiments, the C9orf72 oligonucleotide (a) reduces the expression, activity, and/or level of C9orf72 repeat amplification or a gene product thereof, and (b) does not reduce the expression, activity, and/or level of C9orf72 to a degree sufficient to cause a disease condition. Various disease conditions associated with inadequate production of C9orf72 include inappropriate endosomal migration, robust immune phenotype characterized by bone marrow expansion, T cell activation, plasma cell increase, elevated autoantibodies, immune-mediated glomerulonephropathy, and/or autoimmune responses, as described, for example, in Farg et al 2014 Human mol. 3579-3595; and Atanasio et al Sci Rep.2016, 3 months and 16 days; 6: 23204. doi: 10.1038/srep 23204.
The assessment and testing of the efficacy of the C9orf72 oligonucleotide can be performed in vivo. In some embodiments, the C9orf72 oligonucleotide can be evaluated and/or tested in an animal. In some embodiments, C9orf72 oligonucleotides can be evaluated and/or tested in humans and/or other animals to modulate the alteration or amelioration of levels, activity, expression, allele-specific expression, and/or intracellular distribution and/or to prevent, treat, alleviate, or slow the progression of at least one symptom of a C9orf 72-related disorder or a C9orf 72-related disorder. In some embodiments, such in vivo assessments and/or tests may determine phenotypic changes, such as improved motor function and respiration, following the introduction of C9orf72 oligonucleotides. In some embodiments, motor function may be measured by determining changes in any of a variety of tests known in the art, including: balance poles, grip strength, hind limb footprint test (e.g., in animals), open field performance, pole climbing, and swing poles. In some embodiments, respiration can be measured by determining changes in any of a variety of tests known in the art, including: compliance measurements, resistance to invasion, and whole body plethysmography.
In some embodiments, testing for the efficacy of the C9orf72 oligonucleotide is accomplished by contacting motor neuron cells from an individual with a neurological disease with the C9orf72 oligonucleotide and determining whether the motor neuron cells are degenerating. If the motor neuron cells do not degenerate, the C9orf72 oligonucleotide may be capable of reducing or inhibiting motor neuron degeneration. The motor neuron cell may be derived from a pluripotent stem cell. The pluripotent stem cells may have been reprogrammed from the cells of the subject. The cells from the subject can be, for example, somatic cells. For example, the somatic cell may be a fibroblast, lymphocyte, or keratinocyte. The assessment of whether motor neuron cells are degenerated may be based on comparison with a control. In some embodiments, the control level may be a predetermined or reference value that serves as a benchmark for evaluating measurements and/or visual results. The predetermined or reference value may be the level in a sample from an individual not suffering from a neurological disease (e.g., motor neuron cells) or from a sample from an individual suffering from a neurological disease but in which the motor neuron cells are not contacted with the C9orf72 oligonucleotide. The predetermined or reference value may be the level of a sample from a subject having a neurological disease. In any of these screening methods, cells from an individual with a neurological disease may comprise a (GGGGCC) n-hexanucleotide amplification in C9orf 72.
The efficacy of C9orf72 can also be tested in suitable test animals, such as Peters et al 2015 Neuron [ Neuron ]88(5), as a non-limiting example: 902-9; o' Rourke et al 2015 Neuron [ Neuron ]88 (5): 892-901; and Liu et al 2016 Neuron [ Neuron ]90 (3): 521-34. In some embodiments, the test animal is a C9-BAC mouse. The efficacy of C9orf72 was also tested in C9-BAC transgenic mice with 450 repeat amplifications, also described in Jiang et al 2016 Neuron 90, 1-16.
In some embodiments, the levels of various C9orf72 transcripts can be determined in test animals, but at the level of C9orf72 protein, RNA foci, and DPR (dipeptide repeat protein). The test can be performed on C9orf72 oligonucleotides and compared to reference oligonucleotides. Several C9orf72 oligonucleotides disclosed herein were able to reduce the percentage of cells comprising RNAi lesions and the average number of lesions per cell (data shown below and data not shown). Several of the C9orf72 oligonucleotides disclosed herein were able to reduce the level of DPR, such as polygp. As shown in FIG. 10, the C9orf72 oligonucleotides WV-6408, WV-8009, -8010, WV-8011 and WV-8012 all reduced poly GP (pGP, dipeptide repeat protein) levels in the hippocampal region of C9-BAC mice. In addition, C9orf72 oligonucleotides WV-8549 and WV-8551 also reduced polyGP levels in mouse hippocampus (data not shown).
In some embodiments, the c9orf72 oligonucleotide is capable of reducing the degree or rate of neurodegeneration caused by ALS, FTD, or other c9orf 72-related disorders. In some embodiments, in addition to an improvement, or at least a reduction, in the degree or rate of deterioration of any nervous system tissue in behavioral symptoms, the therapeutic effect of c9orf72 oligonucleotides in an individual or other animal can be monitored under a brain scan (e.g., CAT scan, functional MRI or PET scan, or other methods known in the art).
Various assays for analyzing C9orf72 oligonucleotides are described herein, e.g., in examples 9,13, and 14, and include inter alia reporter assays (luciferase assays), e.g., assays performed in ALS neurons and measuring, e.g., V3/intron expression, activity, and/or levels; measuring the stability; TLR9 assay; performing complementation determination; PD (pharmacodynamics) (C9-BAC, icv or intracerebroventricular injection), e.g., PD and/or efficacy tested in the C9orf72-BAC (C9-BAC) mouse model; in vivo methods, including but not limited to injection into the lateral ventricle or other regions of the central nervous system (including but not limited to the cortex and spinal cord) of a test animal (e.g., a mouse); analysis of the number of lesions and/or the number of cells comprising a lesion; PolyGP (or pGP or DPR assay).
In some embodiments, selection criteria are used to evaluate the data generated by the various assays and to select particularly desired C9orf72 oligonucleotides. In some embodiments, at least one selection criterion is used. In some embodiments, two or more selection criteria are used. In some embodiments, the selection criteria for luciferase assay (e.g., V3/intron knockdown) is at least partial knockdown of the V3 intron and/or at least partial knockdown of the intron transcript. In some embodiments, the selection criteria for luciferase assay (e.g., V3/intron knockdown) are 50% KD for the V3 intron (knockdown) and 50% KD for the intron transcript. In some embodiments, the selection criteria include a comparison of the IC' s50The measurement of (1). In some embodiments, the selection criteria comprise an IC of less than about 10nM, less than about 5nM, or less than about 1nM50. In some embodiments, the selection criterion for the stability assay is at least 50% stability by day 1 [ at least 50% of the level of oligonucleotide remains residual and/or detectable]. In some embodiments, the selection criteria for the stability assay is at least 50% stability on day 2. In some embodiments, the selection criteria for the stability assay is at least 50% stability on day 3. In some embodiments, the selection criteria for the stability assay is at least 50% stability on day 4. In some embodiments, the selection criteria for the stability assay is at least 50% stability on day 5. In some embodiments, the selection criteria for stability analysis is 80% [ at least 80% oligo ] at day 5Nucleotide residues]. In some embodiments, the selection criterion is at least partial knockdown in the number of lesions and/or the number of cells comprising a lesion. In some embodiments, the selection criterion is at least 50% KD (knockdown) in the number of lesions and/or the number of cells comprising lesions. In some embodiments, the selection criteria comprise a lack of activation in a TLR9 assay. In some embodiments, the selection criteria comprises a lack of activation in a complementary assay. In some embodiments, the selection criteria include knockdown in the lateral ventricles or other regions of the central nervous system (including but not limited to the cortex and spinal cord) of a test animal, such as a mouse. In some embodiments, the selection criteria include at least 50% knockdown in the lateral ventricles or other regions of the central nervous system (including, but not limited to, the cortex and spinal cord) of a test animal, such as a mouse. In some embodiments, the selection criteria comprise a knock-down in expression, activity, and/or level of the DPR protein. In some embodiments, the selection criteria comprise a knock-down in expression, activity, and/or level of the DPR protein. In some embodiments, the selection criteria comprise at least 50% knockdown in expression, activity, and/or level of DPR protein. In some embodiments, the selection criteria comprise at least 50% knockdown in expression, activity, and/or level of the DPR protein polygp.
Oligonucleotides that have been evaluated and tested for efficacy in knocking down C9orf72 have various uses, including administration for treating or preventing C9orf 72-related disorders or symptoms thereof.
Assays for detecting target nucleic acids of interest
In some embodiments, the disclosure relates to hybridization assays for detecting and/or quantifying a target nucleic acid (e.g., a target oligonucleotide), wherein the assays utilize a capture probe (which is at least partially complementary to the target nucleic acid) and a detection probe; wherein the detection probe or a complex comprising the capture probe, detection probe and target nucleic acid is capable of being detected. This assay can be used to detect C9orf72 oligonucleotides (e.g., in a tissue or fluid sample), or to detect any target nucleic acid (any target or sequence) in any sample. In some embodiments, the capture probe comprises a primary amine that is capable of reacting with the amino-reactive solid support, thereby immobilizing the probe on the solid support. In some embodiments, the amino-reactive solid support comprises maleic anhydride. The immobility of the probe can be performed using click chemistry methods using alkyne and azide moieties on the probe and solid support. For click chemistry methods, the alkyne or azide can be located, for example, at the 5 'or 3' end of the probe, and can optionally be attached via a linker. For click chemistry methods, the solid support comprises, for example, an alkyne or azide moiety. In some embodiments, the click chemistry method includes, as non-limiting examples, Kolb et al 2011 angelw. chem. int. ed. [ international edition of applied chemistry ] 40: the click chemistry method described in 2004-2021.
In some embodiments, a probe or complex capable of being detected directly or indirectly participates in generating a detectable signal. In some embodiments, the probe or complex is (a) capable of producing a detectable signal in the absence of another chemical component (as a non-limiting example, having a moiety capable of producing a detectable signal, such as a fluorescent dye or a radioactive label), or (b) comprises a ligand, label, or other component that is capable of producing a detectable signal upon binding to an appropriate second moiety. In some embodiments, probe or complex type (b) comprises a label, such as biotin, digoxigenin, a hapten, a ligand, etc., which can be conjugated to a suitable second chemical entity, such as an antibody, which, when conjugated to the label, is capable of generating a signal via radiolabel, chemiluminescence, dye, alkaline phosphatase signal, peroxidase signal, etc.
In some embodiments, the capture probe is immobilized on a solid support. In some embodiments, the capture probe is hybridized, bound, or ligated to the target nucleic acid, and the detection probe is also hybridized, bound, or ligated to the target nucleic acid, and the complex is capable of being detected. Many variations of hybridization assays are known in the art. In some embodiments, in a hybridization assay, the capture and detection probes are the same probe, and a single-stranded nuclease is used to degrade probes that do not bind (or do not fully bind) to the target nucleic acid.
In some embodiments, the disclosure relates to hybridization assays for detecting and/or quantifying a target nucleic acid (e.g., a target oligonucleotide), wherein a probe (e.g., a capture probe) is at least partially complementary to the target nucleic acid and comprises a primary amine, wherein the primary amine is capable of reacting with an amino-reactive solid support, thereby immobilizing the probe on the solid support. The primary amine can be, for example, at the 5 'or 3' end of the probe, and can optionally be attached via a linker. In some embodiments, the amino-reactive solid support comprises maleic anhydride.
The target oligonucleotide may be, for example, a C9orf72 oligonucleotide or an oligonucleotide that reaches any target of interest.
In some embodiments, the assay is a hybridization assay, a sandwich hybridization assay, a competitive hybridization assay, a double-ligation hybridization assay, a nuclease hybridization assay, or an electrochemical hybridization assay.
In some embodiments, the assay is a sandwich hybridization assay, wherein the capture probe is bound to a solid support and is capable of annealing to a portion of the target oligonucleotide; wherein the detection probe is capable of being detected and capable of annealing to another portion of the oligonucleotide; and wherein hybridization of both the capture probe and the detection probe to the target oligonucleotide produces a complex that is capable of being detected.
In some embodiments, the assay is a nuclease hybridization assay and the capture probe is a cleavage probe that is fully complementary to the target oligonucleotide, wherein the cleavage probe bound to the full length target oligonucleotide is capable of being detected; and wherein the cleavage probes of the shorter metabolites or degradation products that are free (not bound to the target oligonucleotide) or bound to the target oligonucleotide are degraded by the S1 nuclease treatment and thus do not generate a detectable signal.
In some embodiments, the assay is a hybrid ligation assay in which the capture probe is a template probe that is fully complementary to the target oligonucleotide and is intended to serve as a substrate for ligase-mediated ligation of the target oligonucleotide and the detection probe.
In some embodiments, the disclosure relates to methods of detecting and/or quantifying a target nucleic acid (e.g., a target oligonucleotide), e.g., in a sample (e.g., a tissue or a liquid), comprising the steps of: (1) providing a capture probe, wherein the capture probe is at least partially complementary to the target nucleic acid and comprises a primary amine, wherein the primary amine is capable of binding to the amino-reactive solid support, thereby immobilizing the probe on the solid support; (2) immobilizing a capture probe on a solid support; (3) providing a detection probe, wherein the detection probe is at least partially complementary to the target nucleic acid (e.g., a region of the target nucleic acid different from the region to which the capture probe binds) and is capable of generating a signal, directly or indirectly; wherein steps (2) and (3) may be performed in either order; (4) contacting the tissue or fluid with the capture probe and the detection probe under conditions suitable for hybridization of the probes to the target nucleic acid; (5) removing detection probes that do not hybridize to the target nucleic acid; and (6) detecting a signal generated directly or indirectly by the detection probe, wherein detection of the signal is indicative of detection and/or quantification of the target nucleic acid.
In some embodiments, the target oligonucleotide is a C9orf72 oligonucleotide. In some embodiments, the target oligonucleotide is not a C9orf72 oligonucleotide. In some embodiments, the target nucleic acid is an oligonucleotide, an antisense oligonucleotide, an siRNA agent, a double-stranded siRNA agent, a single-stranded siRNA agent, or a disease-associated nucleic acid (e.g., a gene or gene product that is expressed or overexpressed in a disease condition, such as a transcript that is substantially increased in a cancer cell, or a nucleic acid thereof that comprises a mutation associated with a disease or condition).
In some embodiments, the amino-reactive solid support comprises maleic anhydride.
Fig. 11A shows an example hybridization ELISA assay for measuring target oligonucleotide (e.g., ASO) levels in, for example, tissues and bodily fluids, including but not limited to animal biopsies. Fig. 11B shows an example chemistry for binding primary amine labeled capture probes to an amino reactive solid support (such as a plate containing maleic anhydride).
The target oligonucleotide is re-annealed to the detection probe and then combined with a capture probe that is attached to the amino-reactive plate via a primary amine label. Generating a double-hybridization (e.g., sandwich hybridization) between the capture probe, the detection probe, and the target oligonucleotide; a gap (not shown in fig. 11A) may be allowed between the capture probe and the detection probe, leaving a single stranded portion of the target oligonucleotide that does not bind to the capture or detection probe. The solid support (e.g., plate surface) comprises maleic anhydride (e.g., maleic anhydride-activated plate) that spontaneously reacts with the primary amine label on the end of the capture probe (e.g., at pH 8 to 9), thereby immobilizing the probe to the solid support. In some embodiments, the solid support is a plate, tube, filter, bead, polymer bead, gold, particle, well, or multi-well plate.
As non-limiting examples, the following conditions may be used:
coating: 500nM in 2.5% Na2CO3, pH 9.0, 50 ul/well, 37 ℃,2 hours
Sample/detection probe: 300nM detection probe as diluent, 4 deg.C, O/N
streptavidin-AP: 1: 2000 in PBST, 50 ul/well, room temperature, 1-2 hours
Substrate AttoPhos: read at 100 ul/well, room temperature, 5 min
For example: target nucleic acids are pre-annealed to detection probes and then combined with capture probes, attached to the plate via click chemistry methods using probes and alkyne (azide) moieties on a solid support. Generating a double-hybrid (e.g., sandwich hybridization) between the capture probe, the detection probe, and the target nucleic acid; the gap may be allowed between the capture probe and the detection probe, leaving a single stranded portion of the target oligonucleotide that does not bind to the capture or detection probe. The solid support (e.g., plate surface) comprises an alkyne (or azide) moiety that is reacted with the azide (or alkyne) moiety label on the end of the capture probe using click chemistry methods to immobilize the probe to the solid support. In some embodiments, the solid support is a plate, tube, filter, bead, polymer bead, gold, particle, well, or multi-well plate.
Non-limiting examples of analyses are provided below:
hybridization ELISA assay to measure target oligonucleotide levels in tissues (including animal biopsies):
the reverse complement of the target oligonucleotide can be divided into 2 segments, each represented by a capture or detection probe. The 5' sequence (of the target oligonucleotide) may be 5-15 nt; the 3' sequence may be 5-15 nt. However, the 5 ' -probe sequence (which hybridizes to the 3 ' portion of the target oligonucleotide) should not overlap with the 3 ' probe sequence when they are all hybridized to the target oligonucleotide. Gaps between the 5 '-probe and the 3' -probe are permissible. Each probe should have a melting temperature (Tm) of at least 25 ℃, preferably > 45 ℃, even more preferably > 50 ℃. To achieve high Tm, modified nucleotides, such as Locked Nucleic Acids (LNA) or Peptide Nucleic Acids (PNA), may be used. The other nucleotides in the probe may be DNA or RNA nucleotides or any other form of modified nucleotide, such as those with 2 ' -OMe, 2 ' -F or 2 ' -MOE modifications.
The 5 '-probe may also be labeled with a detection moiety having a linker at the 5' -position. This probe is a detection probe.
The 5 ' -probe (which hybridizes to the 3 ' -portion of the target oligonucleotide) can be labeled with a primary amine having a linker at the 5 ' -position. The probe is a capture probe. The linker is used to link the primary amine to the probe nucleotide. The linker may be a C6-linker, a C12-linker, PEG, TEG, or any nucleotide sequence unrelated to oligonucleotides such as oligo dT. The 5' primary amine with the linker can be placed during or after synthesis.
The 3 '-probe can also be labeled with a primary amine having a linker sequence at the 3' -position. The probe is a capture probe.
The 3 ' -probe (which hybridizes to the 5 ' portion of the target oligonucleotide) may be labeled with a detection moiety having a linker at the 3 ' -position. This probe is a detection probe. The detection part can be biotin, digoxin,Ligands (Promega), madison, wisconsin) or any other hapten. The detection moiety may also be a sulfonic acid-based label (mesoscale Diagnostics, rockville, maryland). The linker is used to link the detection moiety with the probe nucleotide. The linker may be a C6-linker, a C12-linker, PEG, TEG, or any linker unrelated to oligonucleotides (such as oligo dT)A nucleotide sequence. The 3' -primary amine with the linker can be placed during synthesis or post-synthesis.
Capture probes (with primary amines at the 5 'or 3' ends of the probes) can be immobilized on solid surfaces activated to react with the primary amines, such as maleic anhydride activated plates (Pierce; available from seimer feishel corporation (ThermoFisher), waltham, massachusetts) or N-oxysuccinimide (NOS) activated DNA-BIND plates (corning life Sciences, fig. cosbury, massachusetts). The plate may also be other types of plates activated for amine conjugation, such as MSD plates (Meso Scale Diagnostics, rockvell, maryland), the surface may be a solid support, such as beads, gold particles, carboxylated polystyrene microparticles (MagPlex microspheres, Luminex Corporation; purchased from sefmeisel Corporation (Thermo Fisher), waltham, massachusetts) or donnao beads (Dynabeads) (semmel feishel science (Thermo Fisher Scientific), waltham, massachusetts), so that flow-based analytical platforms, such as flow-based fluorescence detection or bead-array platforms (BD-cytological bead array-CBA, BD Biosciences (Biosciences), jossel, california), may be used.
A biological sample containing the target oligonucleotide, such as a tissue lysate or liquid biological fluid (plasma, blood, serum, CSF, urine or other tissue or fluid), is mixed with the detection probes at the appropriate concentration of oligonucleotide and detection probes, followed by heat modification of the capture probe-coated surface (plate or microparticles) to promote sequence-specific hybridization in an appropriate hybridization buffer for a period of time (hybridization) at room temperature or 4 ℃. Excess detection probes are removed by washing the surface (plate or bead). The surface is then incubated with a reagent that recognizes the detection moiety, such as avidin/streptavidin for biotin, an antibody or hapten against DIG, or a HaloTag against its ligand.
The detection reagent is typically labeled with an enzyme, such as horseradish peroxidase (HRP) or Alkaline Phosphatase (AP), or a fluorophore or a sulfonate group. After extensive washing, the enzyme-labeled detection reagent is detected by adding a corresponding substrate, such as TMB for HRP or AttoPhos for AP, and each plate is read by a plate reader in either absorbance mode or fluorescence mode (fluorogenic substrate). In some embodiments, the label comprises fluorescein, B-phycoerythrin, rhodamine, a cyanine dye, allophycocyanin, or a variant or derivative thereof.
Fluorophore-labeled detection reagents can be used in flow-based detection platforms, such as flow-fluorescent detection or bead array platforms.
The sulfonic acid group-labeled detection reagent can be directly read by an MSD reader (mesoscale discovery).
The amount of oligonucleotide can be calculated using a standard curve of serial dilutions of the test article performed in the same assay.
Another non-limiting example of a hybridization assay is provided in example 14.
Various assays for the utility of oligonucleotides (including but not limited to C9orf72 oligonucleotides) are described herein and/or known in the art.
Administration of the provided oligonucleotides and compositions thereof
In some embodiments, provided oligonucleotides are capable of directing a decrease in the expression and/or level of a target gene or gene product thereof.
In some embodiments, the target gene is C9orf72 comprising a hexanucleotide repeat amplification.
In some embodiments, the provided oligonucleotide compositions are administered at a dose and/or frequency that is lower than that of an otherwise similar reference oligonucleotide composition having a similar effect in improving knockdown of a target (including, as a non-limiting example, a C9orf72 transcript). In some embodiments, the stereocontrolled oligonucleotide composition is administered at a dose and/or frequency that is lower than that of an otherwise similar stereorandom reference oligonucleotide composition having a similar effect in improving knockdown of a target C9orf72 transcript.
In some embodiments, the disclosure contemplates that the properties (e.g., improved knockdown activity, etc.) of oligonucleotides and compositions thereof can be optimized by chemical modification and/or stereochemistry. In some embodiments, the disclosure provides methods for optimizing oligonucleotide properties via chemical modification and stereochemistry.
In some embodiments, the disclosure provides a method of administering an oligonucleotide composition comprising a first plurality of oligonucleotides and having a common nucleotide sequence, the improvement comprising:
administering an oligonucleotide comprising a first plurality of oligonucleotides, characterized in that delivery is improved relative to a reference oligonucleotide composition having the same common nucleotide sequence.
In some embodiments, the provided C9orf72 oligonucleotides, compositions, and methods provide improved delivery. In some embodiments, the provided oligonucleotides, compositions, and methods provide improved cytoplasmic delivery. In some embodiments, the improved delivery is into a cell population. In some embodiments, the improved delivery is into a tissue. In some embodiments, the improved delivery is into an organ. In some embodiments, the improved delivery is into the central nervous system or a portion thereof (e.g., CNS). In some embodiments, the improved delivery is into an organism. Example structural elements (e.g., chemical modifications, stereochemistry, combinations thereof, and the like), oligonucleotides, compositions, and methods that provide improved delivery are detailed in the present invention.
Various dosing regimens may be employed to administer the provided chirally controlled oligonucleotide compositions. In some embodiments, multiple unit doses are administered at intervals. In some embodiments, a given composition has a recommended dosing regimen, which may involve one or more administrations. In some embodiments, the dosing regimen comprises multiple administrations, each of which are separated from each other by a period of the same length; in some embodiments, the dosing regimen comprises multiple administrations and at least two different periods of time spaced apart from the individual administrations. In some embodiments, all administrations within a dosing regimen have the same unit dose. In some embodiments, different administrations within a dosing regimen have different amounts. In some embodiments, a dosing regimen comprises a first administration in a first administered amount followed by one or more additional administrations in a second administered amount different from the first administered amount. In some embodiments, a dosing regimen comprises a first administration of a first dose followed by another administration of a second (or subsequent) dose that is the same or different from the first (or another previous) dose. In some embodiments, the dosing regimen comprises administering at least one unit dose for at least one day. In some embodiments, a dosing regimen comprises administering more than one dose over a period of at least one day, and sometimes more than one day. In some embodiments, the dosing regimen comprises administering multiple doses over a period of at least one week. In some embodiments, the period of time is at least 2, 3, 4,5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2324, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more (e.g., about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more) weeks. In some embodiments, the dosing regimen comprises administering one dose per week for more than one week. In some embodiments, the dosing regimen comprises administering one dose weekly for 2, 3, 4,5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more (e.g., about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more) weeks. In some embodiments, the dosing regimen comprises administering one dose every two weeks for a period of more than two weeks. In some embodiments, the dosing regimen comprises administering one dose every two weeks for a2, 3, 4,5, 6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more (e.g., about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more) week period. In some embodiments, the dosing regimen comprises administering one dose per month for one month. In some embodiments, the dosing regimen comprises administering one dose per month for more than one month. In some embodiments, the dosing regimen comprises administering one dose per month for 2, 3, 4,5, 6,7, 8,9, 10, 11, 12 or more months. In some embodiments, the dosing regimen comprises administering one dose per week for about 10 weeks. In some embodiments, the dosing regimen comprises administering one dose per week for about 20 weeks. In some embodiments, the dosing regimen comprises administering one dose per week for about 30 weeks. In some embodiments, the dosing regimen comprises administering one dose per week for 26 weeks. In some embodiments, the oligonucleotides are administered according to a dosing regimen that is different from the dosing regimen for the same sequence of achiral controlled (e.g., stereorandom) oligonucleotide compositions and/or the dosing regimen for different chirality controlled oligonucleotide compositions of the same sequence. In some embodiments, the oligonucleotide is administered according to a dosing regimen that is reduced compared to a dosing regimen of an achiral controlled (e.g., stereorandom) oligonucleotide composition of the same sequence, which achieves a lower level of total exposure within a given unit time, involves one or more lower unit doses, and/or includes a fewer number of doses within a given unit time. In some embodiments, the oligonucleotide is administered according to a dosing regimen that is extended for a longer period of time as compared to a dosing regimen of an achiral controlled (e.g., stereorandom) oligonucleotide composition of the same sequence. Without wishing to be bound by theory, applicants note that in some embodiments, shorter dosing regimens and/or longer time periods between administrations may be dictated by the improved stability, bioavailability and/or efficacy of the chirally controlled oligonucleotide composition. In some embodiments, the oligonucleotide has a longer dosing regimen than a corresponding achiral controlled oligonucleotide composition. In some embodiments, the oligonucleotide has a shorter time period between at least two administrations as compared to a corresponding achiral controlled oligonucleotide composition. Without wishing to be bound by theory, applicants note that, in some embodiments, longer dosing regimens and/or shorter time periods between doses may be attributed to improved safety of the chirally controlled oligonucleotide compositions.
In some embodiments, with improved delivery (and other characteristics), provided compositions can be administered at lower doses and/or with lower frequency to achieve a biological effect, e.g., clinical efficacy.
A single administration may contain various amounts of the oligonucleotide. In some embodiments, a single administration may contain various amounts of one type of chirally controlled oligonucleotide as appropriate for the application. In some embodiments, a single administration contains about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, or more (e.g., about 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more) mg of one type of chirally controlled oligonucleotide. In some embodiments, a single administration contains about 1mg of one type of chirally controlled oligonucleotide. In some embodiments, a single administration contains about 5mg of one type of chirally controlled oligonucleotide. In some embodiments, a single administration contains about 10mg of one type of chirally controlled oligonucleotide. In some embodiments, a single administration contains about 15mg of one type of chirally controlled oligonucleotide. In some embodiments, a single administration contains about 20mg of one type of chirally controlled oligonucleotide. In some embodiments, a single administration contains about 50mg of one type of chirally controlled oligonucleotide. In some embodiments, a single administration contains about 100mg of one type of chirally controlled oligonucleotide. In some embodiments, a single administration contains about 150mg of one type of chirally controlled oligonucleotide. In some embodiments, a single administration contains about 200mg of one type of chirally controlled oligonucleotide. In some embodiments, a single administration contains about 250mg of one type of chirally controlled oligonucleotide. In some embodiments, a single administration contains about 300mg of one type of chirally controlled oligonucleotide. In some embodiments, the chirally controlled oligonucleotide is administered in a lower amount than the achiral controlled oligonucleotide in a single dose and/or in a total dose. In some embodiments, due to improved efficacy, the chirally controlled oligonucleotide is administered in a lower amount in a single dose and/or in a total dose than the achiral controlled oligonucleotide. In some embodiments, the chirally controlled oligonucleotide is administered in a higher amount in a single dose and/or in a total dose than the achiral controlled oligonucleotide. In some embodiments, due to improved safety, the chirally controlled oligonucleotide is administered in a higher amount in a single dose and/or in a total dose than the achiral controlled oligonucleotide.
Treatment of C9orf72 related disorders or symptoms thereof
In some embodiments, the provided oligonucleotides are capable of directing a decrease in the expression, level, and/or activity of a C9orf72 target gene or gene product thereof. In some embodiments, a C9orf 72-related disorder is a condition that is related to, causes, and/or is associated with: abnormal or excessive activity, level and/or expression of the C9orf72 gene or gene product thereof, deleterious mutations, or abnormal tissue distribution, or intercellular or intracellular distribution. In some embodiments, the C9orf 72-related disorder is Amyotrophic Lateral Sclerosis (ALS), frontotemporal dementia (FTD), corticobasal degeneration syndrome (CBD), atypical parkinsonism, olivopontocerebellar degeneration (OPCD), Primary Lateral Sclerosis (PLS), progressive amyotrophic lateral sclerosis (PMA), pseudophenotypic Huntington's Disease (HD), Alzheimer's Disease (AD), bipolar disorder, schizophrenia, or other non-movement disorder. Symptoms of C9orf 72-related disorders include those described herein and known in the art.
Without wishing to be bound by any particular theory or terminology, the present invention indicates that as the understanding of C9orf 72-related diseases progresses, the exact hallmarks of various C9orf 72-related diseases also progress according to the report. In some embodiments, the C9orf72 oligonucleotide is useful for reducing the level of a mutant allele of C9orf72 that contains a hexanucleotide repeat (at the protein and/or mRNA level), and/or reducing the level of a dipeptide repeat protein produced from a mutant C9orf72 mRNA that contains a hexanucleotide repeat, wherein the oligonucleotide is useful for treating a C9orf72 associated disease.
In some embodiments, the c9orf 72-related obstacle is FTD. In some embodiments, FTD is an abbreviation for frontotemporal dementia or frontotemporal degenerative disorder. In some embodiments, frontotemporal lobar degeneration (FTD) is a disease process affecting the frontal and temporal lobes of the brain. It causes a set of obstacles characterized by behavior, personality, language, and/or action changes. Clinical diagnosis of FTD includes any one or more of the following: behavioral variant ftd (bvftd), Primary Progressive Aphasia (PPA), and dyskinesia Progressive Supranuclear Palsy (PSP), and corticobasal degeneration (CBD). In some embodiments, a patient suffering from or susceptible to PPA, PSP, or CBD does not exhibit dementia or is identified as dementia. In some embodiments, frontotemporal dementia corresponds to or is characterized by symptoms of bvFTD.
The disclosure relates to methods of using the oligonucleotides disclosed herein, which are capable of targeting C9orf72 and are suitable for use in treating a C9orf 72-related disorder and/or in the manufacture of a therapeutic agent for a C9orf 72-related disorder. In some embodiments, the base sequence of the oligonucleotide may comprise or consist of a base sequence having a specified maximum number of mismatches with a specified base sequence.
In some embodiments, the disclosure relates to the use of a composition comprising a C9orf72 oligonucleotide in the manufacture of a medicament for treating a neurodegenerative disease.
In some embodiments, the disclosure relates to a method of treating or ameliorating a C9orf 72-related disorder in a patient, the method comprising the steps of: a therapeutically effective amount of the oligonucleotide is administered to C9orf72 of the patient.
In some embodiments, the disclosure relates to a method comprising administering to an animal a composition comprising a C9orf72 oligonucleotide.
In some embodiments, the animal is a subject, e.g., a human.
In some embodiments, a health care professional can identify or diagnose an individual or patient suitable for treatment of a C9orf 72-related disorder (such as administration of a C9orf72 oligonucleotide). The C9orf72 related disease is one of several neurological diseases. In some embodiments, an individual may be diagnosed as having a neurological disease by assessing one or more symptoms, such as motor neuron degeneration symptoms. In some embodiments, to diagnose neurological disease, a thorough neurological examination may be performed after a physical examination. In some embodiments, the neurological examination may assess motor and sensory skills, neurological function, hearing and speech, vision, coordination and balance, mental state, and emotional or behavioral changes. Non-limiting symptoms of diseases associated with neurological diseases may be: weakness of the arm, leg, foot or ankle; unclear speech; difficulty in lifting the forefoot and toes; weak or clumsy hands; muscle paralysis; muscle stiffness; involuntary shaking or writing movements (chorea); involuntary persistent muscle contractures (dystonia); bradykinesia; loss of spontaneous motility; weakened posture and balance; lack of flexibility; tingling and thorn on the body part; a shock sensation that follows head movements; twitching of the arms, shoulders and tongue; dysphagia; dyspnea; difficulty in chewing; partial or complete loss of vision; double vision; slow or abnormal eye movement; shaking; gait is unstable; fatigue; loss of memory; vertigo; difficulty in thinking or concentration; difficulty reading or writing; misjudging the spatial relationship; loss of direction; depression; anxiety; difficulty in decision making and judgment; loss of impulse control; difficulty in planning and performing familiar tasks; aggressiveness; dysphoria; social withdrawal; mood swings; dementia; a change in sleep habits; absentmindedness; the appetite changes.
In some embodiments, the composition prevents, treats, ameliorates, or slows the progression of at least one symptom of a C9orf 72-related disorder.
In some embodiments, the animal or human is afflicted with a symptom of a C9orf 72-related disorder.
In some embodiments, the disclosure relates to a method for introducing into a cell an oligonucleotide that reduces expression of C9orf72 gene, the method comprising: the cells are contacted with the oligonucleotide or the C9orf72 oligonucleotide.
In some embodiments, the disclosure relates to a method for reducing C9orf72 gene expression in a mammal in need thereof, the method comprising: administering to the mammal a nucleic acid-lipid particle comprising an oligonucleotide directed to C9orf 72.
In some embodiments, the disclosure relates to a method for delivering an oligonucleotide targeting C9orf72 gene expression in vivo, the method comprising: administering to the mammal an oligonucleotide against C9orf 72.
In some embodiments, the present disclosure relates to a method for treating and/or ameliorating one or more symptoms associated with a C9orf 72-related disorder in a mammal in need thereof, the method comprising: administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle comprising an oligonucleotide directed to C9orf 72.
In some embodiments, the disclosure relates to a method of inhibiting C9orf72 expression in a cell, the method comprising: (a) contacting the cell with an oligonucleotide against C9orf 72; and (b) maintaining the resulting cells in step (a) for a period of time sufficient to obtain degradation of the mRNA transcript of the C9orf72 gene, thereby inhibiting expression of the C9orf72 gene in the cells.
In some embodiments, C9orf72 expression is inhibited by at least 30%.
In some embodiments, the disclosure relates to a method of treating a disorder mediated by C9orf72 expression, comprising administering to a human in need of such treatment a therapeutically effective amount of an oligonucleotide against C9orf 72.
In some embodiments, the administration results in a reduction in expression, activity, and/or level of C9orf72 transcript containing repeat amplification or a gene product thereof.
In some embodiments, the disclosure relates to a method of treating a C9orf 72-related disorder.
In some embodiments, the present disclosure relates to a method comprising the steps of: providing a system comprising two or more different splice products having the same mRNA, wherein at least one splice product is disease-related and at least one splice product is non-disease-related; introducing an oligonucleotide into a system, wherein said oligonucleotide is complementary to a sequence that is present in at least one disease-associated splice product but not present in at least one non-disease-associated splice product, wherein said oligonucleotide is capable of reducing the expression, level and/or activity of said disease-associated splice product relative to the expression, level and/or activity of said non-disease-associated splice product.
In some embodiments of the method, the oligonucleotide is complementary to an intron-exon junction present on the disease-associated splice product, but is not complementary to an intron-exon junction present on the non-disease-associated splice product.
In some embodiments of the methods, the oligonucleotide comprises at least one chiral controlled internucleotide linkage.
In some embodiments of the methods, the oligonucleotide is a c9orf72 oligonucleotide and the system is an individual suffering from and/or susceptible to a c9orf 2-related disorder.
In some embodiments, a second therapeutic agent or method is administered to the individual.
In some embodiments, the c9orf72 oligonucleotide and one or more second therapeutic agents or methods are administered to the individual.
In some embodiments, the second therapeutic agent or method is capable of preventing, treating, ameliorating, or slowing the progression of a neurological disease.
In some embodiments, the second therapeutic agent or method is capable of preventing, treating, ameliorating, or slowing the progression of a C9orf 72-related disorder.
In some embodiments, a second therapeutic agent or method capable of preventing, treating, ameliorating, or slowing the progression of a neurological disease is selected from: modulators of endosomal and/or lysosomal trafficking, glutamate receptor inhibitors, PIKFYVE kinase inhibitors, and potassium channel activators.
In some embodiments, the second therapeutic agent or method comprises an antibody directed against a dipeptide repeat protein or an agent (e.g., an antibody or small molecule) that interferes with the substantial formation of RNA lesions or reduces the number of RNA lesions.
In some embodiments, the second therapeutic agent or method indirectly reduces the expression, activity, and/or level of C9orf72 by knocking down a gene or gene product thereof (as a non-limiting example) that increases the expression, activity, and/or level of C9orf 72. In some embodiments, the second therapeutic agent or method knockdown SUPT4H1 (human Spt4 ortholog), which knockdown reduces the production of sense and antisense C9orf72RNA lesions as well as DPR protein. Kramer et al 2016 Science 353: 708. in some embodiments, the second therapeutic agent or method is a nucleic acid, small molecule, gene therapy, or other agent or method described in the literature, including (as a non-limiting example) Mis et al Mol Neurobiol [ molecular neurobiology ]2017, month 8; 54(6): 4466-4476.
In some embodiments, the second therapeutic agent is physically conjugated to the C9orf72 oligonucleotide. In some embodiments, the C9orf72 oligonucleotide is physically conjugated to a second oligonucleotide that reduces (directly or indirectly) the expression, activity, and/or level of C9orf72 or is useful for treating symptoms of a C9orf 72-associated disorder. In some embodiments, the first C9orf72 oligonucleotide is physically conjugated to a second C9orf72 oligonucleotide, which may be the same or different from the first C9orf72 oligonucleotide, and may target a different or the same or overlapping sequence as the first C9orf72 oligonucleotide. In some embodiments, the C9orf72 oligonucleotide is conjugated to or co-administered with or incorporated into the same treatment regimen as the oligonucleotide that knockdown SUPT4H 1. In some embodiments, the C9orf72 oligonucleotide is conjugated or co-administered or incorporated with a second therapeutic agent in the same treatment regimen that improves the expression, activity, and/or level of another (non-C9 orf72) gene or gene product associated with a C9orf 72-related disorder (such as ALS or FTD), such as: SOD1, TARDBP, FUS/TLS, MAPT, TDP-43, SUPT4H1 or FUS/TLS.
In some embodiments, improving the expression, activity, and/or level of such a gene or gene product comprises, inter alia: reducing the expression, activity and/or level of such genes or gene products that are too high in a disease condition; increasing the expression, activity and/or level of such genes or gene products that are too low in a disease condition; and/or reducing the expression, activity and/or level of a mutant and/or disease-associated variant of such a gene or gene product. In some embodiments, the second therapeutic agent is an oligonucleotide. In some embodiments, the second therapeutic agent is an oligonucleotide physically conjugated to a C9orf72 oligonucleotide. In some embodiments, the second therapeutic agent comprises monomethyl fumarate (MMF), which is reported to activate Nrf2, and/or an omega-3 fatty acid. In some embodiments, the second therapeutic agent comprises monomethyl fumarate (MMF) and/or docosahexaenoic acid (DHA), an omega-3 fatty acid reported to inhibit NF- κ B. In some embodiments, the second therapeutic agent comprises a conjugate of monomethyl fumarate (MMF) and docosahexaenoic acid (DHA), an omega-3 fatty acid. In some embodiments, the second therapeutic agent is CAT-4001 (catalytically active Pharmaceuticals, cambridge, massachusetts, usa).
In some embodiments, the second therapeutic agent is capable of preventing, treating, ameliorating, or slowing the progression of a neurological disease, said second therapeutic agent selected from: modulators of endosomal and/or lysosomal trafficking, glutamate receptor inhibitors, PIKFYVE kinase inhibitors, and potassium channel activators are described in WO 2016/210372. In some embodiments, the potassium channel activator is retigabine. In some embodiments, the glutamate receptor is located on a Motor Neuron (MN) or a spinal cord motor neuron. In some embodiments, the glutamate receptor is NMDA, AMPA, or kainite. In some embodiments, the glutamate receptor inhibitor is AP5((2R) -amino-5-phosphorylpentanoic acid; (2R) -amino-5-phosphorylpentanoate), CNQX (6-cyano-7-nitroquinoxaline-2, 3-dione), or NBQX (2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo [ f ] quinoxaline-2, 3-dione).
In some embodiments, the second therapeutic agent is capable of reducing the expression, level, and/or activity of a gene (or gene product thereof) associated with a c9orf 72-related disorder, such as SOD1, TARDBP, FUS/TLS, MAPT, TDP-43, SUPT4H1, or FUS/TLS. In some embodiments, the second therapeutic agent is an agent that reduces the expression, level, and/or activity of a gene (or gene product thereof) associated with Amyotrophic Lateral Sclerosis (ALS) or frontotemporal dementia (FTD), such as SOD1, TARDBP, FUS/TLS, MAPT, TDP-43, SUPT4H1, or FUS/TLS. In some embodiments, the second therapeutic agent is capable of controlling excessive oxidative stress. In some embodiments, the second therapeutic agent is (edaravone). In some embodiments, the second therapeutic agent is ursodeoxycholic acid (UDCA). In some embodiments, the second therapeutic agent is capable of affecting neurons, which reduces neuronal activity via blocking Na + entry into the neuron and blocking the release of chemicals that cause motor neuron activation. In some embodiments, the second therapeutic agent is riluzole. In some embodiments, the second therapeutic agent is capable of: relieving fatigue, relieving muscle cramps, controlling spasms, and/or reducing excess saliva and sputum. In some embodiments, the second therapeutic agent is capable of reducing pain. In some embodiments, the second therapeutic agent is a non-steroid and/or an anti-inflammatory drug and/or an opioid. In some embodiments, the second therapeutic agent is capable of alleviating depression, sleep disorders, dysphagia, spasticity, salivary dysphagia, and/or constipation. In some embodiments, the second therapeutic agent is baclofen or diazepam. In some embodiments, the second therapeutic agent is or comprises trihexyphenidyl, amitriptyline, and/or glycopyrrolate. In some embodiments, the second therapeutic agent is a dsRNA or siRNA, the sequence of the strand of which comprises at least 15 contiguous nt of the sequence of any of the oligonucleotides disclosed herein.
Pharmaceutical composition
In some embodiments, the present disclosure provides pharmaceutical compositions comprising a provided compound (e.g., a provided oligonucleotide), or a pharmaceutically acceptable salt thereof, and a pharmaceutical carrier. In some embodiments, the oligonucleotide is a C9orf72 oligonucleotide.
When used as a therapeutic agent, the provided oligonucleotides or oligonucleotide compositions described herein are administered as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is suitable for administration of the oligonucleotide to an area of the body affected by a disorder, including but not limited to the central nervous system. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the provided oligonucleotide or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable inactive ingredient selected from a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable carrier.
In some embodiments, provided C9orf72 is conjugated to another chemical moiety suitable for delivery to the central nervous system, the chemical moiety selected from the group consisting of: glucose, GluNAc (N-acetylglucosamine), and anisamide, and molecules having any of the following structures:
the chemical moieties are described in more detail in examples 1 and 2.
In some embodiments, the additional chemical moiety conjugated to the oligonucleotide is capable of targeting the oligonucleotide to a cell in the nervous system.
In some embodiments, the additional chemical moiety conjugated to the provided oligonucleotide comprises anisamide or a derivative or analogue thereof and is capable of targeting the provided oligonucleotide to a cell expressing a particular receptor (e.g., a sigma 1 receptor).
In some embodiments, the provided oligonucleotides are formulated for administration to a body cell and/or tissue expressing its target.
In some embodiments, another chemical moiety conjugated to the C9orf72 oligonucleotide is capable of targeting the C9orf72 oligonucleotide to a cell in the nervous system.
In some embodiments, the other chemical moiety conjugated to the C9orf72 oligonucleotide comprises anisamide or a derivative or analog thereof, and is capable of targeting the C9orf72 oligonucleotide to cells expressing a particular receptor (such as a sigma 1 receptor).
In some embodiments, the provided C9orf72 oligonucleotides are formulated for administration to a body cell and/or tissue expressing C9orf 72. In some embodiments, such body cells and/or tissues are neurons or cells and/or tissues of the central nervous system. In some embodiments, the wide distribution of the oligonucleotides and compositions described herein within the central nervous system can be achieved using intraparenchymal, intrathecal, or intraventricular administration.
In some embodiments, the pharmaceutical composition is formulated for intravenous injection, oral administration, inhalation, nasal administration, topical administration, ocular administration, or otic administration. In some embodiments, the pharmaceutical composition is a tablet, pill, capsule, liquid, inhalant, nasal spray solution, suppository, suspension, gel, colloid, dispersion, suspension, solution, emulsion, ointment, lotion, eye drop, or ear drop.
In some embodiments, the present disclosure provides pharmaceutical compositions comprising a chirally controlled oligonucleotide or compositions thereof admixed with a pharmaceutically acceptable excipient. One skilled in the art will recognize that pharmaceutical compositions include pharmaceutically acceptable salts of the chirally controlled oligonucleotides described above, or combinations thereof.
A variety of supramolecular nanocarriers may be used to deliver nucleic acids. Exemplary nanocarriers include, but are not limited to, liposomes, cationic polymer complexes, and various polymers. Complexation of nucleic acids with various polycations is another approach for intracellular delivery; this includes the use of pegylated polycations, Polyvinylamine (PEI) complexes, cationic block copolymers, and dendrimers. Several cationic nanocarriers (including PEI and polyamide dendrimers) help to release the contents from the endosome. Other methods include the use of polymeric nanoparticles, microspheres, liposomes, dendrimers, biodegradable polymers, conjugates, prodrugs, inorganic colloids such as sulfur or iron, antibodies, grafts, biodegradable microspheres, osmotic controlled grafts, lipid nanoparticles, emulsions, oily solutions, aqueous solutions, biodegradable polymers, poly (lactic-co-glycolic acid), poly (lactic acid), liquid reservoirs, polymer micelles, quantum dots, and lipid complexes. In some embodiments, the oligonucleotide is conjugated to another molecule.
In addition to the example delivery strategies described herein, additional nucleic acid delivery strategies are known.
In therapeutic and/or diagnostic applications, the compounds of the present disclosure may be formulated for a variety of modes of administration, including systemic and local (local) administration. Techniques and formulations are commonly found in Remington, The science and Practice of Pharmacy (20 th edition, 2000).
The provided oligonucleotides and compositions thereof are effective over a wide dosage range. For example, in treating adults, dosages of about 0.01 to about 1000mg, about 0.5 to about 100mg, about 1 to about 50mg, and about 5 to about 100mg per day are examples of dosages that may be used. The exact dosage will depend upon the route of administration, the form of the compound administered, the subject to be treated, the weight of the subject to be treated, and the preferences and experience of the attending physician.
Pharmaceutically acceptable salts are generally well known to those of ordinary skill in the art and may include, for example, but are not limited to, acetate, benzenesulfonate (benzanesulfonate), benzenesulfonate (besylate), benzoate, bicarbonate, bitartrate, bromide, calcium ethylenediaminetetraacetate, taurate, carbonate, citrate, ethylenediaminetetraacetate, edisylate, propionate lauryl sulfate (estolate), phenolsulfoethylamine (esylate), fumarate, gluconate, glutamate, glycollylabdanate, hexylresorcinate (hexylorivate), hydrabamine (hydrabamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, methanesulfonate, mucate, naphthalenesulfonate, nitrate, citrate, tartrate, betaine, and acetate, Pamoate/embonate, pantothenate, phosphate/biphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Other pharmaceutically acceptable salts can be found, for example, in Remington, The Science and Practice of Pharmacy (20 th edition, 2000). Preferred pharmaceutically acceptable salts include, for example, acetate, benzoate, bromide, carbonate, citrate, gluconate, hydrobromide, hydrochloride, maleate, methanesulfonate, naphthalenesulfonate, pamoate (pamoate/embonate), phosphate, salicylate, succinate, sulfate or tartrate.
In some embodiments, the provided C9orf72 oligonucleotides are formulated in pharmaceutical compositions described in: U.S. application No. 61/774759; 61/918,175, 61/918,927 filed on 12/19/2013; 61/918,182, respectively; 61/918941, respectively; 62/025224, respectively; 62/046487, respectively; or International application No. PCT/US 04/042911, PCT/EP 2010/070412, or PCT/I B2014/059503.
Such agents may be formulated in liquid or solid dosage forms and administered systemically or locally, depending on the particular condition being treated. The agent may be delivered, for example, in a timed or sustained low release form, as known to those skilled in the art. Techniques for formulation and administration can be found in Remington, The Science and Practice of Pharmacy (20 th edition, 2000). Suitable routes may include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraarticular, intrasternal, intrasynovial, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections, or other modes of delivery.
For injections, the agents of the present disclosure may be formulated and diluted in aqueous solutions, such as in physiologically compatible buffers, e.g., Hank's solutions, Ringer's solutions, or physiological saline buffers. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
It is within the scope of the present disclosure to formulate the compounds disclosed herein for practicing the present disclosure into dosages suitable for systemic administration using a pharmaceutically acceptable inert carrier. By appropriate choice of carrier and appropriate manufacturing methods, the compositions of the present disclosure, particularly those formulated as solutions, may be administered parenterally, for example by intravenous injection.
The compounds can be readily formulated into dosages suitable for oral administration using pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the present disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a subject (e.g., a patient) to be treated.
For nasal or inhalation delivery, the agents of the present disclosure may also be formulated by methods known to those skilled in the art, and may include, for example, but are not limited to, examples of solubilizing, diluting, or dispersing substances (e.g., saline, preservatives (e.g., benzyl alcohol), absorption promoters, and fluorocarbons).
In certain embodiments, the oligonucleotides and compositions are delivered to the CNS. In certain embodiments, the oligonucleotides and compositions are delivered to the cerebrospinal fluid. In certain embodiments, the oligonucleotides and compositions are administered to the brain parenchyma. In certain embodiments, the oligonucleotides and compositions are delivered to the animal/subject by intrathecal or intracerebroventricular administration. The broad distribution of the oligonucleotides and compositions described herein within the central nervous system can be achieved by intraparenchymal administration, intrathecal administration, or intracerebroventricular administration.
In certain embodiments, parenteral administration is by injection, e.g., by syringe, pump, and the like. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly to a tissue, such as the striatum, caudate nucleus, cortex, hippocampus, and cerebellum.
In certain embodiments, the method of specifically localizing an agent (e.g., by bolus injection) reduces the median effective concentration (EC50) by 20, 25, 30, 35, 40, 45, or 50-fold. In certain embodiments, the agent is an antisense compound as further described herein. In certain embodiments, the target tissue is brain tissue. In certain embodiments, the target tissue is striatal tissue. In certain embodiments, it is desirable to reduce EC50 because this reduces the dose required to achieve a pharmacological result in a patient in need thereof.
In certain embodiments, the antisense oligonucleotide is delivered by injection or infusion once every month, every two months, every 90 days, every 3 months, every 6 months; twice a year or once a year.
Pharmaceutical compositions suitable for use in the present disclosure include compositions wherein the composition comprises an effective amount of the active ingredient to achieve its intended purpose. Determination of an effective amount is well within the ability of those skilled in the art, especially in light of the specific disclosure provided herein.
In addition to the active ingredient, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Formulations formulated for oral administration may be in the form of tablets, dragees, capsules or solutions.
Pharmaceutical formulations for oral use can be obtained by the following method: combining the active compound with solid excipients, optionally grinding the resulting mixture, and processing the mixture of granules, if desired after addition of suitable auxiliaries, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, for example maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose (CMC), and/or polyvinylpyrrolidone (PVP: Povidone). If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof (such as sodium alginate).
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbomer, polyethylene glycol (PEG), and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for identifying or characterizing different combinations of active compound doses.
Pharmaceutical preparations for oral use include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Plug-in capsules may contain the active ingredients in admixture with fillers (such as lactose), binders (such as starches) and/or lubricants (such as talc or magnesium stearate) and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols (PEGs). In addition, stabilizers may also be added.
The composition may be obtained by combining the active compound with a lipid. In some embodiments, the lipid is conjugated to an active compound. In some embodiments, the lipid is not conjugated to an active compound. In some embodiments, the lipid comprises C10-C40Linear saturated or partially unsaturated aliphatic chains. In some embodiments, the lipid comprises one or more C optionally1-4Aliphatic radical-substituted C10-C40In some embodiments, the lipid is selected from the group consisting of lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, α -linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), domoic acid (turbinaric acid), and dilinoleyl acidA vitamin; a PEG-lipid; uncharged lipids modified with one or more hydrophilic polymers; a phospholipid; phospholipids such as 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine; stealth lipids; a sterol; cholesterol; and a targeting lipid; and any other lipid described herein or reported in the art. In some embodiments, the composition comprises a lipid and a portion of another lipid capable of mediating at least one function of the other lipid. In some embodiments, the targeting compound or moiety is capable of targeting a compound (e.g., a composition comprising a lipid and an active compound) to a particular cell or tissue or subset of cells or tissues. In some embodiments, the targeting moiety is designed for cell-specific or tissue-specific expression using a specific target, receptor, protein, or other subcellular component. In some embodiments, the targeting moiety is a ligand (e.g., small molecule, antibody, peptide, protein, carbohydrate, aptamer, etc.) that targets the composition to a cell or tissue and/or binds to a target, receptor, protein, or other subcellular component.
Non-limiting illustrative lipids include lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, α -linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), domoic acid, and dilinoleyl.
As described in the present disclosure, lipid conjugation (e.g., to a fatty acid) can improve one or more properties of the oligonucleotide.
In some embodiments, the present disclosure relates to compositions and methods relating to the delivery of active compounds, wherein the compositions comprise an active compound, a lipid, in various embodiments relating to muscle cells or tissues, the lipid is selected from the group consisting of lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, α -linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), domoic acid, and dilinolene.
Depending on the particular disorder to be treated or prevented, additional therapeutic agents that are typically administered to treat or prevent the disorder can be administered with the C9orf oligonucleotides of the present disclosure.
In some embodiments, the second therapeutic agent administered with the first C9orf72 oligonucleotide is a different second C9orf72 oligonucleotide.
In some embodiments, the C9orf72 oligonucleotides disclosed herein are useful in methods for preventing and/or treating a C9orf 72-related disorder, or a symptom thereof, or for making medicaments for use in the methods.
Examples of the invention
Some examples of the provided techniques (compounds (oligonucleotides, reagents, etc.), compositions, methods (methods of preparation, methods of use, methods of evaluation, etc.)) are presented below.
Various techniques for preparing oligonucleotides and oligonucleotide compositions (sterically random and chirally controlled) are known and may be used in accordance with the present disclosure, including, for example, those of WO/2010/064146, WO/2011/005761, WO/2013/012758, WO/2014/010250, US2013/0178612, WO/2014/012081, WO/2015/107425, WO/2017/015555, and WO/2017/062862, the methods and reagents in each of which are incorporated herein by reference.
Example 1.
Conjugation of oligonucleotides.
In some embodiments, the present disclosure provides methods for conjugating oligonucleotides, e.g., for better delivery to the CNS. Examples 1 and 2 show conjugation of oligonucleotides for CNS delivery.
In some embodiments, provided oligonucleotides comprise a chemical moiety attached to the 5' end, optionally via a linker moiety. In some embodiments, provided oligonucleotides comprise a chemical moiety attached to the 5' terminal-OH, optionally via a linker. In some embodiments, the present disclosure provides the following 5' c conjugation strategy:
in some embodiments, provided oligonucleotides comprise a chemical moiety attached to the 5' end, optionally via a linker moiety. In some embodiments, the present disclosure provides the following 3' c conjugation strategy:
various chemical moieties, such as ligands for cellular receptors, such as those described in the following references, may be used in accordance with the present disclosure: juliano et al, j.am.chem.soc. [ journal of the american chemical society ]2010, 132, 8848; banerjee R et al, Int J Cancer [ journal of international Cancer ]2004, 112, 693; med.chem. [ journal of medicinal chemistry ], 2017, 60(10), pages 4161-4172; and the like. In some embodiments, the chemical moiety is selected from:
conjugates of various oligonucleotides
WV-9063(C9orf72, intron), WV-9381(C9orf72, intron), WV-7560(Malat1), WV-8447 (exon 1a), WV-8819 (non-C9 orf72 target), WV-9066 (non-C9 orf72 target), WV-9065 (non-C9 orf72 target)
WV-7558(Malat1)
WV-7559(Malat1)
WV-8448(Malat1), WV-8446 (exon 1a)
WV-8896(Malat1), WV-8445 (exon 1a)
Synthesis of 4,10, 17-trioxo-15, 15-bis ((3-oxo-3- ((3- (4- (((2R, 3R, 4S, 5R, 6R) -3, 4, 5-tris (benzoyloxy) -6- ((benzoyloxy) methyl) tetrahydro-2H-pyran-2-yl) oxy) butyrylamino) propyl) amino) propoxy) methyl) -1- (((2R, 3R, 4S, 5R, 6R) -3, 4, 5-tris (benzoyloxy) -6- ((benzoyloxy) methyl) tetrahydro-2H-pyran-2-yl) oxy) -13-oxa-5, 9, 16-triaza-heneicosane-21-oic acid.
Step 1: a solution of di-tert-butyl 3, 3' - ((2-amino-2- ((3- (tert-butoxy) -3-oxopropoxy) methyl) propane-1, 3-diyl) bis (oxy)) dipropionate 1(4.0g, 7.91mmol) and dihydro-2H-pyran-2, 6(3H) -dione (0.903g, 7.91mmol) in THF (40mL) was stirred at 50 ℃ for 3H and at room temperature for 3H. LC-MS showed the desired product. The solvent was evaporated to give acid 2, which was used in the next step without purification.
Step 2: to a solution of 5- ((9- ((3- (tert-butoxy) -3-oxopropoxy) methyl) -2, 2, 16, 16-tetramethyl-4, 14-dioxo-3, 7, 11, 15-tetraoxaheptadecan-9-yl) amino) -5-oxopentanoic acid 2(4.90g, 7.91mmol) and (bromomethyl) benzene (1.623g, 9.49mmol) in DMF was added anhydrous K2CO3(3.27g, 23.73 mmol). The mixture was stirred at 40 ℃ for 4 hours andstir at room temperature overnight. The solvent was evaporated under reduced pressure. The reaction mixture was diluted with EtOAc, washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a residue which was purified by ISCO (eluting with 10% EtOAc in hexanes to 50% EtOAc in hexanes) to give di-tert-butyl 3, 3' - ((2- (5- (benzyloxy) -5-oxopentanamido) -2- ((3- (tert-butoxy) -3-oxopropoxy) methyl) propane-1, 3-diyl) bis (oxy)) dipropionate 3 as a colorless oil (5.43g, 7.65mmol, 97% yield).1H NMR (400MHz, chloroform-d) δ 7.36-7.28(m, 5H), 6.10(s, 1H), 5.12(s, 2H), 3.70(s, 6H), 3.64(t, J ═ 8.0Hz, 6H), 2.50-2.38(m, 8H), 2.22(t, J ═ 7.3Hz, 2H), 1.95(p, J ═ 7.4Hz, 2H), 1.45(s, 27H); MS, 710.5(M + H) +.
And step 3: a solution of di-tert-butyl 3, 3' - ((2- (5- (benzyloxy) -5-oxopentanamido) -2- ((3- (tert-butoxy) -3-oxopropoxy) methyl) propane-1, 3-diyl) bis (oxy)) dipropionate 3(5.43g, 7.65mmol) in formic acid (50mL) was stirred at room temperature for 48 h. LC-MS showed incomplete reaction. The solvent was evaporated under reduced pressure. The crude product was redissolved in formic acid (50mL) and stirred at room temperature for 6 hours. LC-MS showed the reaction was complete. The solvent was evaporated under reduced pressure, co-evaporated with toluene (3X) under reduced pressure and dried under vacuum to give 3, 3' - ((2- (5- (benzyloxy) -5-oxopentanamido) -2- ((2-carboxyethoxy) methyl) propane-1, 3-diyl) bis (oxy)) dipropionic acid 4(4.22g, 7.79mmol, 102% yield) as a white solid.1H NMR(500MHz，DMSO-d6)δ12.11(s，3H)，7.41-7.27(m，5H)，6.97(s，1H)，5.07(s，2H)，3.55(t，J＝6.4Hz，6H)，3.53(s，6H)，2.40(t，J＝6.3Hz，6H)，2.37-2.26(m，2H)，2.08(t，J＝7.3Hz，2H)，1.70(p，J＝7.4Hz，2H)；MS，542.3(M+H)+。
And 4, step 4: to a solution of 4(4.10g, 7.57mmol) of 3, 3' - ((2- (5- (benzyloxy) -5-oxopentanamido) -2- ((2-carboxyethoxy) methyl) propane-1, 3-diyl) bis (oxy)) dipropionic acid and HOBt (4.60g, 34.1mmol) in DCM (60mL) and DMF (15mL) at 0 deg.C was added tert-butyl (3-aminopropyl) carbamate (5.94g, 34.1mmol), EDAC HCl salt (6.53g,34.1mmol) and DIPEA (10.55ml, 60.6 mmol). The reaction mixture was stirred at 0 ℃ for 15 minutes and at room temperature for 20 hours. LC-MS showed incomplete reaction. EDAC HCl salt (2.0g) and tert-butyl (3-aminopropyl) carbamate (1.0g) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 4 hours. The solvent was evaporated to give a residue which was dissolved in EtOAc (300mL), washed with water (1X), saturated sodium bicarbonate (2X), 10% citric acid (2X) and water, dried over sodium sulfate and concentrated to give a residue which was purified by ISCO (80g gold column) eluting with DCM to 30% MeOH in DCM to give benzyl 15, 15-bis (13, 13-dimethyl-5, 11-dioxo-2, 12-dioxa-6, 10-diazatetrayl) -2, 2-dimethyl-4, 10, 17-trioxo-3, 13-dioxa-5, 9, 16-triazacycloheneicosane-21-oate 5 as a white solid (6.99g, 6.92mmol, 91% yield).1H NMR (500MHz, chloroform-d) δ 7.38-7.33(m, 5H), 6.89(brs, 3H), 6.44(s, 1H), 5.23(brs, 3H), 5.12(s, 2H), 3.71-3.62(m, 12H), 3.29(q, J ═ 6.2Hz, 6H), 3.14(q, J ═ 6.5Hz, 6H), 2.43(dt, J ═ 27.0, 6.7Hz, 8H), 2.24(t, J ═ 7.2Hz, 2H), 1.96(p, J ═ 7.5Hz, 2H), 1.64-1.59(m, 6H), 1.43(d, J ═ 5.8Hz, 27H); ms (esi): 1011.5(M + H) +.
And 5: to a solution of benzyl 15, 15-bis (13, 13-dimethyl-5, 11-dioxo-2, 12-dioxa-6, 10-diazatetetradecyl) -2, 2-dimethyl-4, 10, 17-trioxo-3, 13-dioxa-5, 9, 16-triazacyclononane-21-oate (0.326g, 0.46mmol) in DCM (5mL) was added TFA (2 mL). The reaction mixture was stirred at room temperature for 4 hours. LC-MS showed the reaction was complete. The solvent was evaporated under reduced pressure to give benzyl 5- ((1, 19-diamino-10- ((3- ((3-aminopropyl) amino) -3-oxopropoxy) methyl) -5, 15-dioxo-8, 12-dioxa-4, 16-diazadecan-10-yl) amino) -5-oxopentanoate as a colorless oil. It was used in the next step without purification.
Step 6: to 5- (((2R, 3R, 4R, 5R, 6R) -3-acetamido-4, 5-diacetoxy-6- (acetoxymethyl) tetrahydro-2H-pyran-2-yl) oxy) pentanoic acid (1.10g, 1.61mmol), HBTU (0.558g, 1.4 mmol)7mmol), HOBT (0.062g, 0.46mmol) and DIPEA (1.2mL, 6.9mmol) in DCM (6mL) followed by benzyl 5- ((1, 19-diamino-10- ((3- ((3-aminopropyl) amino) -3-oxopropoxy) methyl) -5, 15-dioxo-8, 12-dioxa-4, 16-diazadecan-10-yl) amino) -5-oxopentanoate in acetonitrile (5 mL). The mixture was stirred at room temperature for 3 hours. The solvent was evaporated under reduced pressure to give a residue which was purified by ISCO (40g gold column) eluting with DCM to 20% MeOH in DCM to give 4,10, 17-trioxo-15, 15-bis ((3-oxo-3- ((3- (4- (((2R, 3R, 4S, 5R, 6R) -3, 4, 5-tris (benzoyloxy) -6- ((benzoyloxy) methyl) tetrahydro-2H-pyran-2-yl) oxy) butyrylamino) propyl) amino) propoxy) methyl) -1- (((2R, 3R, 4S, 5R, 6R) -3, 4, 5-tris (benzoyloxy) -6- ((benzoyloxy) methyl) tetrahydro-2H-pyran-2-yl) as a white solid Oxy) -13-oxa-5, 9, 16-triazacyclononane-21-oic acid benzyl ester (1.14g, 92% yield). Ms (esi): 1353.7(M/2+ H)+。
And 7: to benzyl 4,10, 17-trioxo-15, 15-bis ((3-oxo-3- ((3- (4- (((2R, 3R, 4S, 5R, 6R) -3, 4, 5-tris (benzoyloxy) -6- ((benzoyloxy) methyl) tetrahydro-2H-pyran-2-yl) oxy) butyrylamino) propyl) amino) propoxy) methyl) -1- (((2R, 3R, 4S, 5R, 6R) -3, 4, 5-tris (benzoyloxy) -6- ((benzoyloxy) methyl) tetrahydro-2H-pyran-2-yl) oxy) -13-oxa-5, 9, 16-triaza-heneicosane-21-oate (1.09g, 0.400mmol) in EtOAc (50mL) was added 10% Pd-C (200mg) and methanol (2 mL). The reaction mixture was stirred at room temperature for 3 hours. LC-MS showed the reaction to be complete, diluted with EtOAc and filtered through celite, washed with 20% MeOH in EtOAc and concentrated under reduced pressure to afford 4,10, 17-trioxo-15, 15-bis ((3-oxo-3- ((3- (4- (((2R, 3R, 4S, 5R, 6R) -3, 4, 5-tris (benzoyloxy) -6- ((benzoyloxy) methyl) tetrahydro-2H-pyran-2-yl) oxy) butyramido) propyl) amino) propoxy) methyl) -1- ((((2R, 3R' 4S, 5R, 6R) -3, 4, 5-tris (benzoyloxy) -6- ((benzoyloxy) methyl) tetrahydro-2H-pyran-2-yl) oxy) propana) as a white solid 13-oxa-5-oxygen in the molecule,9, 16-triazaacosane-21-oic acid (1.06g, 100%). Ms (esi): 1308.1(M + H)+。
Example 2
Synthesis of 4-oxo-4- ((4-sulfamoylphenethyl) amino) butanoic acid
To the solid reagent 4- (2-aminoethyl) benzenesulfonamide (2.00g, 9.99mmol) and dihydrofuran-2, 5-dione (0.999g, 9.99mmol) was added THF (30 mL). The reaction mixture was stirred at 60 ℃ for 7 hours. The solvent was evaporated under reduced pressure to give 4-oxo-4- ((4-sulfamoylphenethyl) amino) butyric acid as a white solid (3.00g, 9.99mmol, 100% yield).1H NMR(400MHz，DMSO-d6)δ12.09(s，1H)，7.96(t，J＝5.6Hz，1H)，7.72(d，J＝8.1Hz，2H)，7.38(d，J＝8.1Hz，2H)，7.29(s，2H)，3.26(q，J＝6.8Hz，2H)，2.76(t，J＝7.2Hz，2H)，2.40(t，J＝6.9Hz，2H)，2.27(t，J＝6.9Hz，2H)；MS(ESI)，301.1(M+H)+。
General procedure for conjugation of sulfonamides to WV-7557
(Synthesis of WV-7558 and 7559)
The procedure is as follows: the synthesis of WV-7558 and WV-7559 followed the same procedure as described below. To a solution of sulfonamide (5 equiv) in 2ml DMF was added HATU (4.5 equiv.) and DIPEA (25 equiv.). The mixture was stirred well for 2 minutes (schemes 1 and 2).
To this solution was added a solution of WV-7557(1 eq) in water and shaken well for 60 minutes. The solvent was removed in vacuo and the crude product was purified by RP column (C18) chromatography to afford the product. The purified product was desalted using sodium acetate solution through a C-18 column.
Synthesis of WV-7558: following the general procedure mentioned above, 4-sulfamoylbenzoic acid (11mg, 54.5. mu. mol), HATU (18.6mg, 49. mu. mol) and DIPEA (35mg, 272. mu. mol) were stirred in 2ml DMF for 2 min (scheme 1). This activated HATU intermediate was added to a solution of WV-7557(75mg, 10.9. mu. mol) in 0.75ml of water. The reaction vial was shaken for 60 minutes. The solvent was removed under reduced pressure, purified and desalted as described above. The amount of product obtained was 20 mg. Calculated molecular weight of the product: 7063; the resulting deconvolution quality: 7065
Synthesis of WV-7559: following the general procedure mentioned above, 4-sulfamoylbenzoic acid (16.3mg, 54.5. mu. mol), HATU (18.6mg, 49. mu. mol) and DIPEA (35mg, 272. mu. mol) were stirred in 2ml DMF for 2 minutes (scheme 2). This activated HATU intermediate was added to a solution of WV-7557(75mg, 10.9. mu. mol) in 0.75ml of water. The reaction vial was shaken for 60 minutes. The solvent was removed under reduced pressure, purified and desalted as described above. The amount of product obtained was 13 mg. Calculated molecular weight of the product: 7162; the resulting deconvolution quality: 7165.
general procedure for conjugation of triantenna anisamide to WV-7557 and WV 8444: synthesis of WV-7560 and WV 8447
General procedure: to a solution of triantenna anisamide (2 equiv) in 2ml DMF was added HATU (1.8 equiv.) and DIPEA (10 equiv.). The mixture was stirred well for 2 minutes. To this solution was added a solution of WV-7557(1 eq) in water and shaken well for 60 minutes. The solvent was removed in vacuo and the crude product was purified by RP column (C8) chromatography to afford the product. The purified product was desalted using sodium acetate solution through a C-18 column.
Synthesis of WV-7560: to a solution of triantenna anisamide (11mg, 9.8. mu. mol) in 2ml DMF was added HATU (3.34mg, 8.82. mu. mol) and DIPEA (6.3mg, 9. mu.l, 49. mu. mol). The mixture was stirred well for 2 minutes (scheme 3). To this solution was added a solution of WV-7557(33.7mg, 4.9. mu. mol) in 0.88ml of water and shaken well for 60 minutes. The solvent was removed in vacuo and the crude product was purified by RP column (C8) chromatography to give the product WV-7560(25 mg). The purified product was desalted using sodium acetate solution through a C-18 column. Calculated molecular weight of the product: 7982; the resulting deconvolution quality: 7987.
synthesis of WV-8447: to a solution of triantenna anisamide (13mg, 11.6. mu. mol) in 2ml DMF was added HATU (4mg, 10.4. mu. mol) and DIPEA (7.5mg, 10.3. mu.l, 58. mu. mol). The mixture was stirred well for 2 minutes (scheme 4). To this solution was added a solution of WV-8444(40mg, 5.8. mu. mol) in 1ml of water and shaken well for 60 minutes. The solvent was removed in vacuo and the crude product was purified by RP column (C8) chromatography to give product WV-8447. The purified product was desalted using sodium acetate solution through a C-18 column. Calculated molecular weight of the product: 7970 of a glass fiber; the resulting deconvolution quality: 7975.
general procedure for conjugation of Triantennal glucosamine/glucose derivatives to WV-7557 or WV-8444
To a solution of triantennary glucosamine or a glucose derivative (2 equiv.) in 2ml DMF was added HATU (1.8 equiv.) and DIPEA (10 equiv.). The mixture was stirred well for 2 minutes. To this solution was added a solution of WV-7557 or WV-8444(1 eq) in water and shaken well for 60 minutes. The solvent was removed under vacuum and the crude product was washed with 30% NH at room temperature4The OH solution was treated for 24 hours. The solvent was removed in vacuo and the crude product was purified by RP column (C8) chromatography to afford the product. The purified product was desalted using sodium acetate solution through a C-18 column.
Synthesis of WV-8896: following the general procedure shown above, glucosamine derivative (23.3mg, 11.6. mu. mol), HATU (4mg, 10.44. mu. mol) and DIPEA (7.5mg, 58. mu. mol) were stirred in 2ml DMF (scheme 5). To this solution was added 40mg (5.8. mu. mol) of WV-7557 in 1ml of water. The reaction mixture was stirred for 60 minutes to obtain the desired product. With NH as hereinbefore described4OH treating the product. The amount of product obtained was 20 mg. Calculated molecular weight: 8496; the resulting deconvolution quality: 8494
Synthesis of WV-8448: following the general procedure shown above, glucose derivatives (57mg, 21.8. mu. mol), HATU (7.5mg, 19.6. mu. mol) and DIPEA (14.6mg, 109. mu. mol) were stirred in 2ml DMF (scheme 6). To this solution was added 75mg (10.9. mu. mol) of WV-7557 in 1ml of water. The reaction mixture was stirred for 60 minutes to obtain the desired product. This product was reacted with NH at 40 ℃ as described above4OH are heated together to obtain the product. Calculated molecular weight: 8227; the resulting deconvolution quality: 8233.
synthesis of WV-8446: following the general procedure shown above, glucose derivatives (30mg, 11.6. mu. mol), HATU (4mg, 10.4. mu. mol) and DIPEA (7.5mg, 58. mu. mol) were stirred in 2ml DMF (scheme 7). To this solution was added 40mg (5.8. mu. mol) of WV 8444 in 1ml of water. The reaction mixture was stirred for 60 minutes to obtain the desired product. This product was reacted with NH at 40 ℃ as described above4OH are heated together to obtain the product. Calculated molecular weight: 8214; the resulting deconvolution quality: 8218.
synthesis of WV-8445: following the general procedure shown above, glucosamine derivative (24mg, 12. mu. mol), HATU (4mg, 10.4. mu. mol) and DIPEA (7.5mg, 58. mu. mol) were stirred in 2ml DMF (scheme 8). To this solution was added 40mg (5.8. mu. mol) of WV 8444 in 1ml of water. The reaction mixture was stirred for 60 minutes to obtain the desired product. This product was reacted with NH at 40 ℃ as described above4OH are heated together to obtain the product. Calculated molecular weight: 8477; the resulting deconvolution quality: 8484.
synthesis of GlucNAc linker.
Adding GlucNAc acid under nitrogen at room temperature11(1.88g, 4.2mmol) and HOBT (0.73g, 5.4mmol) were stirred in a mixture of anhydrous DMF-DCM (11+15ml) for 10 min. HBTU (2.05g, 5.4mmol) was added followed by DIPEA (2.17g, 16.8mmol) at 10 ℃. To this solution was added the triamine salt22(1.38g, 1.2mmol) and stirred overnight. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate (200 ml). To this solution was added 100ml of a mixture of saturated ammonium chloride, saturated sodium bicarbonate and water (1: 1). The ethyl acetate layer was initially cloudy. After sufficient shaking, the layers were separated. The aqueous layer was extracted with ethyl acetate (x 2). The combined organic fractions were washed with brine and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to give 490mg of crude product. This product was purified by CC on an ISCO machine. The eluent was DCM-methanol (0-20% methanol in DCM). The amount of product obtained was 1.26g (50%). LC-MS (+ mode):1768(M-1GlucNAc)、1438(M-2 GlucNAc)、1108(M-3 GlucNAc)、1049(M/2+1)。
the procedure is as follows: to a solution of benzyl ester 4(0.25g, 0.119mmol) in 7ml of anhydrous methanol under an argon atmosphere was added dropwise 1.5ml (9.4mmol) Triethylsilane (TES). A vigorous reaction was started and RM3 hours were stirred. LC-MS analysis of the product indicated the reaction was complete. The RM was filtered through celite and the solvent removed in vacuo. The crude product was triturated (X3) with an ether-methanol (3: 1) mixture and dried under vacuum. This product 5 was used in the next reaction without further purification. 1H NMR (500MHz, DMSO-D6): δ 7.90(3H, d, J ═ 10Hz), 7.80(t, 3H), 7.70(t, 3H), 5.03(t, 3H), 4.77(t, 3H), 4.54(3H, d, J ═ 10Hz), 4.14(3H, dd, J ═ 10Hz), and1＝9Hz，J2＝5Hz)，3.97-3.93(m，3H)，3.79-3.74(m，3H)，3.69-3.61(m，6H)，3.51-3.47(m，3H)，3.40-3.35(m，3H)，3.31(d，3H，J＝9Hz)，2.98(m，12H)，2.23(t，3H)，2.13(t，3H)，2.01-1.99(m，3H)，1.97(s，9H)，1.92(s，9H)，1.86(s，9H)，1.71(s，9H)，1.49-1.32(m，22H)，1.18(br s，12H)。
references 1 and 2: us patents WO 2014/025805 a 1; the date is 2014, 2 months and 13 days.
Reference documents:
juliano et al J.Am.chem.Soc. [ J.J.Chem.Soc. [ J.Chem.Soc. ]2010, 132, 8848
Banerjee R et al int.J.cancer. [ J.International journal of cancer ]2004, 112, 693
He et al J.Med.chem. [ journal of medical chemistry ], 2017, 60(10), page 4161-4172
General procedure for deprotection of amines:
15.2g NHBoc amine was dissolved in dry DCM (100ml) followed by dropwise addition of TFA (50ml) at room temperature. Mixing the reactionThe mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and then co-evaporated with toluene (2x50mL) and used in the next step without any further purification. CD (compact disc)3NMR in OD confirmed that NHBoc was deprotected.
General procedure for anisamide formation:
step A: the crude amine from the previous step was dissolved in DCM (100ml) and Et at RT3N (10 equivalents) to a mixture. During this process, the reaction mixture was cooled with a water bath. Then, 4-methoxybenzoyl chloride (4 equivalents) was added dropwise to the reaction mixture under argon atmosphere at room temperature, and stirring was continued for 3 hours. The reaction mixture was diluted with water and extracted with DCM. The organic layer was washed with NaHCO3Aqueous solution, 1N HCl, brine, then dried over magnesium sulfate and evaporated to dryness. The crude product was purified by silica column chromatography using DCM-MeOH as eluent.
And B: the crude amine (0.27 eq.), acid and HOBt (1 eq.) were dissolved in a mixture of DCM and DMF (2: 1) under argon in an appropriately sized RBF. Edac.hcl (1.25 eq) was added to the reaction mixture in portions with constant stirring. After 15 minutes, the reaction mixture was cooled to about 10 ℃, and then DIEA (2.7 equivalents) was added over a period of 5 minutes. The reaction mixture was slowly warmed to ambient temperature and stirred under argon overnight. TLC indicated complete reaction, TLC conditions DCM: MeOH (9.5: 0.5). The solvent was removed under reduced pressure, then water was added to the residue, and a gummy solid was isolated. The clear solution was decanted and the solid residue was dissolved in EtOAc and successively water, 10% aqueous citric acid, aqueous NaHCO3Followed by washing with saturated brine. The organic layer was separated and dried over magnesium sulfate. The solvent was removed under reduced pressure and the crude product was then purified on a silica column to give the pure product.
Anisamide:
using procedure B above, anisamide was obtained from the amine in 2 steps with a yield of 32%: 1H NMR (CDCl)3)：δ＝7.74(d，6H)，7.44(t，2H)，7.34(t，1H)，7.26(m，5H)，7.05(m，3H)，6.83(d，6H)，6.46(s，1H)，5.01(s，2H)，3.75(s，9H)，3.57(m，12H)，3.37(m，6H)，3.25(m，6H)，2.31(m，8H)，2.11(m，2H)，1.84(m，2H)，1.62(m，6H)ppm。
Anisamide:
using the procedure A described above, anisamide was obtained from the amine in 2 steps, with a yield of 57%: 1H NMR (CDCl)3)：δ＝7.75(m，3H)，7.73(d，6H)，7.43(t，3H)，7.25(m，5H)，6.80(d，6H)，6.51(brs，1H)，5.01(s，2H)，3.72(s，9H)，3.58(m，6H)，3.21(m，12H)，2.33(t，3H)，2.25(t，2H)，2.02(t，2H)，1.64(q，6H)，1.52(p，2H)，1.41(q，2H)，1.12(m，12H)ppm。
General procedure for debenzylation:
benzyl ester (10g) was dissolved in a mixture of ethyl acetate (100ml) and methanol (25ml) and then 1g Pd/C (10% palladium content) was added under an argon atmosphere, then the reaction mixture was placed under vacuum and flushed with hydrogen and stirred at room temperature under an H2 atmosphere for 3 hours. TLC indicated completion of the reaction, filtered through a pad of celite and washed with methanol and evaporated to dryness to give a foamy white solid.
Acid:
yield 98%, 1H NMR (CD)3OD)：δ＝8.35(t，1H)，8.01(t，1H)，7.82(d，6H)，7.27(d，1H)，6.99(d，6H)，3.85(s，9H)，3.68(m，12H)，3.41(m，6H)，3.29(m，6H)，2.42(m，6H)，2.31(q，2H)，2.21(td，2H)，1.80(m，8H)ppm。
Acid:
yield 94%, 1H NMR (CD)3OD)：δ＝8.36(t，2H)，8.02(t，2H)，7.82(d，6H)，7.23(d，1H)，6.98(d，6H)，3.85(s，9H)，3.70(s，6H)，3.67(t，6H)，3.41(q，4H)，3.28(m，8H)，2.42(t，6H)，2.27(t，2H)，2.13(t，2H)，1.79(p，6H)，1.54(dp，4H)，1.25(m，12H)ppm。
Other compositions are presented below, comprising oligonucleotides containing analogs of anisamide:
example 3.
Activity of various C9orf72 oligonucleotides in a Dual-luciferase reporter assay
Tables 2A to 2C present data on the activity of various C9orf72 oligonucleotides in the dual luciferase reporter assay.
Each data point in tables 2A through 2C is the Renilla (Renilla) signal compared to WV-993, a control oligonucleotide that does not target C9orf 72. In these tables, ASO is C9orf72 oligonucleotide.
The numbers are duplicate experiments performed simultaneously. The number indicates the amount of remaining gene expression. For example, a repeat of experiment 1 with WV-3677 (in the first column in table 2A) showed 1.522608% residual gene expression, which represents a 98.477392% knock-down.
Construct (a): the C9orf72nt 1-374 (Table 2A), C9orf72nt 158-900 (Table 2B) or C9orf72nt 900-1 (Table 2C) sequences, respectively, were cloned into the NotI site of psiCHECK-2 vector (Promega Corporation, Madison, Wis.) located in the middle of the 3' UTR of hRluc gene. Thus, the new construct comprises a portion of wild-type C9orf72 that does not contain a hexanucleotide repeat amplification.
The sequences used to make these constructs are presented below:
c9, 1-374 (exon 1a and part of intron 1)
C9, 158-900 (intron 1)
C9, 900-1 (antisense RNA)
Each construct expresses two photoproteins: firefly luciferase derived from the hluc gene and Renilla luciferase derived from the hRluc gene.
Constructs (20ng) and oligonucleotides tested (different doses) were co-transfected with Lipofectamine (Lipofectamine)2000 into Cos 7 cells and maintained for 48 hours, and firefly and renilla signals were read with a disk reader.
An effective C9orf72 oligonucleotide targeting the insert should reduce renilla signaling without affecting firefly signaling. Data analysis normalizes the renilla and firefly signals and compares the efficacy of the tested oligonucleotides to the control oligonucleotides. In tables 2A and 2B, the numbers indicate the percentage of the remaining renilla signal. For example, for WV-3677, a 1.5% residual level was detected in duplicate experiments, and a 2.4% residual level was detected in another duplicate experiment at 30nM (which represents 98.5% and 97.6% knockdown, respectively).
Example 4.
The ability of C9orf72 oligonucleotides to knock down C9orf72 transcripts in vitro
Various C9orf72 oligonucleotides were tested for their ability to knock down C9orf72 transcripts in vitro. The oligonucleotides tested had any of 20 different sequences (sequences 1 to 20) and each sequence was tested in each of three different formats (e.g., 2 '-O-methyl full PS, 2' -O-methyl PS/PO, or MOE full PS). The exact sequence and modifications of each C9orf72 oligonucleotide are presented in table 1A.
TABLE 3A C9orf72 oligonucleotides tested in this experiment
C9orf72 oligonucleotides were tested at a concentration of 10uM in iCell neurons at 48 hours. The results are presented below. The numbers indicate the amount of C9orf72 transcripts remaining after in vitro introduction of the oligonucleotide or control into the cells (the measured amount is the total of all C9orf72 transcripts). For example, for water in group a, 0.992 indicates that 99.2% of the C9orf72 transcript level was retained, or there was substantially no knock-down relative to the control. For WV-3675 representing mRNA sequence 19 in group B, 0.316 represents 31.6% of the remaining C9orf72 transcript level or 68.4% of knockdown. Other data representing the remaining transcript levels are presented in this same or similar form unless otherwise mentioned.
TABLE 3B Activity of C9orf72 oligonucleotides tested in this experiment
Example 5.
Activity of various C9orf72 oligonucleotides in various assays
Tables 4A to 4D show that C9orf72 oligonucleotide knockdown C9orf72 transcripts in iPSC neurons in vitro (table 4A, all transcripts; table 4B, V3 only transcript; table 4C, intron/AS transcript; and table 4D, exon 1a only transcript). Figure 4C shows knock-down of intron/AS transcripts (using probes targeting 3' to the region of repeat transcript amplification, the region detected including sense and antisense transcripts of the intron region). The relative fold change for C9orf72/HPRT1 is shown. Three replicates of each C9orf72 oligonucleotide at a concentration of 1. mu.M (panel A) or 10. mu.M (panel B) are shown. Numbers indicate residual transcript levels (all C9orf72 transcripts). For example, three replicates at a concentration of 1 μ M (group A) showed residual C9orf72 transcript levels (all C9orf72 transcripts) of 82.6%, 86.8% and 77.6% for WV-7601, which correspond to knockdowns of 17.4%, 13.2% and 22.3%, respectively. For WV-7601, three replicates at a concentration of 10 μ M (group B) were also performed, showing residual C9orf72 transcript levels (all C9orf72 transcripts) of 76.0%, 68.5% and 75.0%, which correspond to knockdowns of 24.0%, 31.5% and 25.0%, respectively. Delivery of the oligonucleotides was in vitro and cells were tested after 1 week. Controls used included WV-5302 and WV-6493 targeting Malat 1. Malat1 and C9orf72 oligonucleotides were also tested against Malat 1; malat1 oligonucleotide knocked down Malat1 efficiently, but C9orf72 oligonucleotide failed to knock down Malat1 (data not shown). Controls also included WV-2549 and WV-6028 that target gene targets other than C9orf 72.
TABLE 4A. C9orf72 oligonucleotide Activity (residual levels of all c9orf72 transcripts)
TABLE 4B.C9orf72 oligonucleotide activity (residual level of V3C 9orf72 transcript)
TABLE 4 C.C. 9orf72 oligonucleotide activity (remaining levels of intron/AS C9orf72 transcript)
TABLE 4 D.C. 9orf72 oligonucleotide Activity (residual level of exon 1a C9orf72 transcript)
Example 6.
Activity of various C9orf72 oligonucleotides in various assays
Tables 5A to 5D show the activity of various C9orf72 oligonucleotides in knocking down C9orf72 transcripts (table 5A, all transcripts; table 5B, V3 only transcript; table 5C, intron/AS; and table 5D, exon 1a only transcript). The relative fold change for C9orf72/HPRT1 is shown. Three replicates of each C9orf72 oligonucleotide at a concentration of 1. mu.M (panel A) or 10. mu.M (panel B) are shown. With respect to tables 5A to 5D, the numbers indicate residual transcript levels. Delivery of the oligonucleotides was in vitro and cells were tested after 1 week.
TABLE 5A. C9orf72 oligonucleotide Activity (residual levels of all C9orf72 transcripts)
TABLE 5B.C9orf72 oligonucleotide activity (residual level of V3C 9orf72 transcript)
TABLE 5 C.C. 9orf72 oligonucleotide activity (remaining level of intron/AS C9orf72 transcript)
TABLE 5 D.C. 9orf72 oligonucleotide Activity (residual level of exon 1a C9orf72 transcript)
Example 7.
Activity of various C9orf72 oligonucleotides in various assays
Table 6A and Table 6B show the activity of various C9orf72 oligonucleotides in knocking down C9orf72 transcripts (Table 6A, all transcripts; Table 6B, only V3 transcript). The relative fold change for C9orf72/HPRT1 is shown. Three replicates of various C9orf72 oligonucleotides at a concentration of 10. mu.M are shown. With respect to tables 3A to 3D, the numbers indicate residual transcript levels relative to HPRT 1. Delivery of the oligonucleotides was in vitro and cells were tested after 1 week. As a control, C9orf72 oligonucleotide was tested and found to be ineffective at knocking down malt 1 (data not shown). The C9orf72 oligonucleotide was also found to be ineffective against another target, PFN1 (data not shown).
TABLE 6A. C9orf72 oligonucleotide Activity (residual levels of all C9orf72 transcripts)
TABLE 6B C9orf72 oligonucleotide activity (residual level of V3C 9orf72 transcript)
Table 6C below shows the IC50 of various C9orf72 oligonucleotides (delivered in vitro and evaluated after 1 week) tested in ALS MN (motor neurons) in a full dose-response assay. Concentrations of 10, 2.5, 0.625, 0.16, 0.04 and 0.001 μ M were tested.
TABLE 6C IC50 of some C9orf72 oligonucleotides
Example 8.
In vitro screening protocol
This example describes an in vitro screening protocol for C9orf72 oligonucleotides.
In 24-well plates, oligonucleotides were delivered in vitro to ALS neurons and maintained for 48 hours.
RNA extraction
RNA extraction was performed with RNeasy Plus96 kit (Qiagen, Waltham, massachusetts) according to the following protocol: total RNA was purified from cells using vacuum/spin techniques. (gDNA removal is critical).
For each well, total RNA was eluted in 60ul RNase-free water.
Reverse transcription
Using large capacity RNA-to-cDNATMKit (Applied Biosystems, available from Seimer Feishel, ThermoFisher, Waltham, Mass.) for reverse transcription
2XRT buffer mixing
Thing 9ul
RNA sample 13.5ul
Heat denaturation was carried out at 72 ℃ for 5 minutes and the plates were allowed to cool on ice for at least 2 minutes.
To each well with heat-denatured RNA was added:
2XRT buffer mixture 6
20X RT enzyme mixture 1.5ul
The final volume of cDNA was 30 ul.
Real-time PCR
Taqman probe:
all variants of C9orf 72: hs00376619_ m1(FAM), catalog No. 4351368 (Sammer Feishale, Waltham, Mass.)
C9orf 72V 3: hs00948764_ m1(FAM), catalog No. 4351368 (Sammer Feishale, Waltherm, Mass.)
C9orf72 exon 1 a:
forward primer AGATGACGCTTGGTGTGTC
Reverse primer TAAACCCACACCTGCTCTTG
Probe CTGCTGCCCGGTTGCTTCTCTTT
C9orf72 antisense RNA/intron:
forward primer GGTCAGAGAAATGAGAGGGAAAG
Reverse primer CGAGTGGGTGAGTGAGGA
Probe AAATGCGTCGAGCTCTGAGGAGAG
Internal control: human HPRT1(VIC)
Hs02800695_ m1, catalog No. 4448486 (ThermoFisher, Waltherm, Mass.)
And (3) PCR reaction:
2ul cDNA was used for all variant probes. 9ul cDNA was used for the other C9 probes.
Real-time PCR Using Bio-rad CFX96 Touch
Operation information:
195.0C duration 3:00
295.0C continuous 0: 10
360.0C continuous 0:30
+ plate reading
4 GOTO 2, 39 additional times
End up
Example 9.
Activity of various C9orf72 oligonucleotides in various assays
Tables 7A to 7C below present the activity of various C9orf72 oligonucleotides tested in various assays.
Brief description of the various assays performed:
a reporter:
luciferase assay, as described herein. For some oligonucleotides, two numbers are given (e.g., 1.32/2.63 for WV- -6408); these numbers represent duplicate experiments.
ALS neurons:
neuronal differentiation of ipscs: ipscs derived from fibroblasts from ALS patients (female, 64 years) related to C9orf72 were obtained from RUCDR Infinite Biologics. Ipscs were maintained as colonies on Corning Matrigel matrix (Sigma Aldrich, st. louis, MO) in mTeSR1 medium (stem cell Technologies, Vancouver, British Columbia (BC)) in mTeSR1 medium. Neural progenitor cells were generated using the STEMdiff nervous system (stem cell technologies, wengover, british columbia). Ipscs were suspended in AggreWell800 plates and embryoid bodies were grown over 5 days in STEMdiff neural induction medium, with 75% of the medium changed daily. Embryoid bodies were collected using a 37 μm cell filter and plated onto Matrigel-coated plates in STEMdiff neural induction medium. The medium was changed daily for 7 days, and 85% -95% of the embryoid bodies developed rosettes (rosette) 2 days after plating. Rosettes were picked manually and transferred to STEMdiff nerve induction medium (stem cell technologies, wengowski, british columbia) in plates coated with poly-L-ornithine and laminin. The medium was changed daily for 7 days until the cells reached 90% confluence and were considered Neural Progenitor Cells (NPCs). NPC were isolated using TrypLE (Gibco, available from Seimer Feishale, Waltham, Mass.) and supplemented with growth factors (20ng/ml FGF2, 20 n)g/ml EGF, 5. mu.g/ml heparin) were passaged on poly-L-ornithine/laminin plates at a ratio of 1: 2 or 1: 3 in nerve maintenance medium (NMM, 70% DMEM, 30% Ham's F12, 1X B27 supplement). For maturation into neurons, NPCs were maintained and expanded for less than five passages and passaged on poly-L-ornithine/laminin coated plates at > 90% confluence in NMM supplemented with growth factors at 1: 4. The next day, day 0 of differentiation, the medium was changed to fresh NMM without growth factors. Neurons undergoing differentiation were maintained in NMM for 4 or more weeks with 50% media changed twice weekly. Using TrypLE to 125,000 cells/cm as required2Replating the cells at the density of (1).
V3/intron: knock-down (KD) of V3 RNA transcript and intron RNA transcript was measured in ALS neurons. Both wild type and repeated sequence-containing V3 transcripts (designated "healthy allele" V3 and "pathological allele" V3 in fig. 1) were knocked down. However, it should be noted that, while the present disclosure is not bound by any particular theory, transcripts containing repeated sequences may be retained in the nucleus for longer periods of time and thus may be preferentially knocked down. The intron transcript is indicated by the AS arrow backwards in figure 1. Two numbers represent V3 and intron knockdown; for example, for WV-6408, V3 was knocked down 59% and introns were knocked down 65%.
Stability:
in vitro stability assays were performed using mouse (Ms) brain homogenates.
TLR9：
TLR9 reporter assay protocol: determination using human TLR 9or mouse TLR9 reporter (HEK-Blue)TMTLR9 cells, Invivoge (InvivoGen), San Diego (San Diego), Calif.) were analyzed for induction of NF-. kappa.B (NF-. kappa.B-inducible SEAP) activity. Oligonucleotides at a concentration of 50 μ M (330 μ g/mL) and 2-fold serial dilutions were plated into 96-well plates at a final volume of 20 μ L in water. HEK-BlueTMTLR9 cells at 7.2x104Density of Individual cells to 180. mu.L volume of HEK Blue per wellTMDetecting in the culture medium. Final work of oligonucleotides in these wellsConcentrations were 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078 and 0.0375 μ M. HEK-BlueTMTLR9 cells with oligonucleotides at 37 ℃ and 5% CO2Incubate for 16 hours. At the end of the incubation, the absorbance at 655nM was measured by Spectramax. Water is the negative control. Positive controls were WV-2021 and ODN 2359(CpG oligonucleotide). Results are expressed as fold change in NF-. kappa.B activation relative to vehicle control treated cells. Reference: human TLR9 agonist kit (invitrogen, san diego, ca). In this assay, an oligonucleotide is considered to be "cleared" if no or substantially no activity is detected. In some experiments, WV-8005, WV-8006, WV-8007, WV-8008, WV-8009, WV-8010, WV-8011, WV-8012, and WV-8321 did not show appreciable hTLR9 activity, but some showed less mTRL9 activity.
Complement
In some embodiments, complement is evaluated in a cynomolgus monkey serum complement activation ex vivo assay. The effect of oligonucleotides on complement activation was measured in ex vivo cynomolgus monkey serum. Serum samples from 3 individual male cynomolgus monkeys were pooled and the pool used for the study.
The time course of C3a production was measured by incubating oligonucleotide or water control at a final concentration of 330. mu.g/mL in freshly thawed cynomolgus monkey serum (1: 30 ratio, v/v) at 37 ℃. Specifically, 9.24 μ L of 10mg/mL oligonucleotide stock in vehicle or vehicle alone was added to 270.76 μ L of pooled serum and the resulting mixture was incubated at 37 ℃. At 0, 5, 10 and 30 minutes, 20- μ L aliquots were collected and the reaction was immediately stopped by the addition of 2.2 μ L of 18mg/mL EDTA.
The concentration of C3a was measured at a dilution of 1: 3000 using a MicroVue C3a plus enzyme immunoassay. The results are presented as an increase in the concentration of complement lysis products following treatment of pooled sera with oligonucleotides compared to vehicle control treatment.
PD (pharmacodynamics) (C9-BAC, icv or intracerebroventricular injection):
PD and efficacy were tested in a C9orf72-BAC (C9-BAC) mouse model:
transgenic mice for in vivo pharmacological studies have been described in O' Rourke et al 2015 Neuron. [ neurons ]88 (5): 892-. Briefly, a Bacterial Artificial Chromosome (BAC) clone derived from fibroblasts from patients with Amyotrophic Lateral Sclerosis (ALS) was used to design a transgene construct such that the open reading frame 72 gene of human chromosome 9 (C9orf72) has a hexanucleotide repeat expansion (GGGGCC) in an alternatively spliced intron between non-coding first exons 1a and 1b (variant 3). BAC isolated a sequence of approximately 166kbp (human C9orf72 genomic sequence of approximately 36kbp, with approximately 110kbp being the upstream sequence and approximately 20kbp being the downstream sequence). After amplification of different BAC subclones, one subclone restricted to 100-fold 1000 GGGGCC repeats was used. The Tg (C9orf72_3) line 112 mice (JAX stock No. 023099, Jackson Laboratories, Barr Harbor, Maine) had several tandem copies of the C9orf72_3 transgene, each copy having 100-1000 repeats ([ GGGGGGCC ] 100-1000). However, only mice expressing 500 or more repeats were selected for in vivo studies as used herein.
In vivo procedure:
for the injection of oligonucleotides into the lateral ventricle, mice were anesthetized and placed on rodent stereotaxic equipment; a stainless steel guide cannula (coordinates: posterior to bregma-0.3 mm, transverse +1.0mm and vertical-2.2 mm) was then implanted in one of its lateral ventricles and fixed in place using dental cement. Mice were allowed to spend a one week recovery period prior to compound injection. A typical pharmacological study involves the injection of 50. mu.g of oligonucleotide in a volume of up to 2.5. mu.l on day 1, followed by another injection of the same amount and volume on day 8. Euthanasia was performed on day 15; mice were anesthetized deeply with avermectin and perfused transcardially with saline. The brain was quickly removed from the skull, one hemisphere was processed for histological analysis, and the other hemisphere was dissected and frozen on dry ice for biochemical analysis. Similarly, the spinal cords were dissected and either frozen on dry ice (lumbar spinal cord) or processed for histological analysis (cervical/thoracic spinal cord).
Efficacy (C9-BAC): focus:
tissue preparation and histological analysis
The brains and spinal cords were fixed in 4% paraformaldehyde for 24 hours, then transferred to 30% sucrose for 24-48 hours and frozen in liquid nitrogen. Successive sagittal sections 20- μm thick were excised at-18 ℃ in a cryostat and placed on Superfrost slides.
Efficacy (C9-BAC): PolyGP (DPR assay):
tissue preparation for protein and PolyGP quantification:
brain and spinal cord samples were processed using a 2-step extraction procedure; after each step, centrifugation was carried out at 10,000rpm for 10 minutes at 4 ℃. The first step consists of: samples were incubated in RIPA (50mM Tris, 150mM NaCl, 0.5% DOC, 1% NP40, 0.1% SDS and Complete)TMpH 8.0). The second step consists of: the pellet was resuspended in 5M guanidine-HCl.
PolyGP was quantified in each pool using a mesoscale-based assay. Briefly, polyclonal antibody AB1358 (Millipore, available from Millipore Sigma, billeric, Billerica, massachusetts) was used as the capture and detection antibody. The multi-array 96Sm drop-on SECTOR plates were coated overnight at 4 ℃ with 1. mu.l of 10ug/ml purified anti-polyGP antibody (available from Millibo Sigma, Billerica, Mass.) in PBS directly on a small dot. After 3 washes with PBST (0.05% Tween-20 in PBS), the plates were blocked with MSD blocker A kit (R93AA-2) or 10% FBS/PBS for 1 hour at room temperature. Poly-GP purified from HEK-293 cells (by anti-FLAG affinity purification after plasmid transfection, customized by Genescript) was serially diluted with 10% FBS/PBS and used as standards. Mu.l of the standard poly-GP and sample (diluted or undiluted) were added to each well and incubated for 1-2 hours at room temperature. After 3 washes with PBST, 25ul of sulfo-labeled anti-GP (AB1358) was added per well and incubated for an additional hour at room temperature. The plates were then washed 3 times, 150. mu.l/well of MSD reading buffer T (1X) (R92TC-2, MSD) were added to each well and read by MSD (MESO QUICKPLEX SQ 120) according to the manufacturer's default settings.
Expression of C9orf72 protein was determined by western blotting. Briefly, proteins from RIPA extracts were size fractionated by 4% -12% SDS-PAGE (standard gel, burle corporation (Bio-Rad)) and transferred to PVDF membranes. To detect C9orf72, membranes were immunoblotted with the mouse monoclonal anti-C9 orf72 antibody GT779 (1: 2000; GeneTex, Irvine, Calif.) followed by DyLight-conjugated secondary antibody. Visualization was performed using an Odyssey/Li-Cor imaging system.
Some other abbreviations:
cx: cortex (cortex)
HP: sea horse body
KD: knock down
SC: spinal cord
Str: striatum body
TABLE 8 Activity of various c9orf72 oligonucleotides.
In tables 8A to 8X, 10 μ M of each c9orf72 oligonucleotide was tested in ALS Motor Neurons (MN). These oligonucleotides differ in, inter alia, base sequence, chemical pattern (e.g., pattern of 2' sugar modifications), backbone internucleotide linkage pattern, and/or stereochemical pattern. In tables 8A to 8X, the remaining levels of various c9orf72 transcripts (e.g., all transcripts or only V3, V1, intron 1, etc.) relative to HPRT1 after treatment with c9orf72 oligonucleotides are shown, where 1.000 would represent 100% relative transcript levels (no knockdown) and 0.000 would represent 0% relative transcript levels (e.g., 100% knockdown). In tables 8A to 8X, the results of the repeated experiments are shown.
TABLE 8A. Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript)
| WV-3688 | 0.619 | 0.817 | 0.806 |
| WV-7124 | 0.800 | 0.641 | 0.711 |
| WV-6408 | 0.646 | 0.574 | 0.582 |
| WV-7130 | 0.344 | 0.321 | 1.070 |
| WV-8550 | 0.310 | 0.253 | 0.316 |
| WV-8011 | 0.113 | 0.144 | 0.111 |
| WV-8012 | 0.157 | 0.185 | 0.153 |
| WV-2376 | 1.188 | 1.108 | 1.180 |
| WV-9491 | 1.034 | 1.027 | 1.108 |
| WV-5302 | 1.140 | 1.101 | 1.078 |
| WV-6493 | 1.056 | 1.049 | 1.063 |
| WV-8552 | 1.300 | 1.140 | 0.932 |
| Water (W) | 0.834 | 1.041 | 0.985 |
TABLE 8B Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts)
TABLE 8C activity of various C9orf72 oligonucleotides (residual level of V1C 9orf72 transcript)
| WV-3688 | 0.901 | 0.829 | 0.655 |
| WV-7124 | 0.594 | 0.829 | 0.702 |
| WV-6408 | 0.784 | 0.732 | 0.888 |
| WV-7130 | 0.476 | 0.539 | 0.972 |
| WV-8550 | 0.379 | 0.341 | 0.466 |
| WV-8011 | 0.207 | 0.279 | 0.216 |
| WV-8012 | 0.250 | 0.241 | 0.291 |
| WV-2376 | 0.993 | 0.864 | 0.920 |
| WV-9491 | 1.156 | 0.946 | 1.049 |
| WV-5302 | 0.920 | 1.101 | 0.933 |
| WV-6493 | 1.056 | 0.858 | 1.071 |
| WV-8552 | 0.901 | 1.148 | 1.140 |
| Water (W) | 1.197 | 0.846 | 0.999 |
TABLE 8D Activity of various C9orf72 oligonucleotides (remaining level of intron 1C 9orf72 transcript)
TABLE 8E Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript)
| WV-3688 | 0.619 | 0.817 | 0.806 |
| WV-6408 | 0.646 | 0.574 | 0.582 |
| WV-8550 | 0.310 | 0.253 | 0.316 |
| WV-3662 | 0.105 | 0.121 | 0.119 |
| WV-7188 | 0.065 | 0.074 | 0.062 |
| WV-9494 | 0.009 | 0.006 | 0.009 |
| WV-6936 | 0.795 | 0.972 | 0.800 |
| WV-7027 | 0.741 | 0.882 | 0.900 |
| WV-8595 | 0.926 | 0.741 | 0.919 |
| WV-2376 | 1.188 | 1.108 | 1.180 |
| WV-9491 | 1.034 | 1.027 | 1.108 |
| WV-5302 | 1.140 | 1.101 | 1.078 |
| Water (W) | 0.834 | 1.041 | 0.985 |
TABLE 8F Activity of various c9orf72 oligonucleotides (residual levels of all V C9orf72 transcripts)
TABLE 8G Activity of various C90rf72 oligonucleotides (residual level of V1C 9orf72 transcript)
| WV-3688 | 0.901 | 0.829 | 0.655 |
| WV-6408 | 0.784 | 0.732 | 0.888 |
| WV-8550 | 0.379 | 0.341 | 0.466 |
| WV-3662 | 0.185 | 0.099 | 0.182 |
| WV-7188 | 0.114 | 0.128 | 0.106 |
| WV-9494 | 0.023 | 0.018 | 0.026 |
| WV-6936 | 0.913 | 0.939 | 0.907 |
| WV-7027 | 0.702 | 0.757 | 0.926 |
| WV-8595 | 0.952 | 0.959 | 0.959 |
| WV-2376 | 0.993 | 0.864 | 0.920 |
| WV-9491 | 1.156 | 0.946 | 1.049 |
| WV-5302 | 0.920 | 1.101 | 0.933 |
| Water (W) | 1.197 | 0.846 | 0.999 |
TABLE 8H Activity of various C9orf72 oligonucleotides (remaining level of intron 1C 9orf72 transcript)
TABLE 8I Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript)
| WV-8550 | 0.310 | 0.253 | 0.316 |
| WV-8011 | 0.113 | 0.144 | 0.111 |
| WV-8012 | 0.157 | 0.185 | 0.153 |
| WV-9493 | 1.013 | 0.978 | 1.034 |
| WV-9492 | 0.784 | 0.811 | 0.741 |
| WV-3536 | 0.789 | 0.510 | 0.678 |
| WV-2376 | 1.188 | 1.108 | 1.180 |
| Water (W) | 0.834 | 1.041 | 0.985 |
TABLE 8J Activity of various c9orf72 oligonucleotides (residual levels of all V C9orf72 transcripts)
| WV-8550 | 0.686 | 0.538 | 0.667 |
| WV-8011 | 0.440 | 0.446 | 0.495 |
| WV-8012 | 0.597 | 0.509 | 0.565 |
| WV-9493 | 1.122 | 1.084 | 1.069 |
| WV-9492 | 1.107 | 0.816 | 0.924 |
| WV-3536 | 0.991 | 0.783 | 0.977 |
| WV-2376 | 1.092 | 1.012 | 0.944 |
| Water (W) | 1.122 | 0.950 | 1.122 |
TABLE 8K Activity of various C9orf72 oligonucleotides (residual level of V1C 9orf72 transcript)
TABLE 8L Activity of various C9orf72 oligonucleotides (remaining level of intron 1C 9orf72 transcript)
| WV-8550 | 0.443 | 0.350 | 0.298 |
| WV-8011 | 0.336 | 0.378 | 0.434 |
| WV-8012 | 0.446 | 0.446 | 0.475 |
| WV-9493 | 0.917 | 0.838 | 0.917 |
| WV-9492 | 1.075 | 0.714 | |
| WV-3536 | 0.710 | 0.969 | 1.061 |
| WV-2376 | 0.685 | 0.681 | 0.714 |
| Water (W) | 1.315 | 1.193 | 0.990 |
TABLE 8M Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript)
TABLE 8N activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts)
| WV-3688 | 0.940 | 0.847 | 0.813 |
| WV-7124 | 0.737 | 0.764 | 1.022 |
| WV-6408 | 0.774 | 0.717 | 0.646 |
| WV-7130 | 0.591 | 0.559 | 0.525 |
| WV-8550 | 0.567 | 0.536 | 0.555 |
| WV-8011 | 0.451 | 0.421 | 0.421 |
| WV-8012 | 0.451 | 0.429 | 0.470 |
| WV-2376 | 1.182 | 1.029 | 1.058 |
| WV-3542 | 0.966 | 0.902 | 0.871 |
| WV-9491 | 1.087 | 0.973 | 0.933 |
| WV-5302 | 0.902 | 0.966 | 0.980 |
| WV-6493 | 1.043 | 0.966 | 0.947 |
| WV-8552 | 1.149 | 1.087 | 0.947 |
| Water (W) | 0.895 | 1.029 | 0.987 |
TABLE 8O activity of various C9orf72 oligonucleotides (residual level of V1C 9orf72 transcript)
TABLE 8P Activity of various C9orf72 oligonucleotides (remaining level of intron 1C 9orf72 transcript)
| WV-3688 | 0.324 | 0.481 | 0.626 |
| WV-7124 | 0.734 | 0.354 | 0.181 |
| WV-6408 | 0.261 | 0.340 | 0.548 |
| WV-7130 | 0.452 | 0.288 | 0.449 |
| WV-8550 | 0.484 | 0.382 | 0.424 |
| WV-8011 | 0.391 | 0.296 | |
| WV-8012 | 0.461 | 0.508 | 0.375 |
| WV-2376 | | 1.038 | 1.269 |
| WV-3542 | 1.184 | 0.879 | 0.600 |
| WV-9491 | 1.060 | 1.023 | 1.674 |
| WV-5302 | 1.217 | 1.295 | 1.097 |
| WV-6493 | 1.136 | 1.418 | |
| WV-8552 | 1.128 | 1.332 | 0.776 |
| Water (W) | 0.968 | 0.903 | 0.685 |
TABLE 8Q activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript)
Table 8r. activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts)
| WV-3688 | 0.940 | 0.847 | 0.813 |
| WV-6408 | 0.774 | 0.717 | 0.646 |
| WV-8550 | 0.567 | 0.536 | 0.555 |
| WV-3662 | 0.261 | 0.235 | 0.238 |
| WV-7118 | 0.276 | 0.263 | 0.291 |
| WV-9494 | 0.046 | 0.043 | 0.047 |
| WV-6936 | 1.014 | 1.007 | 1.007 |
| WV-7027 | 1.065 | 0.966 | 0.947 |
| WV-8595 | 0.994 | 0.818 | 0.830 |
| WV-2376 | 1.182 | 1.029 | 1.058 |
| WV-3542 | 0.966 | 0.902 | 0.871 |
| WV-9491 | 1.087 | 0.973 | 0.933 |
| Water (W) | 0.895 | 1.029 | 0.987 |
TABLE 8S Activity of various C9orf72 oligonucleotides (residual level of V1C 9orf72 transcript)
TABLE 8T Activity of various C9orf72 oligonucleotides (remaining level of intron 1C 9orf72 transcript)
| WV-3688 | 0.324 | 0.481 | 0.626 |
| WV-6408 | 0.261 | 0.340 | 0.548 |
| WV-8550 | 0.484 | 0.382 | 0.424 |
| WV-3662 | 0.995 | 0.831 | 0.891 |
| WV-7118 | 0.596 | 0.724 | 0.584 |
| WV-9494 | 0.699 | 0.455 | 0.556 |
| WV-6936 | | 1.144 | 0.948 |
| WV-7027 | 0.729 | 1.176 | 1.260 |
| WV-8595 | 1.045 | 0.837 | 1.209 |
| WV-2376 | | 1.038 | 1.269 |
| WV-3542 | 1.184 | 0.879 | 0.600 |
| WV-9491 | 1.060 | 1.023 | |
| Water (W) | 0.968 | 0.903 | 0.685 |
TABLE 8U activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript)
| WV-8550 | 0.297 | 0.286 | 0.260 |
| WV-8011 | 0.135 | 0.123 | 0.097 |
| WV-8012 | 0.111 | 0.162 | 0.106 |
| WV-9493 | 0.761 | 0.705 | 0.649 |
| WV-9492 | 0.506 | 0.520 | 0.478 |
| WV-3536 | 0.663 | 0.606 | 0.805 |
| WV-2376 | 0.833 | 1.033 | 1.092 |
| Water (W) | 0.899 | 1.122 | 1.122 |
TABLE 8V. Activity of various c9orf72 oligonucleotides (residual levels of all V C9orf72 transcripts)
| WV-8550 | 0.567 | 0.536 | 0.555 |
| WV-8011 | 0.451 | 0.421 | 0.421 |
| WV-8012 | 0.451 | 0.429 | 0.470 |
| WV-9493 | 1.014 | 0.824 | 0.807 |
| WV-9492 | 0.859 | 0.818 | 0.801 |
| WV-3536 | 0.830 | 0.790 | 1.126 |
| WV-2376 | 1.182 | 1.029 | 1.058 |
| Water (W) | 0.895 | 1.029 | 0.987 |
TABLE 8W Activity of various C9orf72 oligonucleotides (residual level of V1C 9orf72 transcript)
| WV-8550 | 0.562 | 0.426 | 0.384 |
| WV-8011 | 0.213 | 0.235 | 0.272 |
| WV-8012 | 0.368 | 0.283 | 0.351 |
| WV-9493 | 1.049 | 0.870 | 0.586 |
| WV-9492 | 0.993 | 0.795 | 0.758 |
| WV-3536 | 0.683 | 0.697 | 1.021 |
| WV-2376 | 1.086 | 0.835 | 0.858 |
| Water (W) | 1.079 | 0.870 | 0.889 |
TABLE 8X Activity of various C9orf72 oligonucleotides (remaining level of Intron 1C 9orf72 transcript)
| WV-8550 | 0.484 | 0.382 | 0.424 |
| WV-8011 | 0.391 | 0.296 | 0.781 |
| WV-8012 | 0.461 | 0.508 | 0.375 |
| WV-9493 | 0.391 | 0.942 | 0.724 |
| WV-9492 | 0.481 | 0.989 | 0.942 |
| WV-3536 | 0.729 | 0.948 | 0.580 |
| WV-2376 | | 1.038 | 1.269 |
| Water (W) | 0.968 | 0.903 | 0.685 |
TABLE 9 Activity of various c9orf72 oligonucleotides.
In tables 9A to 9D, 1 μ M of each c9orf72 oligonucleotide was tested in ALS Motor Neurons (MN). These oligonucleotides differ in, inter alia, base sequence, chemical pattern (e.g., pattern of 2' sugar modifications), backbone internucleotide linkage pattern, and/or stereochemical pattern. In tables 9A to 9D, the remaining levels of various c9orf72 transcripts (e.g., all transcripts or only V3, V1, intron 1, etc.) relative to HPRT1 after treatment with c9orf72 oligonucleotides are shown, where 1.000 would represent 100% relative transcript levels (no knockdown) and 0.000 would represent 0% relative transcript levels (e.g., 100% knockdown). In tables 9A to 9D, the results of the repeated experiments are shown.
TABLE 9A. Activity of various C9orf72 oligonucleotides (residual levels of V3C 9orf72 transcripts)
| WV-8550 | 0.557 | 0.672 |
| WV-8011 | 0.389 | 0.417 |
| WV-9505 | 0.370 | 0.378 |
| WV-9506 | 0.465 | 0.446 |
| WV-9507 | 0.799 | 0.822 |
| WV-9508 | 0.502 | 0.478 |
| WV-9509 | 0.428 | 0.397 |
| WV-9510 | 0.589 | 0.478 |
| WV-2376 | 1.047 | 1.018 |
| Water (W) | 0.899 | 1.122 |
TABLE 9B Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts)
TABLE 9C activity of various C9orf72 oligonucleotides (residual level of V1C 9orf72 transcript)
| WV-8550 | 0.758 | 0.979 |
| WV-8011 | 1.000 | 0.818 |
| WV-9505 | 0.702 | 0.603 |
| WV-9506 | 0.476 | 0.972 |
| WV-9507 | 0.993 | 1.265 |
| WV-9508 | 0.870 | 0.926 |
| WV-9509 | 0.907 | 0.806 |
| WV-9510 | 1.109 | 1.049 |
| WV-2376 | 1.301 | 1.310 |
| Water (W) | 1.079 | 0.870 |
TABLE 9D Activity of various C9orf72 oligonucleotides (remaining level of intron 1C 9orf72 transcript)
| WV-8550 | 0.781 | 0.533 |
| WV-8011 | 1.002 | 0.600 |
| WV-9505 | 1.009 | 0.916 |
| WV-9506 | 0.910 | 0.765 |
| WV-9507 | 0.634 | 0.843 |
| WV-9508 | 0.724 | 0.657 |
| WV-9509 | 0.512 | 0.873 |
| WV-9510 | 0.245 | 1.045 |
| WV-2376 | 1.128 | 1.226 |
| Water (W) | 0.968 | 0.903 |
TABLE 10 Activity of various c9orf72 oligonucleotides.
In tables 10A to 10B, various c9orf72 oligonucleotides were tested at various concentrations between 0.01 μ M to 10 μ M in ALS Motor Neurons (MN). These oligonucleotides differ in inter-backbone internucleotide linkage patterns and/or stereochemical patterns, among others. In the DNA core, various oligonucleotides contain one or two SSOs [ 5 '-PS (phosphorothioate) in Sp configuration, PS in Sp configuration, PO (phosphodiester) -3' ] or one or two SSRs [ 5 '-PS (phosphorothioate) in Sp configuration, PS in Rp configuration-3'). In tables 10A to 10B, the remaining levels of various c9orf72 transcripts (e.g., all transcripts or V3 only) relative to HPRT1 after treatment with c9orf72 oligonucleotides are shown, where 1.000 would represent 100% relative transcript levels (no knockdown) and 0.000 would represent 0% relative transcript levels (e.g., 100% knockdown). In tables 10A to 10B, the results of the repeated experiments are shown.
TABLE 10A. Activity of various c9orf72 oligonucleotides (residual levels of all V C9orf72 transcripts)
TABLE 10B Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript)
TABLE 11 Activity of various c9orf72 oligonucleotides.
In Table 11A and 11B, 10. mu.M of each c9orf72 oligonucleotide was tested in ALS Motor Neurons (MN). These oligonucleotides differ in, inter alia, base sequence, internucleotide linkage pattern, and chemical pattern (e.g., 2' modification pattern of sugars), with some oligonucleotides having a symmetric form (e.g., table 11B) and some oligonucleotides having an asymmetric form (e.g., table 11A). In table 11A and table 11B, the remaining levels of V3 c9orf72 transcripts relative to HPRT1 after treatment with c9orf72 oligonucleotides are shown, where 1.000 would represent 100% relative transcript levels (no knockdown) and 0.000 would represent 0% relative transcript levels (e.g., 100% knockdown). In table 11A and table 11B, the results of the repeated experiments are shown. In this and other tables, not all positive and negative controls performed in the various experiments are necessarily shown.
TABLE 11A. Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript)
TABLE 11B Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript)
TABLE 12 Activity of various c9orf72 oligonucleotides.
In Table 12A and Table 12B, 2.5. mu.M or 10. mu.M of each c9orf72 oligonucleotide was tested in ALS Motor Neurons (MN). These oligonucleotides differ in, inter alia, base sequence, internucleotide linkage pattern, and chemical pattern (e.g., 2' modification pattern of sugars), some of which have a symmetric form and some of which have an asymmetric form. In table 12A and table 12B, the remaining levels of V3 or all V c9orf72 transcripts relative to HPRT1 after treatment with c9orf72 oligonucleotides are shown, where 1.000 would represent 100% relative transcript levels (no knockdown) and 0.000 would represent 0% relative transcript levels (e.g., 100% knockdown). In table 12A and table 12B, the results of the repeated experiments are shown. In this and other tables, not all positive and negative controls performed in the various experiments are necessarily shown.
TABLE 12A. Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript)
TABLE 12B Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts)
TABLE 13 Activity of various c9orf72 oligonucleotides.
In tables 13A to 13F, various c9orf72 oligonucleotides were tested in c9 BAC mice; the c9orf72 oligonucleotide was administered to mice ICV at 50 μ g each, two doses divided by one week, and tissues were collected one week after the second dose. These oligonucleotides differ in, inter alia, base sequence, internucleotide linkage pattern, and chemical pattern (e.g., 2' modification pattern of sugars), some of which have a symmetric form and some of which have an asymmetric form. In tables 13A to 13F, the remaining levels of c9orf72 transcripts [ e.g., all transcripts (all V) or only V3] relative to HPRT1 after treatment with c9orf72 oligonucleotides are shown, where 1.000 would represent 100% relative transcript levels (no knockdown) and 0.000 would represent 0% relative transcript levels (e.g., 100% knockdown). Results of repeated experiments are shown. The tissue evaluated: SC, spinal cord; and CX, cerebral cortex.
TABLE 13A Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts in CX)
TABLE 13B Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript in CX)
TABLE 13C Activity of various C9orf72 oligonucleotides (remaining level of Intron 1/AS C9orf72 transcript in CX)
| PBS | WV-8548 | WV-12482 | WV-12483 | WV-12444 | WV-12448 |
| 0.426 | 1.124 | 1.248 | 0.619 | 2.712 | 1.256 |
| 0.441 | 0.840 | 1.944 | 2.113 | 2.344 | 2.280 |
| 0.852 | 0.846 | | 3.072 | 0.993 | 2.377 |
| 1.213 | 0.646 | | 3.137 | 0.888 | |
| 1.433 | 0.325 | | 3.704 | 1.109 | 2.693 |
| 1.230 | | 1.453 | 3.247 | | 1.568 |
| 1.180 | 0.673 | | | 1.740 |
| 1.404 | 1.827 | 0.301 | 1.931 | 2.218 | 0.864 |
TABLE 13D Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts in SC)
| PBS | WV-8548 | WV-12482 | WV-12483 | WV-12444 | WV-12448 |
| 1.635 | 0.747 | 0.692 | 0.603 | 0.528 | 0.747 |
| 0.999 | 1.042 | 0.747 | 1.504 | 0.673 | 0.507 |
| 1.525 | 0.768 | 0.692 | 0.536 | 0.779 | 0.659 |
| 0.742 | 0.835 | 0.721 | 0.598 | 0.806 | 0.727 |
| 0.779 | 0.717 | 0.603 | 0.632 | 0.551 | 0.712 |
| 0.678 | 1.172 | 0.615 | 1.515 | 0.574 | 0.517 |
| 0.697 | 0.727 | 0.795 | 0.558 | 0.574 | 0.586 |
| 0.945 | 0.939 | 0.578 | 0.582 | 0.795 | 0.558 |
TABLE 13E Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript in SC)
| PBS | WV-8548 | WV-12482 | WV-12483 | WV-12444 | WV-12448 |
| 1.325 | 0.681 | 0.686 | 0.465 | 0.513 | 0.805 |
| 1.122 | 1.307 | 0.735 | 0.816 | 0.746 | 0.355 |
| 1.382 | 0.725 | 0.827 | 0.408 | 0.905 | 0.725 |
| 0.788 | 0.772 | 0.799 | 0.389 | 0.856 | 0.475 |
| 0.874 | 0.499 | 0.672 | 0.416 | 0.557 | 0.833 |
| 0.761 | 0.887 | 0.527 | 0.931 | 0.550 | 0.309 |
| 0.777 | 0.715 | 1.069 | 0.443 | 0.557 | 0.301 |
| 0.970 | 0.950 | 0.431 | 0.482 | 0.816 | 0.499 |
TABLE 13F Activity of various C9orf72 oligonucleotides (remaining level of Intron 1/AS C9orf72 transcript in SC)
| PBS | WV-8548 | WV-12482 | WV-12483 | WV-12444 | WV-12448 |
| 1.812 | 0.054 | 1.070 | 0.065 | 0.070 | 0.869 |
| 1.942 | 1.545 | 0.998 | | 0.241 | 0.074 |
| 0.055 | 1.163 | 0.258 | 0.131 | 0.438 |
| 0.528 | 0.075 | 1.503 | 0.281 | 0.072 | 0.721 |
| 0.789 | 0.149 | 0.124 | 0.381 | 0.099 | 0.091 |
| 0.701 | 2.293 | 0.015 | | 0.057 | 0.058 |
| 0.757 | 0.450 | 0.206 | 0.129 | 0.358 | 0.016 |
| 0.472 | | 0.027 | 0.287 | | 0.021 |
TABLE 14 Activity of various c9orf72 oligonucleotides.
In tables 14A-14B, various c9orf72 oligonucleotides were tested in motor neurons, with the oligonucleotides being delivered in vitro at concentrations between 0.003 μ M and 10 μ M (concentrations provided in the form of exp 10). The c9orf72 oligonucleotide WV-11532 tested contained three neutral internucleotide linkages. In table 14A and table 14B, the remaining levels of c9orf72 transcripts [ e.g., all transcripts (all V) or only V3] relative to HPRT1 after treatment with c9orf72 oligonucleotides are shown, where 1.000 would represent 100% relative transcript levels (no knockdown) and 0.000 would represent 0% relative transcript levels (e.g., 100% knockdown). Results of repeated experiments are shown.
TABLE 14A. Activity of various c9orf72 oligonucleotides (residual levels of all V C9orf72 transcripts)
TABLE 14B Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript)
TABLE 15 Activity of various c9orf72 oligonucleotides.
In tables 15A to 15H, various c9orf72 oligonucleotides targeting AS (antisense strand) were tested in c9 BAC mice; the c9orf72 oligonucleotide was administered to mice ICV at 50 μ g each, two doses divided by one week, and tissues were collected one week after the second dose. In table 15A and table 15H, the remaining levels of AS (antisense strand) c9orf72 transcripts relative to HPRT1 after treatment with c9orf72 oligonucleotides are shown, where 1.000 would represent 100% relative transcript levels (no knockdown) and 0.000 would represent 0% relative transcript levels (e.g., 100% knockdown). Results of repeated experiments are shown. The tissue evaluated: SC, spinal cord; and CX, cerebral cortex.
TABLE 15A Activity of various C9orf72 oligonucleotides (residual level of AS C9orf72 transcript in CX)
TABLE 15B Activity of various C9orf72 oligonucleotides (residual level of AS C9orf72 transcript in SC)
| PBS | 0.932 | 1.048 | 1.020 | | |
| WV-3542 | 1.238 | 1.131 | 0.965 | 1.155 | 0.882 |
| WV-7117 | 0.645 | 0.472 | 0.687 | 0.389 | 0.363 |
| WV-5969 | 1.108 | 0.971 | 1.213 | 1.247 | 1.213 |
| WV-5979 | 1.264 | 0.965 | 1.085 | 0.846 | 0.913 |
| WV-5980 | 0.397 | 1.070 | 1.027 | 0.823 | 1.355 |
| WV-5981 | 0.876 | 0.992 | 1.085 | 0.823 | 1.155 |
| WV-5982 | 1.238 | 1.171 | 0.806 | 0.811 | 0.664 |
| WV-5985 | 0.741 | 0.817 | 0.925 | 0.773 | 0.789 |
| WV-5987 | 0.659 | 0.517 | 0.602 | 0.757 | 0.566 |
TABLE 15C Activity of various C9orf72 oligonucleotides (residual level of AS C9orf72 transcript in CX)
TABLE 15D Activity of various C9orf72 oligonucleotides (residual level of AS C9orf72 transcript in SC)
| PBS | 0.843 | 0.860 | 1.037 | | 1.260 |
| WV-3542 | 1.313 | 1.217 | 1.304 | 1.260 | 1.037 |
| WV-7117 | 0.781 | 0.362 | 0.357 | 0.458 | 1.151 |
| WV-5967 | 1.437 | 1.097 | 0.909 | 1.151 | 1.175 |
| WV-5970 | 1.313 | 1.030 | 0.808 | 1.175 | 1.097 |
| WV-5971 | 1.628 | 3.102 | 1.313 | 1.417 | 1.794 |
| WV-5972 | 1.572 | 2.757 | 1.819 | 1.794 | 2.060 |
| WV-5973 | 1.143 | 0.922 | 1.192 | 1.313 | 1.304 |
| WV-5974 | 1.467 | 1.674 | 1.175 | 1.037 | 1.407 |
| WV-5978 | 0.975 | 1.104 | 1.081 | 0.848 | 1.030 |
TABLE 15E Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts in CX)
| PBS | 1.061 | 0.943 | 0.997 | | |
| WV-3542 | 1.068 | 1.025 | 1.061 | 1.137 | 1.235 |
| WV-7117 | 0.443 | 0.388 | 0.787 | 0.509 | 0.561 |
| WV-5969 | 1.270 | 1.083 | 1.137 | 1.113 | 1.046 |
| WV-5979 | 1.161 | 1.083 | 1.010 | 1.137 | 1.010 |
| WV-5980 | 1.046 | 1.253 | 1.129 | 1.053 | 1.297 |
| WV-5981 | 0.917 | 0.983 | 0.815 | 0.892 | 1.053 |
| WV-5982 | 1.017 | 1.039 | 0.886 | 1.068 | 1.075 |
| WV-5985 | 1.075 | 1.169 | 1.177 | 1.161 | 0.868 |
| WV-5987 | 0.990 | 1.032 | 1.153 | 1.010 | 0.868 |
TABLE 15F Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts in SC)
Table 15G Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts in CX)
| PBS | 1.073 | 0.859 | 1.095 | 0.818 | 0.973 |
| WV-3542 | 0.813 | 0.824 | 0.947 | 1.149 | 0.830 |
| WV-7117 | 0.470 | 0.274 | 0.703 | 0.563 | 1.001 |
| WV-5967 | 0.404 | 0.818 | 0.973 | 1.065 | 0.896 |
| WV-5970 | 0.987 | 1.095 | 0.960 | 0.973 | 1.118 |
| WV-5971 | 0.830 | 0.953 | 1.087 | 0.947 | 1.134 |
| WV-5972 | 1.182 | 0.960 | 0.987 | 1.065 | 1.103 |
| WV-5973 | 0.973 | 0.896 | 0.987 | 0.902 | 1.103 |
| WV-5974 | 1.142 | 1.058 | 0.967 | 0.967 | 1.043 |
| WV-5978 | 0.980 | 0.836 | 1.001 | 0.953 | 0.973 |
TABLE 15H Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts in SC)
In tables 15i.1 to 15i.6 and tables 15J.1 to 15j.6, various C9orf72 oligonucleotides were tested in C9 BAC mice. The c9orf72 oligonucleotides tested had different base sequences and different numbers and positions of non-negatively charged internucleotide linkages. The remaining levels of c9orf72 transcripts [ e.g., all transcripts (all V) or only V3] relative to HPRT1 after treatment with c9orf72 oligonucleotides are shown, where 1.000 would represent 100% relative transcript levels (no knockdown) and 0.000 would represent 0% relative transcript levels (e.g., 100% knockdown).
In tables 15i.1 to 15M.3, as well as in each of the other tables herein, C9orf72 transcript levels relative to HPRT1 are shown, and data from repeated experiments are shown.
TABLE 15I.1 Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts in CX)
| PBS | WV-12441 | WV-13803 | WV-13804 | WV-13805 |
| 0.94 | 0.89 | 0.70 | 0.91 | 0.90 |
| 1.02 | 0.71 | 0.76 | 0.71 | 0.77 |
| 0.94 | 0.77 | 0.54 | 0.70 | 0.72 |
| 0.98 | 0.81 | 0.60 | 0.66 | 0.75 |
| 1.09 | 0.62 | 0.76 | 0.66 | 0.73 |
| 1.08 | 0.75 | 0.63 | 0.78 | 0.68 |
| 0.94 | 0.73 | 0.39 | 0.81 | 0.81 |
| 0.58 | 0.63 | 0.57 | 0.80 |
TABLE 15I.2 Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript in CX)
TABLE 15I.3 Activity of various c90rf72 oligonucleotides (remaining level of Intron 1 transcript in CX)
| PBS | WV-12441 | WV-13803 | WV-13804 | WV-13805 |
| 0.45 | 0.62 | 0.86 | 0.29 | 0.87 |
| 1.54 | 0.61 | 0.99 | 0.40 | 0.84 |
| 0.83 | 0.35 | 0.31 | 0.54 | 0.70 |
| 0.90 | 0.44 | 0.55 | 0.68 | 0.55 |
| 0.82 | 0.18 | 0.86 | 0.73 | 0.31 |
| 1.76 | 0.64 | 0.67 | 0.70 | 0.75 |
| 0.70 | 0.60 | 0.32 | | 1.15 |
| 0.55 | 0.47 | 0.36 | 1.20 |
TABLE 15I.4 Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts in SC)
| PBS | WV-12441 | WV-13803 | WV-13804 | WV-13805 |
| 0.67 | 0.49 | 0.63 | 0.71 |
| 0.88 | 0.99 | 0.44 | 0.62 | 0.60 |
| 0.94 | 0.52 | 0.53 | 0.51 | 0.78 |
| 1.03 | 0.91 | 0.47 | 0.85 | 0.64 |
| 1.16 | 0.78 | 0.59 | 0.43 | 0.99 |
| 1.03 | 0.59 | 0.47 | 0.74 | 0.63 |
| 0.96 | 0.72 | 0.50 | 0.79 | 0.64 |
| 0.43 | 0.49 | 0.51 | 0.82 |
TABLE 15I.5 Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript in SC)
TABLE 15I.6 Activity of various C9orf72 oligonucleotides (remaining level of intron 1C 9orf72 transcript in SC)
| PBS | WV-12441 | WV-13803 | WV-13804 | WV-13805 |
| 0.16 | 0.11 | 0.13 | 0.20 |
| 0.67 | 1.40 | 0.07 | 0.22 | 0.16 |
| 1.72 | 0.22 | 0.04 | 0.09 | 0.64 |
| 1.10 | 0.99 | 0.06 | 0.22 | 0.11 |
| 1.58 | 0.09 | 0.25 | 0.17 | 1.33 |
| 0.45 | 0.27 | 0.08 | 0.44 | 0.15 |
| 0.48 | 0.52 | 0.12 | 0.60 | 0.19 |
| 0.12 | 0.25 | 0.12 | 0.41 |
TABLE 15J.1 Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts in CX)
| PBS | WV-12483 | WV-13806 | WV-13807 | WV-13808 |
| 0.86 | 0.84 | 0.80 | 0.76 | 0.90 |
| 1.03 | 0.91 | 0.92 | 0.60 | 0.89 |
| 0.93 | 0.79 | 0.83 | 0.77 | 0.98 |
| 1.00 | 0.74 | 0.96 | 0.83 | 0.76 |
| 1.05 | 0.73 | 0.68 | 0.71 | 0.85 |
| 1.15 | 0.76 | 0.90 | 0.85 | 0.78 |
| 0.75 | 0.96 | 0.98 | 0.82 |
| 0.79 | 0.79 | 0.86 | 0.81 |
TABLE 15J.2 Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript in CX)
| PBS | WV-12483 | WV-13806 | WV-13807 | WV-13808 |
| 0.98 | 0.71 | 0.79 | 0.88 | 0.94 |
| 1.18 | 0.95 | 1.08 | 0.58 | 0.77 |
| 0.91 | 0.83 | 0.78 | 0.92 | 0.94 |
| 1.03 | 0.82 | 1.08 | 0.82 | 0.73 |
| 1.03 | 0.80 | 0.64 | 0.61 | 1.08 |
| 0.87 | 1.06 | 1.08 | 0.74 | 1.00 |
| 0.83 | 1.19 | 0.94 | 1.11 |
| 0.89 | 0.94 | 0.72 | 0.78 |
TABLE 15J.3 Activity of various c9orf72 oligonucleotides (remaining level of Intron 1 transcript in CX)
| PBS | WV-12483 | WV-13806 | WV-13807 | WV-13808 |
| 0.46 | 0.67 | 1.31 | 1.04 | 1.45 |
| 1.50 | 2.15 | 1.39 | 0.42 | 1.40 |
| 0.95 | 1.13 | 0.75 | 0.63 | 1.98 |
| 1.24 | 1.66 | 1.51 | 1.33 | 1.29 |
| 0.78 | 0.88 | 0.87 | 0.89 | 1.58 |
| 1.08 | 0.45 | 1.16 | 0.88 | 0.96 |
| 0.80 | 0.83 | 1.56 | 1.54 |
| 1.37 | 1.37 | 0.53 | 1.49 |
TABLE 15J.4 Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts in SC)
TABLE 15J.5 Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript in SC)
| PBS | WV-12483 | WV-13806 | WV-13807 | WV-13808 |
| 0.95 | 0.77 | 0.47 | 0.87 | 0.51 |
| 1.06 | 0.49 | 0.49 | 0.60 | 0.54 |
| 1.10 | 0.42 | 0.51 | 0.75 | 0.48 |
| 0.91 | 0.37 | 0.95 | 0.80 | 0.54 |
| 0.95 | 0.48 | 0.40 | 0.48 | 0.76 |
| 1.04 | 0.54 | 0.40 | 0.54 | 0.79 |
| 0.49 | 0.52 | 0.53 | 1.02 |
| 0.56 | 0.49 | 0.37 | 0.79 |
TABLE 15J.6 Activity of various C9orf72 oligonucleotides (remaining level of intron 1C 9orf72 transcript in SC)
| PBS | WV-12483 | WV-13806 | WV-13807 | WV-13808 |
| 0.97 | 0.42 | 0.67 | 0.50 | 0.75 |
| 1.81 | 0.32 | 0.60 | 0.15 | 0.75 |
| 1.10 | 0.35 | 0.74 | 0.24 | 0.59 |
| 0.94 | 0.60 | 1.10 | 1.14 | 0.76 |
| 0.77 | 0.57 | 0.56 | 0.68 | 0.59 |
| 0.41 | 0.12 | 0.72 | 0.58 | 0.39 |
| 0.27 | 0.45 | 0.58 | 0.14 |
| 2.11 | 0.37 | 0.69 | 0.67 |
Tables 15k.1 to 15L.2 show the activity of various C9orf72 oligonucleotides in ALS motor neurons in vitro.
TABLE 15K.1 Activity of various c9orf72 oligonucleotides (residual levels of all V C9orf72 transcripts/HPRT 1)
| WV-13312(1uM) | 0.61 | 0.69 | 0.66 |
| WV-13312(0.2uM) | 0.90 | 0.97 | 0.92 |
| WV-8007(1uM) | 0.69 | 0.80 | 0.71 |
| WV-8007(0.2uM) | 1.05 | 0.83 | 0.92 |
| WV-13313(1uM) | 0.68 | 0.67 | 0.64 |
| WV-13313(0.2uM) | 0.93 | 0.89 | 0.90 |
| WV-8008(1uM) | 0.63 | 0.76 | 0.66 |
| WV-8008(0.2uM) | 0.90 | 0.99 | 0.98 |
| WV-13305(1uM) | 0.63 | 0.68 | 0.71 |
| WV-13305(0.2uM) | 0.72 | 0.96 | 0.88 |
| WV-13308(1uM) | 0.60 | 0.75 | 0.62 |
| WV-13308(0.2uM) | 0.77 | 0.79 | 0.90 |
| WV-13309(1uM) | 0.67 | 0.63 | 0.66 |
| WV-13309(0.2uM) | 0.84 | 0.77 | 0.79 |
| WV-14552(1uM) | 0.70 | 0.71 | 0.65 |
| WV-14552(0.2uM) | 0.79 | 0.73 | 0.81 |
| WV-14553(1uM) | 0.79 | 0.58 | 0.62 |
| WV-14553(0.2uM) | 0.81 | 0.83 | 0.75 |
| WV-14554(1uM) | 0.63 | 0.64 | 0.69 |
| WV-14554(0.2uM) | 0.81 | 0.64 | 0.63 |
| WV-14555(1uM) | 0.70 | 0.66 | 0.62 |
| WV-14555(0.2uM) | 0.65 | 0.78 | 0.88 |
| WV-8550(1uM) | 0.81 | 0.67 | 0.76 |
| WV-8550(0.2uM) | 0.86 | 0.75 | 0.80 |
| Water (W) | 1.17 | 1.13 | 1.02 |
TABLE 15K.2 Activity of various C9orf72 oligonucleotides (V3C 9orf72 transcript/residual level of HPRT 1)
Table 15L.1 to table 15M.3 show the activity of various C9orf72 oligonucleotides.
The remaining levels of c9orf72 transcripts [ e.g., all transcripts (all V) or only V3] relative to HPRT1 after treatment with c9orf72 oligonucleotides are shown, where 1.000 would represent 100% relative transcript levels (no knockdown) and 0.000 would represent 0% relative transcript levels (e.g., 100% knockdown). In these and other tables, not all controls are shown.
In these and various other tables, the C9orf72 oligonucleotides tested differed in base sequence, form (e.g., some had an asymmetric form), pattern of internucleotide linkages, and/or stereochemical pattern of internucleotide linkages.
TABLE 15L.1 Activity of various c9orf72 oligonucleotides (residual levels of all V C9orf72 transcripts/HPRT 1)
TABLE 15L.2 Activity of various C9orf72 oligonucleotides (V3C 9orf72 transcript/residual level of HPRT 1)
TABLE 15M.1 Activity of various c9orf72 oligonucleotides (residual levels of all V C90rf72 transcripts/HPRT 1)
TABLE 15M.2 Activity of various C9orf72 oligonucleotides (residual levels of V3C 9orf72 transcript/HPRT 1)
TABLE 15M.3 Activity of various C9orf72 oligonucleotides (remaining levels of intron/AS C9orf72 transcript/HPRT 1)
TABLE 16 Activity of various c9orf72 oligonucleotides.
In tables 16A to 16H, various combinations of S and AS (sense and antisense) c9orf72 oligonucleotides were tested in c9 BAC mice; the c9orf72 oligonucleotide was administered to mouse ICV two doses one week apart, and tissues were collected one week after the second dose. Administering WV-7117 in two doses, each 50 μ g; administering WV-5987 in two administrations, each at 50 μ g; administering WV-5987 in two administrations, each at 100 μ g; and a combination of WV-7117(50 μ g) and WV-5987(50 μ g) was administered in two administrations. In tables 16A to 16H, the remaining levels of c9orf72 transcripts relative to HPRT1 after treatment with c9orf72 oligonucleotides are shown, where 1.000 would represent 100% relative transcript level (no knockdown) and 0.000 would represent 0% relative transcript level (e.g., 100% knockdown). Results of repeated experiments are shown. The tissue evaluated: SC, spinal cord; and CX, cerebral cortex.
TABLE 16A Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts in CX)
TABLE 16B Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript in CX)
TABLE 16C Activity of various C9orf72 oligonucleotides (remaining levels of intron in CX/AS C9orf72 transcript)
TABLE 16D Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts in SC)
TABLE 16E Activity of various C9orf72 oligonucleotides (residual level of V3C 9orf72 transcript in SC)
TABLE 16F Activity of various C9orf72 oligonucleotides (residual level of AS C9orf72 transcript in SC)
Example 10.
Activity of various oligonucleotides
Various other chemical moieties useful for constructing the C9orf72 oligonucleotide were tested for potency in an oligonucleotide targeting another gene target, Malat 1. Data is provided in fig. 7A-7D and described herein.
Single-stranded Malat1 oligonucleotide WV-3174 was conjugated with any of the various conjugates (Mod027, Mod028, or Mod007) to yield WV-7558, WV-7559, and WV-7560, which are detailed in table 17 below and illustrated in example 1. WV-3174 is a cross-species oligonucleotide because its base sequence has no mismatch with the corresponding sequences in human, dog (Canis luma micromiliaris) (mm10), mouse (Mus musculus) (mm10), rat (Rattus norvegicus) (m6), and monkey rhesus macaque (Macaca mulatta) (rheMa 8) and cynomolgus monkey (Macaca fascicularis) (macFas 5).
Table 17 below provides information for some Malat1 oligonucleotides. Included in table 17 is WV-8448 described elsewhere herein.
These experiments demonstrated that sulfonamide or anisamide conjugated WV-3174 has greater biodistribution and higher knockdown. The animals tested were: male C57BL/6 mice, 10-12 weeks old, 5 groups, 50 mice. ICV cannulation was performed. Stage 1: n is 10; ICV was injected with PBS, 50mg, 100mg, 150mg or 250mg ICV (2 mice per group) and observed clinically for 2 days. Stage 2: n-40; on day 1, conscious animals were injected with PBS or oligonucleotides via ICV. Necropsy was performed 7 days after injection. Autopsy: PBS was perfused systemically. The procedure is as follows: dissect the lumbar spinal cord (PD) and place the thoracic/cervical spinal cord in formalin (histology); one of the brains (cortex, hippocampus, striatum, cerebellum) was dissected and frozen very rapidly (exposure/transcript). The second half-brain was fixed in formalin, cryoprotected and frozen at extreme speed (Malat1 KD/oligo visualization).
The phase 2 parameters were as follows:
TABLE 18.2 parameters of the Malat1 animal test
| Group of | Test article | Dosage form | Dosing regimens | Volume of dose | Number of mice |
| 1 | PBS | NA | ICV | 2.5ml | 8 |
| 2 | WV-3174 | 50mg | ICV | 2.5ml | 8 |
| 3 | WV-7558 | 50mg | ICV | 2.5ml | 8 |
| 4 | WV-7559 | 50mg | ICV | 2.5ml | 8 |
| 5 | WV-7560 | 50mg | ICV | 2.5ml | 8 |
Fig. 7B-7D show MALAT1 knockdown in the spinal cord. Triantennamide-conjugated Malat1 oligonucleotide (WV-3174) showed significant Malat1 knockdown (about 70%). Triantennanilide-conjugated Malat1 oligonucleotide (WV-3174) was also shown to accumulate in large amounts in the spinal cord.
Fig. 7E, 7F, and 7G show MALAT1 knockdown in cortex. Triantennamide-conjugated Malat1 oligonucleotide (WV-3174) showed a knockdown of Malat1 (about 34%). Triantennamide-conjugated Malat1 oligonucleotide (WV-3174) also showed higher accumulation in the cortex.
Example 11.
In vivo Effect of C9orf72 oligonucleotides on C9orf72 transcripts in c9-BAC mice
Pharmacodynamic studies were performed to compare the effect of C9orf72 oligonucleotide on C9orf72 transcript knockdown in C9-BAC mice.
The C9orf72 oligonucleotides tested were: WV-6408, WV-8009, WV-8010, WV-8011 and WV-8012. Negative controls were PBS (phosphate buffered saline) and WV-2376 not targeted to C9orf 72.
The animals used were: male and female C9-BAC mice, 12 weeks old, 7 groups, 50 mice.
ICV cannulation was performed. PBS or 50. mu.g of oligonucleotide was injected ICV in conscious animals on day 1. On day 8, a second dose of PBS or 50. mu.g oligonucleotide. The dose volume was 2.5. mu.l. Necropsy was performed 2 weeks after the first injection.
Autopsy: PBS was perfused systemically. Dissect the lumbar spinal cord (PD) and place the thoracic/cervical spinal cord in formalin (histology); one of the brains (cortex, hippocampus, striatum, cerebellum) was dissected and frozen very rapidly (exposure/transcript). The second half-brain was fixed in formalin, cryoprotected and frozen very rapidly (RNA foci/oligonucleotide visualization).
The results are shown in fig. 8A to 8H.
Transcripts were analyzed in cerebral cortex (fig. 8A to 8D) and spinal cord (fig. 8E to 8H). The transcripts analyzed were: all transcripts (fig. 8A and 8E); v3 (fig. 8B and 8F); v3 (exon 1a) (fig. 8C and 8G); and intron 1/AS (FIGS. 8D and 8H).
Several C9orf72 oligonucleotides were shown to be able to knock down C9orf72 transcripts (including V3) in the cortex and spinal cord of C9-BAC mice.
Example 12.
In vivo distribution of C9orf72 oligonucleotides in spinal cord and cerebral cortex of C9-BAC mice
Pharmacokinetic studies were performed to examine the in vivo distribution of C9orf72 oligonucleotides in the spinal cord and cerebral cortex of C9-BAC mice.
The C9orf72 oligonucleotides tested were: WV-6408, WV-8009, WV-8010, WV-8011 and WV-8012. Negative controls were PBS (phosphate buffered saline) and WV-2376 not targeted to C9orf 72.
The results are shown in fig. 9A (spinal cord) and fig. 9B (cerebral cortex). Red indicates points outside the standard curve.
Several C9orf72 oligonucleotides were shown to accumulate in large amounts in the spinal cord and cortex.
Example 13.
In vivo Effect of C9orf72 oligonucleotides on Poly GP levels in hippocampal regions of C9-BAC mice
A study was conducted to evaluate the in vivo effect of C9orf72 oligonucleotide on poly GP (dipeptide repeat protein) levels in the hippocampus of C9-BAC mice.
The C9orf72 oligonucleotides tested were: WV-6408, WV-8009, WV-8010, WV-8011 and WV-8012. Negative controls were PBS (phosphate buffered saline) and WV-2376 not targeted to C9orf 72. WT is control.
The method comprises the following steps:
tissues were homogenized in RIPA buffer and clarified by centrifugation. The concentration of the dissolved product was determined by Pierce Protein660nm analysis (available reagents such as Cat. No.: 22660, Sammer Feishel, Waltham, Mass.) and normalized in RIPA dissolution and extraction buffer (available reagents such as Cat. No.: 89900, Sammer Feishel, Waltham, Mass.). MSD 96 well small spot standard plates were coated with anti-poly GP (Millipore ABN1358, available from milli-bosch sigma, bellerica, ma) at 4C overnight, washed in PBST, and blocked with PBS containing 10% fetal bovine serum. Lysate samples were diluted 1: 4 in PBS/10% FBS and loaded 75ug per well and incubated at room temperature. The standard curve included affinity purified Flag-poly GP diluted in wild-type mouse brain RIPA lysate (GenScript, piscativy, new jersey). Detection was performed with sulfo-label conjugated anti-polyGP and read in an MSD QuickPlex SQ 120 (mesoscale diagnostics, Rockville, Md.) instrument with MSD read buffer T with surfactant.
The data shown in figure 10 were obtained from standard curve quantification of GenScript Flag-polygp diluted in wild type mouse brain lysate.
The data show that C9orf72 oligonucleotide was able to reduce poly GP (dipeptide repeat protein) levels in hippocampus in C9-BAC mice.
Example 14.
Other arrangements
Other protocols for the experiments are presented below.
Non-limiting examples of hybridization assays for detecting a target nucleic acid are described herein. Such an assay may be used to detect and/or quantify the C9orf72 oligonucleotide, or any other nucleic acid or oligonucleotide directed against any target, including targets other than C9orf 72.
Pharmacokinetic studies:
tissue preparation for oligonucleotide quantification and transcript quantification:
the tissue was dissected and freshly frozen in pre-weighed microcentrifuge tubes. The tissue weight was calculated by re-weighing the tube. 4 volumes of Trizol or lysis buffer (4M guanidine; 0.33% N-lauryl sarcosine; 25mM sodium citrate; 10mM DTT) (1mg of tissue plus 4. mu.l buffer). Tissue lysis was performed by a Precellys Evolution tissue homogenizer (Bertin Technologies, Montigner (Montigy-le-Bretonneux), France) at 4C until all tissue blocks were lysed. 30-50 μ l of tissue lysate were stored in 96-well plates for PK measurements, and the remaining lysate was stored at-80C (if it was in lysis buffer) or continued for RNA extraction (if it was in Trizol buffer).
Transcript quantification:
hybridization probes (IDT-DNA)
And (3) capturing a probe: "C9-intron-cap"/5 AmMC12/TGGCGAGTGG
Detecting a probe: "C9-intron-Det": GTGAGTGAGG/3BioTEG @
5AmC12 is a compound having C125' -amine of linker.
3BioTEG is a biotinylated probe.
Maleic anhydride-activated 96-well culture plates (Pierce 15110) were coated with 2.5% NaHCO3(Gibco, 25080-094) containing 500nM of 50. mu.l capture probe and maintained for 2 hours at 37C. Plates were then washed 3 times with PBST (PBS + 0.1% tween-20) and blocked with 5% skim milk-PBST for 1 hour at 37C. Payload oligonucleotides were serially diluted into the matrix. This standard was diluted with lysis buffer (4M guanidine; 0.33% N-lauryl sarcosine; 25mM sodium citrate; 10mM DTT) together with the initial sample so that the amount of oligonucleotide in all samples was less than 50 ng/ml. Mu.l of the diluted sample was mixed with 180. mu.l of 333nM detection probe diluted in PBST and then denatured in a PCR instrument (65C, 10 min; 95C, 15 min; 4C ∞). 50 μ l of denatured sample was dispensed in triplicate in blocked ELISA plates and incubated overnight at 4C. After 3 washes with PBST, 50. mu.l of 1: 2000 streptavidin-AP in PBST (southern Biotech, 7100-04) was added per well and incubated at room temperature for 1 hour. After extensive washing with PBST, 100 μ l AttoPhos (PromegaS1000) was added, incubated at room temperature for 10 minutes in the dark, and read on the plate reader (molecular instrumentation, M5) fluorescence channel: ex435nm, Em555 nm. Oligonucleotides in the samples were calculated by 4-parameter regression according to the standard curve.
FISH protocols against GGGGCC and GGCCCC RNA foci
Fixing:
slides were allowed to dry at room temperature for 30 minutes and then fixed in 4% PFA for 20 minutes. After fixation, slides were washed 3 times in PBS and then stored in 70% pre-cooled ethanol at 4 ℃ for at least 30 minutes.
Pre-hybridization:
the slides were rehydrated in FISH wash buffer (40% formamide, 2XSSC in DEPC water) for 10 min. Hybridization buffer (40% formamide, 2XSSC, 0.1mg/ml BSA, 0.1g/ml dextran sulfate, 1% vanadyl sulfate complex, 0.25mg/ml tRNA in DEPC water) was added to the slides and incubated at 55 ℃ for 30 minutes.
Preparing a probe:
the Cy3- (GGCCCC)3 probe (to detect sense repeat amplifications) and Cy3- (GGGGCC)3 probe (to detect antisense repeat amplifications) were denatured at 95 ℃ for 10 minutes. After cooling on ice, the probe was diluted to 200ng/ml with cold hybridization buffer.
And (3) hybridization:
the slides were simply washed with FISH wash buffer and the diluted probes were added to the slides. The slides were incubated at 55 ℃ for 3 hours in a hybridization apparatus. After hybridization, the slides were washed 3 times with FISH wash buffer at 55 ℃ for 15 minutes each. The slides were then simply washed once with 1 XPBS.
Immunofluorescence staining of neuronal nuclei:
slides were blocked with blocking solution (2% normal goat serum in PBS) for 1 hour. anti-NeuN antibody (MAB377, Michibo) was diluted 1: 500 in blocking solution and applied to the slides overnight at 4 ℃. The slides were then washed 3 times with PBS and incubated with 1: 500 diluted goat anti-mouse secondary antibody (Life technologies) with Alexa Fluor 488 for 1 hour at room temperature. The slides were then washed 3 times with PBS. Finally, the slides were mounted with DAPI for imaging.
Imaging and lesion quantification:
images were taken with a 40 × magnification RPI rotating disc confocal microscope (Zeiss). The 488, CY3 and DAPI channels were collected. RNA foci were quantified using ImageJ software (NIH).
Although various embodiments have been described and illustrated herein, it will be apparent to those of ordinary skill in the art that various other methods and/or structures for performing the functions described in the present disclosure and/or obtaining the results and/or one or more advantages described in the present disclosure, as well as each of such variations and/or modifications, are deemed to be included. More generally, those of ordinary skill in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be examples, and that the actual parameters, dimensions, materials, and/or configurations will depend upon the particular application or applications for which the teachings of the present disclosure are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described in the disclosure. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the claimed technology may be practiced otherwise than as specifically described and claimed. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually incompatible, is included within the scope of the present disclosure.