The present invention claims priority from chinese patent application CN202311365471.8 filed 10/20/2023, the contents of which are incorporated in and form a part of the original description of the present invention, as well as the description of the specification, the drawings of the specification and the claims. The applicant further states that the applicant has the right to modify the description and claims of the invention based on this priority file.
5. Detailed description of the preferred embodiments
Definition of the definition
"Antisense oligonucleotide" or "ASO" means an oligonucleotide having a nucleobase sequence complementary to a target nucleic acid or region or segment thereof. The antisense oligonucleotide can specifically hybridize to a target nucleic acid or region or segment thereof, which hybridization results in rnase H mediated cleavage of the target nucleic acid.
Throughout the present application, the term "about" is used to indicate that the value includes inherent variations in the error of the method/apparatus employed to determine the value, or variations that exist between study subjects. Typically, the term "about" is intended to encompass approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% variation, depending on the particular situation.
The term "or" is used in the claims to mean "and/or" unless explicitly indicated to mean only alternatives or that the alternatives are mutually exclusive, although the disclosure supports definitions of only alternatives and "and/or".
"2' -Deoxyfuranosyl sugar moiety" or "2' -deoxyfuranosyl sugar" means a furanosyl sugar moiety having two hydrogens at the 2' -position. The 2 '-deoxyfuranosyl sugar moiety may be unmodified or modified and may be substituted or unsubstituted at a position other than the 2' -position. In the context of oligonucleotides, the β -D-2 '-deoxyribose moiety is an unsubstituted, unmodified 2' -deoxyfuranosyl, and is found in naturally occurring deoxyribonucleic acid (DNA).
"2 '-Deoxynucleoside" means a nucleoside comprising a 2' -H (H) furanosyl sugar moiety, as found in naturally occurring deoxyribonucleic acid (DNA). In certain embodiments, the 2' -deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil).
"2 '-O-methoxyethyl" (also known as 2' -MOE) refers to 2'-O (CH 2) 2-OCH3 instead of the 2' -OH group of the ribosyl ring. The 2' -O-methoxyethyl modified sugar is a modified sugar.
"2' -MOE nucleoside" (2 ' -O-methoxyethyl nucleoside) means a nucleoside comprising a 2' -MOE modified sugar moiety.
"2 '-Substituted nucleoside" or "2' -modified nucleoside" means a nucleoside comprising a2 '-substituted or 2' -modified sugar moiety. As used herein, "2' -substituted" or "2' -modified" with respect to a sugar moiety means a sugar moiety comprising at least one 2' -substituent group other than H or OH.
"Spacer" or "gap" means an antisense oligonucleotide comprising an internal region having a plurality of nucleosides that support rnase H cleavage positioned between an external region having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically different from the one or more nucleosides comprising the external region. The inner region may be referred to as a "gap" or "spacer" and the outer region may be referred to as a "wing". In certain embodiments, the antisense oligonucleotide is a spacer.
"Conjugate group" means a group of atoms attached directly to a polynucleotide. In certain embodiments, the conjugate group includes a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the polynucleotide.
The term "complementary" is used to describe a relationship between nucleotide bases and/or polynucleotides capable of hybridizing to each other, e.g., when two nucleotide sequences are aligned in opposite directions, the nucleotide sequence of such polynucleotide or one or more regions thereof matches the nucleotide sequence of another polynucleotide or one or more regions thereof. As described herein, nucleobase matching or complementary nucleobases include pairs of adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine (m C) and guanine (G). Complementary polynucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside, and may include one or more nucleobase mismatches. Thus, the disclosure also includes isolated polynucleotides complementary to sequences as disclosed or used herein, as well as those substantially similar nucleic acid sequences. The degree to which two polynucleotides have matching nucleobases can be expressed in terms of "percent complementarity" or "percent complementarity". Unless otherwise indicated, percent complementarity is the percentage of nucleobases of a shorter sequence that are complementary to a longer sequence.
"Mismatched" or "non-complementary" means that the nucleobase of the first polynucleotide is not complementary to the corresponding nucleobase of the second polynucleotide or target nucleic acid when the first and second polynucleotides are aligned. For example, nucleobases (including but not limited to universal nucleobases, inosine, and hypoxanthine) can hybridize to at least one nucleobase, but remain mismatched or non-complementary relative to the nucleobase to which it hybridizes. As another example, when the first and second polynucleotides are aligned, the nucleobase of the first polynucleotide that is incapable of hybridizing to the corresponding nucleobase of the second polynucleotide or target nucleic acid is a mismatched or non-complementary nucleobase.
Nucleobases may be naturally occurring or synthetic. The nucleobases and the glycosyls can each independently be modified or unmodified. "modified nucleoside" means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety. Modified nucleosides can include abasic nucleosides lacking nucleobases.
"5-Methylcytosine" means cytosine having a methyl group attached to the 5-position. 5-methylcytosine is a modified nucleobase.
In the context of oligonucleotides, "contiguous" refers to a nucleoside, nucleobase, sugar moiety, or internucleoside linkage in close proximity to one another. For example, "contiguous nucleobases" means nucleobases that are immediately adjacent to each other in sequence.
"Linked nucleosides" means adjacent nucleosides that are linked together by internucleoside linkages.
An "internucleoside linkage" is a covalent linkage between adjacent nucleosides in a polynucleotide. As used herein, "modified internucleoside linkage" means any internucleoside linkage other than a phosphodiester internucleoside linkage.
"Phosphorothioate linkage" means a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. "methanesulfonyl-phosphoramidate (MsPA) linkage" refers to a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced by a methanesulfonyl-amino group, the structure of which is as follows:
both the methanesulfonyl-phosphoramidate (MsPA) linkage and the phosphorothioate internucleoside linkage are modified internucleoside linkages.
"Polynucleotide" means a polymer of linked nucleosides, each of which can be modified or unmodified independently of the other. Unless otherwise indicated, a polynucleotide consists of 8-80 linked nucleosides. "modified polynucleotide" means a polynucleotide in which at least one sugar, nucleobase, or internucleoside linkage is modified. "unmodified polynucleotide" means a polynucleotide that does not comprise any sugar, nucleobase, or internucleoside modifications.
Oligonucleotides
In some embodiments, the disclosure provides oligonucleotides consisting of 8to 50 linked nucleosides and having at least 90% sequence complementarity to the equal length portion of SEQ ID NOS: 1-3. In some embodiments, the oligonucleotide consists of 10 to 30 linked nucleosides and has at least 90% sequence complementarity to the equivalent length portion of SEQ ID NOS: 1-3. In some embodiments, the oligonucleotide consists of 17 to 23 linked nucleosides and has at least 90% sequence complementarity to the equivalent length portion of SEQ ID NOS: 1-3.
In some embodiments, the oligonucleotide has a nucleobase sequence which is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or about 100% complementary to the equivalent length of SEQ ID NOs 1-3.
In some embodiments, the oligonucleotide comprises at least one modification selected from the group consisting of at least one modified internucleoside linkage, at least one modified sugar moiety, and at least one modified nucleobase.
In some embodiments, the oligonucleotide comprises at least one modified internucleoside linkage. Naturally occurring internucleoside linkages in RNA and DNA are 3 'to 5' phosphodiester linkages. In some embodiments, oligonucleotides with one or more modified (i.e., non-naturally occurring) internucleoside linkages are generally selected to be superior to oligonucleotides with naturally occurring internucleoside linkages due to desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for the target nucleic acid, and increased stability in the presence of nucleases.
In some embodiments, any internucleoside linkage can be used to link together the nucleosides of the modified polynucleotides. Two main classes of internucleoside linkages are defined by the presence or absence of phosphorus atoms. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphate esters containing a phosphodiester linkage ("O") (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates, phosphoramidates, substituted phosphoramidates (e.g., methanesulfonyl-phosphoramidates), and phosphorothioates ("S") and phosphorodithioates ("HS-S"). Representative non-phosphorus containing internucleoside linkages include, but are not limited to, methyleneimino (-CH2-N(CH3)-O-CH2 -), thiodiester, thiourethane (-O-C (=O) (NH) -S-)), siloxane (-O-SiH2 -O-)), and N, N' -dimethylhydrazine (-CH2-N(CH3)-N(CH3) -. Modified internucleoside linkages can be used to alter (typically increase) nuclease resistance of the polynucleotide compared to naturally occurring phosphate linkages. In some embodiments, the internucleoside linkages having chiral atoms are prepared as a racemic mixture, or as separate enantiomers. Methods for preparing phosphorus-containing internucleoside linkages and non-phosphorus-containing internucleoside linkages are known to those skilled in the art.
Representative chiral internucleoside linkages include, but are not limited to, methanesulfonyl-aminophosphonate and phosphorothioate. Modified polynucleotides disclosed herein comprising internucleoside linkages having a chiral center can be prepared as a population of polynucleotides comprising a stereorandom internucleoside linkage, or as a population of polynucleotides comprising phosphorothioate linkages in a particular stereochemical configuration. In certain embodiments, the population of polynucleotides comprises phosphorothioate internucleoside linkages, wherein all phosphorothioate internucleoside linkages are stereotactic. Such polynucleotides can be produced using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate linkage. Nonetheless, each individual phosphorothioate of each individual polynucleotide molecule has a defined steric configuration. In certain embodiments, the population of polynucleotides is enriched in polynucleotides comprising one or more specific phosphorothioate internucleoside linkages in specific, independently selected stereochemical configurations. Unless otherwise indicated, chiral internucleoside linkages of RNAi polynucleotides described herein may be stereorandom or in a particular stereochemical configuration. Methods for preparing phosphorus-containing internucleoside linkages and non-phosphorus-containing internucleoside linkages are known to those skilled in the art.
In certain embodiments, the polynucleotide (e.g., antisense RNAi polynucleotide and/or sense RNAi polynucleotide) comprises one or more inverted nucleosides.
In certain embodiments, the inverted nucleoside is a terminal inverted nucleoside (i.e., the last nucleoside on one end of the oligonucleotide), and thus only one internucleoside linkage depicted above will be present. In certain such embodiments, additional features (e.g., conjugate groups) can be attached to the inverted nucleoside. Such terminal inverted nucleosides can be attached to either or both ends of the polynucleotide.
In certain embodiments, such groups lack nucleobases and are referred to herein as inverted sugar moieties. In certain embodiments, the inverted sugar moiety is a terminal inverted sugar moiety (i.e., the last nucleoside attached to one end of the polynucleotide), and thus only one internucleoside linkage above will be present. In certain such embodiments, additional features (e.g., conjugate groups) may be attached to the inverted sugar moiety. Such terminal inverted sugar moieties may be attached to either or both ends of the polynucleotide.
In certain embodiments, the nucleic acid may be a 2 'to 5' linkage rather than a standard 3 'to 5' linkage.
In some embodiments, the polynucleotide comprises modified internucleoside linkages arranged in a defined pattern or modified internucleoside linkage motif along the polynucleotide or region thereof. In some embodiments, the internucleoside linkages are arranged as gapped (gap) motifs. In such embodiments, the internucleoside linkage in each of the two wing regions can be different from the internucleoside linkage in the notch region. In some embodiments, the internucleoside linkage in the wing is a phosphodiester and the internucleoside linkage in the gap is a phosphorothioate. The wing and notch lengths may or may not be the same. In some embodiments, the gap further comprises n number of split ions, further split into n+1 sub-gaps. Wherein each isolate comprises a 1-3 linked nucleoside composition and each sub-notch consists of 1-10 linked deoxynucleosides, such as those described in CN114507663 and WO 2023/131098.
In some embodiments, the polynucleotide comprises one or more methanesulfonyl-phosphoramidate (MsPA) linkages. In some embodiments, the polynucleotide having a spacer nucleoside motif comprises all phosphorothioate linked linking motifs except for 1-5 methanesulfonyl-phosphoramidate (MsPA) linkages. In some embodiments, 1-5 consecutive methylphosphonates are ligated in the nicks of an oligonucleotide having a nick body nucleotide motif. In some embodiments, 1-5 consecutive methylphosphonates are linked in the wings of an oligonucleotide having a notch nucleoside motif. In some embodiments, 1-5 consecutive methylphosphonates are ligated in the nicks and wings of an oligonucleotide having a nick body nucleoside motif.
In some embodiments, the number and positions of phosphorothioate internucleoside linkages, methanesulfonyl-phosphoramidate (MsPA) and phosphodiester internucleoside linkages can be arranged in a manner that maintains nuclease resistance.
In some embodiments, the modified polynucleotide comprises at least one modified sugar moiety. In some embodiments, the at least one modified sugar is a bicyclic sugar, a 2' -O-methoxyethyl, a 2' -F, or a 2' -O-methyl.
Nucleosides comprising a modified sugar moiety (e.g., a non-bicyclic modified sugar moiety) are referred to by one or more positions of one or more substitutions on the sugar moiety of the nucleoside. For example, nucleosides comprising 2 '-substituted or 2-modified sugar moieties are referred to as 2' -substituted nucleosides or 2-modified nucleosides. Examples of suitable 2 '-substituent groups for non-bicyclic modified sugar moieties include, but are not limited to, 2' -F, 2'-OCH3 ("OMe" or "O-methyl") and 2' -O (CH2)2OCH3 ("MOE"). In some embodiments, the 2' -substituent group is selected from the group consisting of halo, allyl, amino, azido, SH, CN, OCN, CF3、OCF3、O-C1-C10 alkoxy, O-C1-C10 substituted alkoxy, O-C1-C10 alkyl, O-C1-C10 substituted alkyl, S-alkyl, N (Rm) -alkyl, O-alkenyl, S-alkenyl, N (Rm) -alkenyl, O-alkynyl, S-alkynyl, N (Rm) -alkynyl, O-alkylene-O-alkyl, alkynyl, alkylaryl, arylalkyl, O-alkylaryl, O-arylalkyl, O (CH2)2SCH3、O(CH2)2 ON (Rm) (Rn) or OCH2 C (=O) -N (Rm) (Rn), wherein each Rm and Rn is independently H, Amino protecting groups, or substituted or unsubstituted C1-C10 alkyl 、-O(CH2)2ON(CH3)2("DMAOE")、2'-OCH2OCH2N(CH2)2("DMAEOE")、, and 2' -substituent groups described in U.S.6,531,584, U.S.5,859,221, U.S.6,005,087. Some embodiments of these 2' -substituent groups may be further substituted with one or more substituent groups independently selected from the group consisting of hydroxy, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl. In some embodiments, the 2' -substituted nucleoside or the 2' -non-bicyclic modified nucleoside comprises a sugar moiety :F、NH2、N3、OCF3、OCH3、O(CH2)3NH2、CH2CH2、OCH2CH2、OCH2CH2OCH3、O(CH2)2SCH3、O(CH2)2ON(Rm)(Rn)、O(CH2)2O(CH2)2N(CH3)2、 comprising a linear 2' -substituent group selected from the group consisting of OCH2 C (=o) -N (Rm) (Rn)), wherein each Rm and Rn is independently H, an amino protecting group, or a substituted or unsubstituted C1-C10 alkyl group.
In certain embodiments, the non-bicyclic modified sugar moiety comprises a substituent group at the 4' position. Examples of suitable 4' -substituent groups include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in WO 2015/106128. In certain embodiments, the non-bicyclic modified sugar moiety comprises a substituent group at the 3' position. Examples of substituent groups suitable for the 3' position of the modified sugar moiety include, but are not limited to, alkoxy (e.g., methoxy), alkyl (e.g., methyl, ethyl). In certain embodiments, the non-bicyclic modified sugar moiety comprises a substituent group at the 5' position. Examples of substituent groups suitable for the 5 'position of the modified sugar moiety include, but are not limited to, alkyl (e.g., methyl (R or S)), vinyl, and 5' -alkoxy (e.g., methoxy). In some embodiments, the non-bicyclic modified sugar comprises more than one non-bridging sugar substituent, e.g., a 2'-F-5' -methyl sugar moiety and modified sugar moieties and modified nucleosides described in Migawa et al, WO 2008/101157 and Rajeev et al, US 2013/0203836.
In naturally occurring nucleic acids, the sugars are linked to each other 3 'to 5'. In certain embodiments, the polynucleotide comprises one or more nucleoside or sugar moieties linked at alternative positions (e.g., at 2' or inverted 5' to 3 '). For example, when attached at the 2' position, the 2' -substituent group may be conversely at the 3' position.
Some modified sugar moieties contain bridging sugar substituents that form a second ring, thereby producing a bicyclic sugar moiety. Nucleosides comprising such bicyclic sugar moieties have been referred to as Bicyclic Nucleosides (BNA), locked nucleosides, or conformationally constrained nucleosides (CRN). Some such compounds are described in U.S. patent publication No. 2013/0190383, and PCT publication No. WO 2013/036868. In some such embodiments, the bicyclic sugar moiety comprises a bridge between the 4 'and 2' furanose ring atoms. In certain such embodiments, the furanose ring is a ribose ring. Examples of such 4 'to 2' bridging sugar substituents include, but are not limited to, :4'-CH2-2'、4'-(CH2)2-2'、4'-(CH2)3-2'、4'-CH2-O-2'("LNA")、4'-CH2-S-2'、4'-(CH2)2-O-2'("ENA")、4'-CH(CH3)-O-2'( referred to as "constrained ethyl" or "cEt", when in the S configuration), 4'-CH2-O-CH2-2'、4'-CH2-N(R)-2'、4'-CH(CH2OCH3) -O-2' ("constrained MOE" or "cMOE") and analogs thereof (see, e.g., seth et al, u.s.7,399,845, bhat et al, u.s.7,569,686, swayze et al, u.s.7,741,457 and Swayze et al, u.s.8,022, 193), and the like, 4'-C (CH3)(CH3) -O-2' and analogs thereof (see, e.g., seth et al, U.S.8,278,283), 4'-CH2-N(OCH3) -2' and analogs thereof (see, e.g., prakash et al, U.S.8,278,425), 4'-CH2 -O-N (CH 3) -2' (see, e.g., allerson et al, U.S.7,696,345 and Allerson et al, U.S.8,124,745), 4'-CH2-C(H)(CH3) -2' (see, e.g., zhou et al, J.Org.chem. [ J.Organchem., 2009,74,118-134), 4'-CH2-C(＝CH2) -2' and analogs thereof (see, e.g., seth et al, U.S.8,278,426), 4'-C (RaRb) -N (R) -O-2' 4'-C (RaRb) -O-N (R) -2', 4'-CH2 -O-N (R) -2', and 4'-CH2 -N (R) -O-2', wherein each R, ra and Rb is independently H, a protecting group, or C1-C12 alkyl (see, e.g., imanishi et al, U.S.7,427,672).
In some embodiments, such 4 'to 2' bridges independently comprise 1 to 4 attached groups independently selected from :-[C(Ra)(Rb)]n-、-[C(Ra)(Rb)]n-O-、-C(Ra)＝C(Rb)-、-C(Ra)＝N-、-C(＝NRa)-、-C(＝O)-、-C(＝S)-、-O-、-Si(Ra)2-、-S(＝O)x-、 and-N (Ra) -, wherein x is 0,1, or 2;n is 1, 2,3, or 4, each Ra and Rb is independently H, a protecting group, hydroxy, C1-C12 alkyl, substituted C1-C12 alkyl, c2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, Substituted C5-C20 aryl, heterocyclyl, substituted heterocyclyl, heteroaryl, substituted heteroaryl, C5-C7 alicyclic, substituted C5-C7 alicyclic, halogen, OJ1、NJ1J2、SJ1、N3、COOJ1, Acyl (C (=O) -H), substituted acyl, CN, sulfonyl (S (=O)2-J1), or sulfoxylate (sulfoxyl, S (=O) -J1), and each J1 and J2 is independently H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, Substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C (=O) -H), substituted acyl, heterocyclyl, substituted heterocyclyl, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.
Additional bicyclic sugar moieties are known in the art, see, for example: freier et al, nucleic AcidsResearch [ nucleic acids research ],1997,25 (22), 4429-4443, albaek et al, J.org.chem. [ journal of organic chemistry ],2006,71,7731-7740, singh et al, chem.Commun. [ chemical communication ],1998,4,455-456; koshkin et al, tetrahedron ],1998,54,3607-3630; wahlstedt et al, proc.Natl.Acad.Sci.U.S.A. [ Proc.Natl.Sci.Sci.USA. ] [ Proc.Sci.USA., 2000,97,5633-5638; kumar et al, bioorg.Med.chem.Lett. [ journal of biological chemistry and medical chemistry ],1998,8,2219-2222; singh et al, J.org.chem. [ journal of organic chemistry ],1998,63,10035-10039; srivastava et al, J.am. chem. Soc. [ U.S. chemical society ],20017,129,8362-8379; elayadi et al, curr. Opinion Invens. Drugs [ recent views of study drugs ],2001,2,558-561; braasch et al, chem. Biol. [ chemical biology ],2001,8,1-7; orum 10 et al, curr. Opinion mol. Ther. [ recent views of molecular therapy ],2001,3,239-243; wengel et al, U.S.7,053,207, imanishi et al, U.S.6,268,490, imanishi et al, U.S.6,770,748, imanishi et al, U.S. RE44,779; wengel et al U.S.6,794,499, wengel et al U.S.6,670,461, wengel et al U.S.7,034,133, wengel et al U.S.8,080,644, wengel et al U.S.8,034,909, wengel et al U.S.8,153,365, wengel et al U.S.7,572,582, and Ramasamy et al U.S.6,525,191, torsten et al, WO 2004/106356, wengel et al, WO 91999/014226, seth et al, WO/134181, seth et al U.S.7,547,684, seth et al U.S.7,666,854, U.S.8,8,746, seth et al U.S.7,08980, U.S. 131,131, U.S.7,572,582, and U.S. 5,012,2008, U.S. 5,805, U.S. 5,2008, U.S. Pat. 6,012, and U.S. 5,012, U.S. 5,2008, U.S. Pat. No. 5,2008, and U.S. 5,2008, U.S.6,2008, and U.S. Pat. 5,2008, respectively.
In some embodiments, the bicyclic sugar moiety and nucleosides incorporating such bicyclic sugar moiety are further defined by isomeric configurations. For example, LNA nucleosides (described herein) can be in the α -L configuration or in the β -D configuration.
In some embodiments, the sugar substitute comprises a ring having not 5 atoms. For example, in some embodiments, the sugar substitute comprises six-membered tetrahydropyran ("THP"). Such tetrahydropyran may be further modified or substituted.
In some embodiments, the sugar substitute comprises a non-cyclic moiety. Examples of nucleosides and polynucleotides (e.g., polynucleotides) that include such acyclic sugar substitutes include, but are not limited to, peptide nucleic acids ("PNA"), acyclic butyl nucleic acids (see, e.g., kumar et al, org.Biomol.chem. [ organic chemistry and biomolecular chemistry ],2013,11,5853-5865), and nucleosides and polynucleotides described in Manoharan et al, WO 2011/133876.
In certain embodiments, the sugar substitute is an "unlocked" sugar structure of a UNA (unlocked nucleic acid) nucleoside. UNA is an unlocked acyclic nucleic acid in which any bonds of the sugar have been removed, thereby forming an unlocked sugar substitute. Representative U.S. disclosures teaching preparation of UNA include, but are not limited to, U.S. patent number 8,314,227, and U.S. patent publication numbers 2013/0096289, 2013/0011922, and 201I/0313020, each of which is hereby incorporated by reference in its entirety.
In certain embodiments, the sugar substitute is GNA (glycol nucleic acid) as depicted below:
(S)-GNA
wherein Bx represents any nucleobase.
Modified nucleobases
In some embodiments, the polynucleotide comprises at least one modified nucleobase. In some embodiments, the at least one modified nucleobase is a 5-methylcytosine. In certain embodiments, the polynucleotide comprises one or more inosine nucleosides (i.e., nucleosides comprising a hypoxanthine nucleobase). Nucleobase (or base) modification or substitution can be structurally distinguished from, but functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications may confer nuclease stability, binding affinity, or some other beneficial biological property to the antisense compound.
In some embodiments, a polynucleotide described herein comprises a modification, i.e., a modified polynucleotide. In some embodiments, the modified polynucleotide comprises one or more nucleosides that comprise an unmodified nucleobase. In some embodiments, the modified polynucleotide comprises one or more nucleosides that comprise a modified nucleobase. In some embodiments, the modified polynucleotide comprises one or more nucleosides that do not comprise a nucleobase (referred to as abasic nucleosides).
In some embodiments, the modified nucleobase is selected from the group consisting of 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6, and O-6 substituted purines. In some embodiments, the modified nucleobases are selected from the group consisting of 2-aminopropyl adenine, 5-hydroxymethyl cytosine, 5-methylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyl adenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (C-CH 3) uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-ribosyl uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, 8-aza and other 8-substituted purines, 5-halo (particularly 5-bromo), 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-uracil, 7-azaguanine, 4-azabenzoyl-N, 4-azabenzoyl-N-azaadenine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, enlarged size bases, and fluorinated bases. Additional modified nucleobases include tricyclic pyrimidines such as 1, 3-diazaphenoxazin-2-one, 1, 3-diazaphenothiazin-2-one, and 9- (2-aminoethoxy) -1, 3-diazaphenoxazin-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced by other heterocycles, for example, 7-deaza-adenine, 7-deaza-guanosine, 2-aminopyridine, and 2-pyridone.
Sugar motif
In some embodiments, a polynucleotide provided herein comprises one or more types of modified sugar and/or unmodified sugar moieties arranged in a defined pattern or sugar motif along the polynucleotide or region thereof. In some embodiments, such sugar motifs include, but are not limited to, any of the sugar modifications discussed herein.
In some embodiments, the modified polynucleotide comprises or consists of a region having a notch motif comprising two outer regions or "wings" and a central or inner region or "notch". These three regions of the notch motif (5 '-wing, notch, and 3' -wing) form a contiguous sequence of nucleosides, wherein at least some of the sugar moieties of the nucleosides of each of the wings are different from at least some of the sugar moieties of the nucleosides of the notch. Specifically, at least the sugar portion of the nucleoside closest to the notch (the 3 '-most nucleoside of the 5' -wing and the 5 '-most nucleoside of the 3' -wing) of each wing is different from the sugar portion of the adjacent notch nucleoside, thus defining a boundary between the wing and the notch (i.e., a wing/notch junction). In some embodiments, the sugar moieties within the notch are identical to each other. In some embodiments, a notch includes one or more nucleosides having a sugar moiety that is different from a sugar moiety of one or more other nucleosides of the notch. In some embodiments, the sugar motifs of the two wings are identical to each other (symmetrical notch). In some embodiments, the 5 '-wing sugar motif is different from the 3' -wing sugar motif (asymmetric notch).
In some embodiments, the wings of the notch comprise 1-5 nucleosides. In some embodiments, the wings of the notch comprise 2-5 nucleosides. In some embodiments, the wings of the notch comprise 3-5 nucleosides. In some embodiments, the nucleosides of the notch are all modified nucleosides.
In some embodiments, the notch of the notch body comprises 7-12 nucleosides. In some embodiments, the notch of the notch body comprises 7-10 nucleosides. In some embodiments, the notch of the notch body comprises 8-10 nucleosides. In some embodiments, the notch of the notch body comprises 10 nucleosides. In a certain embodiment, each nucleoside of the notch body is an unmodified 2' -deoxynucleoside.
In some embodiments, the notch is a deoxidizing notch. In such embodiments, the nucleoside on the nick side of each wing/nick junction is an unmodified 2' -deoxynucleoside and the nucleoside on the wing side of each wing/nick junction is a modified nucleoside. In some such embodiments, each nucleoside of the gap is an unmodified 2' -deoxynucleoside. In some such embodiments, each nucleoside of each wing is a modified nucleoside.
In some embodiments, the modified polynucleotide has a fully modified sugar motif, wherein each nucleoside of the modified polynucleotide comprises a modified sugar moiety. In some embodiments, the modified polynucleotide comprises or consists of a region having a fully modified sugar motif, wherein each nucleoside of the region comprises a modified sugar moiety. In some embodiments, the modified polynucleotide comprises or consists of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety (referred to herein as a conformationally modified sugar motif). In some embodiments, the fully modified polynucleotide is a consistently modified polynucleotide. In some embodiments, each nucleoside of a consistently modified polynucleotide comprises the same 2' -modification.
In some embodiments, the compounds provided herein comprise or consist of polynucleotides (modified or unmodified) and optionally one or more conjugate groups and/or terminal groups. The conjugate group consists of one or more conjugate moieties and a conjugate linker that links the conjugate moiety to the polynucleotide. The conjugate groups may be attached to either or both ends of the polynucleotide and/or at any internal position. In some embodiments, the conjugate group is attached to the 2' position of the nucleoside of the modified polynucleotide. In some embodiments, the conjugate groups attached to either or both ends of the polynucleotide are terminal groups. In certain such embodiments, the conjugate group or terminal group is attached at the 3 'and/or 5' end of the polynucleotide. In certain such embodiments, the conjugate group (or terminal group) is attached at the 3' end of the polynucleotide. In some embodiments, the conjugate group is attached near the 3' end of the polynucleotide. In some embodiments, the conjugate group (or terminal group) is attached at the 5' end of the polynucleotide. In some embodiments, the conjugate group is attached near the 5' end of the polynucleotide.
In some embodiments, the conjugate/terminal group of the polynucleotide comprises a capping group, a phosphate moiety, a protecting group, and a modified or unmodified nucleoside. In some embodiments, the conjugate/terminal group includes an intercalator, a reporter, a polyamine, a polyamide, a peptide, a carbohydrate (e.g., galNAc), a vitamin, a polyethylene glycol, a thioether, a polyether, a folic acid, a lipid, a phospholipid, a biotin, a phenazine, a phenanthridine, an anthraquinone, an adamantane, an acridine, a fluorescein, a rhodamine, a coumarin, a fluorophore, and a dye.
In some embodiments, the conjugate/terminal group of the polynucleotide comprises a targeting moiety. In some embodiments, the targeting moiety is at the 5' end of the polynucleotide. In some embodiments, the targeting moiety is at the 3' end of the polynucleotide. In some embodiments, the targeting moiety targets the polynucleotide to a specific subcellular location and/or to a specific cell or tissue type. In some embodiments, the targeting moiety comprises a ligand for a receptor. In some embodiments, the receptor is specific for a certain type of cell and/or tissue. In some embodiments, recognition of the targeting moiety (e.g., ligand) by the receptor mediates endocytosis of the polynucleotide conjugated to the targeting moiety.
In some embodiments, the targeting moiety targets a liver cell (also referred to herein as a hepatocyte). In some embodiments, the liver cell is a human liver cell. In some embodiments, the liver cell expresses an asialoglycoprotein receptor (ASGPr) on its cell surface. In some embodiments, the targeting moiety is a ligand of ASGPr. In some embodiments, the targeting moiety comprises an N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the targeting moiety comprises 1 to 5 GalNAc moieties. In some embodiments, the targeting moiety comprises 1, 2, 3, 4, or 5 GalNAc moieties. In some embodiments, the targeting moiety comprises 3 GalNAc moieties. In some embodiments, the targeting moiety comprises 3 GalNAc moieties (triple-antenna GalNAc) arranged in a triple-antenna (TRIANTENNARY) pattern. In some embodiments, the polynucleotide comprises a triple-antenna GalNAc at the 5' of the polynucleotide.
The compounds described herein include non-radioactive isotopes or radioisotope-substituted variants in which one or more atoms are replaced with a designated element. For example, a compound herein that contains a hydrogen atom encompasses all possible deuterium substitutions for each1 H hydrogen atom. Isotopic substitutions encompassed by the compounds herein include, but are not limited to2 H or3 H instead of1H、13 C or14 C instead of12C、15 N instead of14N、17 O or18 O instead of16 O, and33S、34S、35 S or36 S instead of32 S. In certain embodiments, non-radioisotope substitution may impart novel properties to oligomeric compounds that are useful as therapeutic or research tools. In certain embodiments, radioisotope substitution may render the compound suitable for research or diagnostic purposes, such as imaging.
By "hepatitis b-related condition" or "HBV-related condition" is meant any disease, biological symptom, medical symptom, or event caused by, associated with, or attributable to a hepatitis b infection, exposure, or disease. The term hepatitis b-associated condition includes chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocyte inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, transplantation associated with liver disease.
Example 1
General method for preparing Gap antisense oligonucleotides by solid phase technique
All reagents and solutions for synthesizing the oligomeric compounds were purchased from commercial sources unless otherwise indicated. Standard phosphoramidite building blocks and solid supports are used to incorporate nucleoside residues including, for example, T, A, G and mC residues. The phosphoramidite solution of all monomers used (β -D-2 '-deoxyribonucleoside and β -D-2' - (MOE) ribonucleoside) was a 0.06M solution in anhydrous acetonitrile.
A500 nmol synthesis column made of Universal CPG solid support was packed on LK-48E synthesizer and phosphoramidite coupling method was used to perform the designated sequence synthesis. For the coupling step, the phosphoramidite monomer was delivered in an amount exceeding 4 times the loading on the solid support and phosphoramidite condensation was performed for 10 min. All other steps follow the standard protocols supplied by the manufacturer. A solution of 3% trichloroacetic acid in dichloromethane was used to remove Dimethoxytrityl (DMT) from the 5' -hydroxy group of the nucleotide. BTT (0.35M, 0.5% nmi) in anhydrous acetonitrile was used as activator during the coupling step. Phosphorothioate linkages were introduced by sulfiding with a 0.2M solution of diphenylacetyl disulfide (PADS) in 1:1 pyridine/acetonitrile for a contact time of 3 minutes.
After the specified sequence is synthesized. The solid support bound designated sequence was suspended in ammonia (25 wt% to 30 wt%) and heated at 85 ℃ for 2h. The solid support is then filtered off and the ammonia is removed under reduced pressure. The residue was purified by high pressure liquid chromatography to prepare Gap antisense oligonucleotides as shown in table 1.
The Gap antisense oligonucleotides in Table 1 were 20 nucleotides in length and were designed as 5-10-5 spacers. Wherein the gap comprises 102 ' -deoxynucleosides and is flanked on both sides (in the 5' and 3' directions) by wings each comprising 5 nucleosides. Each nucleoside in the 5 'wing and each nucleoside in the 3' wing are modified with a 2'-MOE sugar, each nucleoside in the gap is modified with a 2' -deoxy sugar, the internucleoside linkage in the entire spacer is a phosphorothioate (p=s) linkage, and all cytosine residues in the entire spacer are 5-methylcytosine.
TABLE 1 solid phase technique for preparing Gap antisense oligonucleotides
Example 2
General procedure for preparation of MsPA modified antisense oligonucleotides by solid phase techniques
All reagents and solutions for synthesizing the oligomeric compounds were purchased from commercial sources unless otherwise indicated. Standard phosphoramidite building blocks and solid supports are used to incorporate nucleoside residues including, for example, T, A, G and mC residues. The phosphoramidite solution of all monomers used (β -D-2 '-deoxyribonucleoside and β -D-2' - (MOE) ribonucleoside) was a 0.06M solution in anhydrous acetonitrile.
A500 nmol synthesis column made of Universal CPG solid support was packed on LK-48E synthesizer and phosphoramidite coupling method was used to perform the designated sequence synthesis. For the coupling step, the phosphoramidite monomer was delivered in an amount exceeding 4 times the loading on the solid support and phosphoramidite condensation was performed for 10 min. All other steps follow the standard protocols supplied by the manufacturer. A solution of 3% trichloroacetic acid in dichloromethane was used to remove Dimethoxytrityl (DMT) from the 5' -hydroxy group of the nucleotide. BTT (0.35M, 0.5% nmi) in anhydrous acetonitrile was used as activator during the coupling step. Phosphorothioate linkages were introduced by sulfiding with a 0.2M solution of diphenylacetyl disulfide (PADS) in 1:1 pyridine/acetonitrile for a contact time of 3 minutes. The methanesulfonyl-phosphoramidate (MsPA) linkage was introduced by a 2 minute contact time with a 1M solution of methanesulfonyl azide (MsN) in acetonitrile.
After the specified sequence is synthesized. The solid support bound designated sequence was suspended in ammonia (25 wt% to 30 wt%) and heated at 85 ℃ for 2h. The solid support is then filtered off and the ammonia is removed under reduced pressure. The residue was purified by high pressure liquid chromatography to give MsPA modified antisense oligonucleotides.
Example 3
Antiviral Effect of Luciferase detection of antisense oligonucleotides of different sequences in HepG2-CMV-HBV-Luciferase cell line
The cells used in the experiment are HepG2 HBV-Luciferase stably transformed cell lines capable of stably expressing HBV. After three generations of cells were plated in 96-well plates at 3x104 cells/well, and after 12 hours different sequence oligonucleotides were transfected into the corresponding wells with Lipofectamine RNAiMax (thermo fisher) transfection reagent at a final concentration of 100nM, with the blank group being transfected with DEPC water. After culturing for 48h at 37℃under 5% CO2, the medium was changed, 50. Mu.L of fresh medium was added to each well, and 50. Mu.L of Bright-Glo Luciferase detection reagent was added to each well. After 1 hour incubation at room temperature, the Luciferase signal was detected with a microplate reader. The knockdown efficiency of the oligonucleotides was calculated compared to the group of cells treated with DEPC water. The knockdown effect of the oligonucleotides of different sequences on HBV gene is shown in FIG. 1, and each antisense oligonucleotide used is shown in Table 2.
TABLE 2 Effect of antisense oligonucleotides of different sequences on reducing HBV Gene expression
As shown in FIG. 1, 121 antisense oligonucleotides with different sequences used in the example reduce the expression of HBV genes in cells, the knocking down efficiency is different from 20% -90%, and the antisense oligonucleotides selected by the invention successfully inhibit the expression of corresponding mRNA.
Example 4
Real-time fluorescent quantitative PCR (polymerase chain reaction) detection of antiviral effect of antisense oligonucleotides with different concentrations in HepG2-CMV-HBV-Luciferase cell line
To further verify the inhibition of the antisense oligonucleotides to HBV genes in the present invention, we randomly selected 4-9 sequences from antisense oligonucleotides derived from different nucleotide fragments, respectively, and concentration gradient detection was performed on the selected sequences, i.e., to detect the antiviral effect of antisense oligonucleotides of different concentrations in cells.
The cells used in this example were HepG2-CMV-HBV-Luciferase cell line stably expressing HBV gene. The cells were seeded in 24-well plates, 1.5x105 cells per well, and after 12 hours different sequence oligonucleotides were transfected into the corresponding wells with Lipofectamine RNAiMax (ThermoFisher) transfection reagent at final concentrations of 33nM and 100nM, with the blank group being transfected with DEPC water. After further culturing at 37℃for 24 hours under 5% CO2, the cells were treated with Trizol (Invitrogen) to extract RNA, and the obtained RNA was used as a template for reverse transcription with mmlv reverse transcriptase (Promega) to obtain cDNA. And then detecting the expression of HBV genes by using cDNA as a template and ACTB as an internal reference gene and using real-time fluorescence quantitative PCR, and carrying out data analysis by adopting a2--ΔΔCT method. The knockdown efficiency of the oligonucleotides at different concentrations was calculated as compared to the group of cells treated with DEPC water and the results are shown in fig. 2 and table 3.
The detection results are shown in FIG. 2. In this example, a total of 20 antisense oligonucleotides were included, each at three different concentrations of 0nM,33nM and 100 nM. According to the real-time fluorescence quantitative PCR result, the oligonucleotide has dose dependency on HBV knockdown effect, wherein SG12-73, SG12-79 and SG12-85 have better antiviral effect, and higher HBV knockdown efficiency is shown at the final transfection concentration of 33 nM.
TABLE 3 antiviral Effect of antisense oligonucleotides at different concentrations on HBV
Example 5
Antiviral effect of different numbers MsPA bond antisense oligonucleotides at different sites in HepG2-CMV-HBV-Luciferase cell line by real-time fluorescence PCR detection
2,3 Or 4 MsPA bonds were introduced at different sites in SG12-73, SG12-79 and SG12-85, respectively, and the above antisense oligonucleotides containing MsPA modifications were tested for antiviral effect in cells.
The cells used in this example were HepG2-CMV-HBV-Luciferase cell line stably expressing HBV gene. The cells were seeded in 24-well plates, 1.5x105 cells per well, and after 12 hours different sequence oligonucleotides were transfected into the corresponding wells with Lipofectamine RNAiMax (ThermoFisher) transfection reagent at a final concentration of 100nM, with the blank group being transfected with DEPC water. After further culturing at 37℃for 24 hours under 5% CO2, the cells were treated with Trizol (Invitrogen) to extract RNA, and the obtained RNA was used as a template for reverse transcription with mmlv reverse transcriptase (Promega) to obtain cDNA. And then detecting the expression of HBV genes by using cDNA as a template and ACTB as an internal reference gene and using real-time fluorescence quantitative PCR, and carrying out data analysis by adopting a 2--ΔΔCT method. The knockdown efficiency of the oligonucleotides was calculated as compared to the group of cells treated with DEPC water, and the results are shown in FIG. 3, and the specific MsPA modification sites are shown in Table 4.
Table 4MsPA knock-down efficiency of modified antisense oligonucleotides
As a result, as shown in FIG. 3, the antisense oligonucleotides modified at the positions MsPA of the total of 58 different sequences in this example all showed a certain antiviral effect. Compared with antisense oligonucleotides without MsPA modification, the antisense oligonucleotides modified by MsPA on SG12-73 and SG12-85 sequences show better effects, but most of antiviral effects are obviously reduced. Surprisingly, antisense oligonucleotides modified MsPA on the SG12-79 sequence have a stable overall effect and exhibit a stronger or comparable antiviral effect.
Example 6
MsPA antiviral Effect of modified antisense oligonucleotide in AAV-HBV mouse model
In this example, a mouse model of persistent HBV infection was obtained by intravenous injection of rAAV8-1.3HBV into the tail of C57 mice. AAV virus injection dose is 1E+11vg/mouse, blood is taken after 3 weeks of virus injection to detect HBsAg content in serum, and mice are randomly divided into 3 groups after HBV virus stable replication in individuals is determined according to the detection result of the HBsAg in the serum of the mice. The first group is a normal saline control group, which comprises 4 mice, the second group is a positive control group, which comprises 3 mice, the positive control drug comprises SG12-79, and the third group is provided with antisense oligonucleotide (SG 12-79-2) modified at positions MsPA and 3, which comprises 4 mice. Each group of mice was dosed singly by subcutaneous injection at a dose of 100mg/kg and after the dosing was completed, blood was taken every 5 days to detect HBsAg content in serum. That is, each group of mice was dosed at D0, defined as the day of dosing, while serum HBsAg levels of each model mice were tested at D5, D10, D15, D20, D25, D30 and D35 by blood sampling and compared to HBsAg levels in D-2 serum to determine the anti-HBV effect of the different oligonucleotides in the mouse model. As shown in fig. 4 and table 5.
AAV-HBV model mice showed a significant decrease in HBsAg levels in serum after receiving a single dose. Wherein, msPA modified antisense oligonucleotide sequence has similar antiviral effect with positive control drug in mice, but the duration of drug effect is longer. As shown in FIG. 4, the antiviral effect of the positive drug control group was gradually reduced after completion of the single dose administration, the HBsAg content in the serum of the model mice was gradually recovered, the HBsAg content in the serum of the model mice at 30 days after the administration had been recovered to the pre-administration level, the HBsAg content in the serum of the model mice of the group was raised by 0.11log10 as compared with that before the administration at 35 days after the administration, and the antisense oligonucleotide containing the MsPA modification at 3,4 positions showed a more durable antiviral effect in the model mice, and the HBsAg content in the serum of the model mice was still reduced by 0.52log10 as compared with that before the administration after 30 days of the drug treatment, and the HBsAg content in the serum of the model mice was still reduced by 0.32log10 as compared with that before the administration after 35 days of the drug treatment.
TABLE 5MsPA antiviral Effect of modified antisense oligonucleotides on HBV in vivo