Detailed Description
The following describes specific embodiments of the present disclosure in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.
Definition of the definition
In the present disclosure, HAV viral RNA refers to the sequence shown by Genbank accession NC-001489.1.
In the foregoing and in the following, unless otherwise specified, uppercase letters C, G, U, A denote the base composition of the nucleotide, lowercase letters m denote that one nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide, lowercase letters f denote that one nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide, lowercase letters s denote that a phosphorothioate subunit linkage is between two nucleotides adjacent to the left and right of the letter s, P1 denote that one nucleotide adjacent to the right of the P1 is a 5' -phospho nucleotide or a 5' -phosphoanalogue-modified nucleotide, and in some embodiments P1 denotes a specifically modified VP, ps or P, wherein the letter combination VP denotes that one nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate (5 ' - (E) -vinylphosphonate, E-VP) -modified nucleotide, and uppercase letters P denote that one nucleotide adjacent to the right of the letter combination Ps is a phosphorothioate-modified nucleotide.
In the above and below, the "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group of the nucleotide is substituted with fluorine, and the "non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorine group. "nucleotide analog" refers to a group that is capable of replacing a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Such as an iso-nucleotide, a bridged nucleotide (bridged nucleic acid, abbreviated as BNA) or an acyclic nucleotide. The "methoxy-modified nucleotide" refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is replaced with a methoxy group.
In the present context, the expressions "complementary" or "reverse complementary" are used interchangeably and have the meaning known to the person skilled in the art, i.e. in a double stranded nucleic acid molecule the bases of one strand are each paired with a base on the other strand in a complementary manner. In DNA, the purine base adenine (a) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); in contrast, "mismatch" in the art means that in a double-stranded nucleic acid, the bases at corresponding positions do not pair in a complementary fashion, when adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary to each other, and the sequence of the strand can be deduced from the sequence of its complementary strand.
In the above and below, unless otherwise specified, "substantially reverse complement" refers to the presence of no more than 3 base mismatches between the two nucleotide sequences involved, "substantially reverse complement" refers to the presence of no more than 1 base mismatch between the two nucleotide sequences, and "fully reverse complement" refers to the absence of base mismatches between the two nucleotide sequences.
In the above and below, the "nucleotide difference" between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position is changed as compared with the former, for example, when one nucleotide base is A, when the corresponding nucleotide base at the same position of the former is U, C, G or T, it is determined that there is a nucleotide difference between the two nucleotide sequences at the position. In some embodiments, a nucleotide difference is also considered to occur at an original position when the nucleotide is replaced with an abasic nucleotide or its equivalent. An abasic nucleotide refers to a monomeric compound formed after the nucleobase in the nucleotide has been replaced by other groups or hydrogen atoms, including but not limited to substituted or unsubstituted aromatic or heteroaromatic groups.
In the foregoing and in the following, and particularly in describing the methods of preparing siRNA, pharmaceutical compositions or siRNA conjugates of the present disclosure, unless otherwise indicated, the nucleoside monomer (nucleoside monomer) refers to a modified or unmodified nucleoside phosphoramidite monomer (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites also referred to as Nucleoside phosphoramidites) used in phosphoramidite solid phase synthesis, depending on the type and order of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphoramidite solid phase synthesis is a method well known to those skilled in the art for use in RNA synthesis. Nucleoside monomers useful in the present disclosure are all commercially available.
In the context of the present disclosure, unless otherwise indicated, "conjugate" refers to a covalent linkage between two or more chemical moieties each having a particular function, and accordingly, "conjugate" refers to a compound formed by a covalent linkage between the respective chemical moieties. Further, "siRNA conjugate" means a compound formed by covalently attaching one or more chemical moieties having specific functions to an siRNA. siRNA conjugates are understood to be, depending on the context, the collective term of multiple siRNA conjugates or siRNA conjugates of a certain chemical formula. In the context of the present disclosure, a "conjugate molecule" is understood to be a specific compound that can be conjugated to an siRNA by reaction, ultimately forming the presently disclosed siRNA conjugate.
Those skilled in the art will appreciate that for any group comprising one or more substituents, these groups are not intended to introduce any substitution or pattern of substitution that is sterically impractical, synthetically infeasible, and/or inherently unstable.
As used herein, "alkyl" refers to straight and branched chains having the indicated number of carbon atoms, typically 1 to 20 carbon atoms, for example 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms. For example, the C1-C6 alkyl groups contain straight and branched alkyl groups of 1 to 6 carbon atoms. When referring to alkyl residues having a particular number of carbons, it is intended to cover all branched and straight chain forms having that number of carbons, and thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl, and "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl groups and refers to a divalent radical that is the same as alkyl groups but has two points of attachment.
As used herein, "alkenyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double bond obtained by removing a molecule of hydrogen from adjacent carbon atoms of the parent alkyl group. The group may be in the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to, ethenyl, propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl, butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylpropan-1-en-1-yl, but-2-en-2-yl, but-1, 3-dien-1-yl, but-1, 3-dien-2-yl, and the like. In certain embodiments, alkenyl groups have 2 to 20 carbon atoms, and in other embodiments 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl groups and refers to residues that are identical to alkenyl groups but have two points of attachment.
As used herein, "alkoxy" refers to an alkyl group of the specified number of carbon atoms attached through an oxygen bridge, e.g., methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-methylpentyloxy, and the like. Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected by an oxygen bridge.
Various hydroxyl protecting groups may be used in the present disclosure. In general, the protecting group renders the chemical functional group insensitive to specific reaction conditions and this functional group can be added and removed in the molecule without substantially damaging the rest of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al, tetrahedron 1992,48,2223-2311, and Greeneand Wuts,Protective Groups in Organic Synthesis,Chapter 2,2d ed,John Wiley&Sons,New York,1991, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under alkaline conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include Dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl), and 9- (p-methoxyphenyl) xanthen-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4, 4 '-dimethoxytrityl), and TMTr (4, 4',4 "-trimethoxytrityl).
The term "subject" as used herein refers to any animal, such as a mammal or a pouched animal. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, rabbits, sheep, rats, and any variety of poultry.
As used herein, "treatment" refers to a method of achieving a beneficial or desired result, including but not limited to therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is obtained by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.
In the siRNA or siRNA conjugates described in the present disclosure, each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate bond, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the phosphorothioate bond carries a negative charge, and the siRNA or siRNA conjugate can exist in a form of hydroxyl group or sulfhydryl group, and hydrogen ions in the hydroxyl group or sulfhydryl group can also be partially or completely replaced by cations. The cation may be any cation, such as one of a metal cation, ammonium ion NH4+, organic ammonium cation, in some embodiments selected from one or more of an alkali metal ion, a tertiary amine formed ammonium cation, and a quaternary ammonium cation, for improved solubility. The alkali metal cations may be K+ and/or Na+, and the tertiary amine cations may be triethylamine ammonium ions and/or N, N-diisopropylethylamine ammonium ions. Thus, the siRNA or siRNA conjugates of the present disclosure may exist at least partially in salt form. In some embodiments, the non-bridging oxygen or sulfur atoms in the phosphodiester or phosphorothioate linkages are at least partially bound to sodium ions, and the siRNA or siRNA conjugates of the present disclosure are in the form of sodium salts or partial sodium salts. Thus, references to an siRNA or siRNA conjugate described in the present disclosure, including but not limited to an siRNA conjugate of any structural formula described in the present disclosure, are intended to encompass the sodium salt or partial sodium salt form of the siRNA or siRNA conjugate.
SiRNA of the present disclosure
In one aspect, the present disclosure provides an siRNA capable of inhibiting HAV viral RNA.
The siRNA of the present disclosure contains a nucleotide group as a basic structural unit, which is well known to those skilled in the art, and the nucleotide group contains a phosphate group, a ribose group, and a base, and is not described herein.
The siRNA of the present disclosure contains a sense strand and an antisense strand, the sense strand and the antisense strand being the same or different in length, the sense strand being 19-23 nucleotides in length, and the antisense strand being 19-26 nucleotides in length. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure may be 19/19、19/20、19/21、19/22、19/23、19/24、19/25、19/26、20/20、20/21、20/22、20/23、20/24、20/25、20/26、21/20、21/21、21/22、21/23、21/24、21/25、21/26、22/20、22/21、22/22、22/23、22/24、22/25、22/26、23/20、23/21、23/22、23/23、23/24、23/25 or 23/26. In some embodiments, the siRNA has a length ratio of sense strand to antisense strand of 19/21, 21/23 or 23/25.
According to the present disclosure, the siRNA comprises a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a stretch of nucleotide sequence I and the antisense strand comprises a stretch of nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse-complementary to form a double-stranded region.
In some embodiments, the siRNA with stabilized modified nucleotides of the present disclosure may be the following first to fourth sirnas, each of which is described below, respectively.
First siRNA
In some embodiments, the siRNA of the present disclosure, the nucleotide sequence I is equal in length to the nucleotide sequence set forth in SEQ ID No. 1 and is NO more than 3 nucleotides different, and the nucleotide sequence II is equal in length to the nucleotide sequence set forth in SEQ ID No. 2 and is NO more than 3 nucleotides different:
5'-CCAGUUUGGGAAUUGACAZa1-3'(SEQ ID NO:1);
5'-Za2UGUCAAUUCCCAAACUGG-3'(SEQ ID NO:2),
Wherein Za1 is A and Za2 is U, nucleotide sequence I comprises nucleotide Za3 corresponding in position to Za1, nucleotide sequence II comprises nucleotide Za4 corresponding in position to Za2, and Za4 is the first nucleotide at the 5' -end of the antisense strand.
In the context of the present disclosure, "position corresponds" means that, from the same end of the nucleotide sequence, the first nucleotide at the 3 'end of the nucleotide sequence I is the nucleotide at the position corresponding to the 1 st nucleotide at the 3' end of SEQ ID NO. 1, for example, at the same position in the nucleotide sequence.
In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II. In some embodiments, the nucleotide sequence I differs from the nucleotide sequence set forth in SEQ ID NO.1 by NO more than 1 nucleotide, and/or the nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO.2 by NO more than 1 nucleotide.
In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO. 2 comprises a difference at position Za4, and Za4 is selected from A, C or G. In some embodiments, the Za3 is a nucleotide complementary to Za4.
In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary, wherein substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences, wherein substantially reverse complementary means that there are no more than 1 base mismatch between the two nucleotide sequences, and wherein fully reverse complementary means that there are no base mismatches between the two nucleotide sequences.
In some embodiments, the sense strand of the siRNA comprises a nucleotide sequence as set forth in SEQ ID NO. 3 and the antisense strand of the siRNA comprises a nucleotide sequence as set forth in SEQ ID NO. 4:
5'-CCAGUUUGGGAAUUGACAZa3-3'(SEQ ID NO:3);
5'-Za4 UGUCAAUUCCCAAACUGG-3'(SEQ ID NO:4),
Wherein, Za4 is the first nucleotide at the 5' end of the antisense strand, Za3 is selected from A, U, G or C, and Za4 is a nucleotide complementary to Za3;
In some embodiments, Za3 is a and Za4 is U.
In some embodiments, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, the nucleotide sequence III and the nucleotide sequence IV are each 1-4 nucleotides in length, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is attached to the 5 'end of the nucleotide sequence I, and the nucleotide sequence IV is attached to the 3' end of the nucleotide sequence II.
In some embodiments, the nucleotide sequence IV is substantially reverse-complementary or fully reverse-complementary to a second nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO. 1 and is the same length as the nucleotide sequence IV in the HAV viral RNA.
In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are each 1 nucleotide in length in the 5'-3' direction, the nucleotide sequence III is C, the nucleotide sequence IV is G, the ratio of the length of the sense strand to the length of the antisense strand is 20/20, or the nucleotide sequence III and the nucleotide sequence IV are each 2 nucleotides in length, the nucleotide sequence III is AC in the 5'-3' end direction, the nucleotide sequence IV is GU in the nucleotide sequence IV is 21/21 in the sense strand to the length of the antisense strand, or the nucleotide sequence III and the nucleotide sequence IV are each 3 nucleotides in length, the nucleotide sequence III is UAC in the 5'-3' end direction, the nucleotide sequence IV is GUA in the nucleotide sequence III is 22/22 in the sense strand to the antisense strand is AUAC in the 5'-3' end direction, and the nucleotide sequence III is 5323 in the nucleotide sequence IV is 5323/3923 in the sense strand to the antisense strand is 5323.
In some embodiments, nucleotide sequence III and nucleotide sequence IV are fully reverse-complementary, thus, the base of nucleotide sequence III is given, and the base of nucleotide sequence IV is also determined.
Second siRNA
In some embodiments, the siRNA of the present disclosure, the nucleotide sequence I is equal in length to the nucleotide sequence set forth in SEQ ID NO. 33 and is not more than 3 nucleotides different, and the nucleotide sequence II is equal in length to the nucleotide sequence set forth in SEQ ID NO. 34 and is not more than 3 nucleotides different:
5'-GAUAUGUCUUGUGUUGAUZb1-3'(SEQ ID NO:33);
5'-Zb2AUCAACACAAGACAUAUC-3'(SEQ ID NO:34),
Wherein Zb1 is U and Zb2 is A, nucleotide Zb3 corresponding in position to Zb1 is contained in nucleotide sequence I, nucleotide Zb4 corresponding in position to Zb2 is contained in nucleotide sequence II, and Zb4 is the first nucleotide at the 5' -end of the antisense strand.
In some embodiments, the nucleotide sequence I differs from the nucleotide sequence set forth in SEQ ID NO. 33 by NO more than 1 nucleotide and/or the nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO. 34 by NO more than 1 nucleotide.
In some embodiments, the difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO 34 includes a difference at position Zb4, and Zb4 is selected from U, G or C.
In some embodiments, the Zb3 is a nucleotide complementary to Zb4. In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary, wherein substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences, wherein substantially reverse complementary means that there are no more than 1 base mismatch between the two nucleotide sequences, and wherein fully reverse complementary means that there are no base mismatches between the two nucleotide sequences.
In some embodiments, the sense strand of the siRNA comprises a nucleotide sequence as set forth in SEQ ID NO:35 and the antisense strand of the siRNA comprises a nucleotide sequence as set forth in SEQ ID NO: 36:
5'-GAUAUGUCUUGUGUUGAUZb3 -3'(SEQ ID NO:35);
5'-Zb4AUCAACACAAGACAUAUC-3'(SEQ ID NO:36),
Wherein, Zb4 is the first nucleotide at the 5' end of the antisense strand, Zb3 is selected from A, U, G or C, and Zb4 is a nucleotide complementary to Zb3.
In some embodiments, wherein Zb3 is U and Zb4 is a.
In some embodiments, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, the nucleotide sequence III and the nucleotide sequence IV are each 1-4 nucleotides in length, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is attached to the 5 'end of the nucleotide sequence I, and the nucleotide sequence IV is attached to the 3' end of the nucleotide sequence II.
In some embodiments, the nucleotide sequence IV is substantially reverse-complementary or fully reverse-complementary to a second nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO. 33 and is the same length as the nucleotide sequence IV in the HAV viral RNA.
In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are each 1 nucleotide in length in the 5'-3' direction, the nucleotide sequence III is A, the nucleotide sequence IV is U, the ratio of the length of the sense strand to the length of the antisense strand is 20/20, or the nucleotide sequence III and the nucleotide sequence IV are each 2 nucleotides in length, the nucleotide sequence III is AA in the 5'-3' direction, the nucleotide sequence IV is UU in the 5'-3' direction, the ratio of the length of the sense strand to the length of the antisense strand is 21/21, or the nucleotide sequence III and the nucleotide sequence IV are each 3 nucleotides in length, the nucleotide sequence III is AAA in the 5'-3' direction, the nucleotide sequence IV is UU in the length ratio of the sense strand to the antisense strand, the ratio of the length of the sense strand to the length of the antisense strand is 22/22, or the nucleotide sequence III and the length of the nucleotide sequence IV are each 4 nucleotides in the 5'-3' direction, the nucleotide sequence III is AA in the 5'-3' direction, the nucleotide sequence III is 5723, and the nucleotide sequence IV is CA23 in the ratio of the sense strand to the length of the antisense strand is 5723.
In some embodiments, nucleotide sequence III and nucleotide sequence IV are fully reverse-complementary, thus, the base of nucleotide sequence III is given, and the base of nucleotide sequence IV is also determined.
Third siRNA
In some embodiments, the siRNA of the present disclosure, the nucleotide sequence I is equal in length to the nucleotide sequence set forth in SEQ ID NO. 65 and is not more than 3 nucleotides different, and the nucleotide sequence II is equal in length to the nucleotide sequence set forth in SEQ ID NO. 66 and is not more than 3 nucleotides different:
5'-GGUGGAACUUACUAUUCAZc1-3'(SEQ ID NO:65);
5'-Zc2UGAAUAGUAAGUUCCACC-3'(SEQ ID NO:66),
Wherein Zc1 is A and Zc2 is U, nucleotide sequence I comprises nucleotide Zc3 corresponding in position to Zc1, nucleotide sequence II comprises nucleotide Zc4 corresponding in position to Zc2, and Zc4 is the first nucleotide at the 5' -end of the antisense strand.
In some embodiments, the nucleotide sequence I differs from the nucleotide sequence set forth in SEQ ID NO. 65 by NO more than 1 nucleotide and/or the nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO. 66 by NO more than 1 nucleotide;
In some embodiments, the difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:66 comprises a difference at position Zc4, and Zc4 is selected from A, G or C.
In some embodiments, the Zc3 is a nucleotide complementary to Zc4.
In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary, wherein substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences, wherein substantially reverse complementary means that there are no more than 1 base mismatch between the two nucleotide sequences, and wherein fully reverse complementary means that there are no base mismatches between the two nucleotide sequences.
In some embodiments, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 67 and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 68:
5'-GGUGGAACUUACUAUUCA Zc3 -3'(SEQ ID NO:67);
5'-Zc4 UGAAUAGUAAGUUCCACC-3'(SEQ ID NO:68),
Wherein, Zc4 is the first nucleotide at the 5' end of the antisense strand, Zc3 is selected from A, U, G or C, and Zc4 is a nucleotide complementary to Zc3.
In some embodiments, wherein Zc3 is a and Zc4 is U.
In some embodiments, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, the nucleotide sequence III and the nucleotide sequence IV are each 1-4 nucleotides in length, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is attached to the 5 'end of the nucleotide sequence I, and the nucleotide sequence IV is attached to the 3' end of the nucleotide sequence II.
In some embodiments, the nucleotide sequence IV is substantially reverse-complementary or fully reverse-complementary to a second nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO. 65 and is the same length as the nucleotide sequence IV in the HAV viral RNA.
In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are each 1 nucleotide in length in the 5'-3' direction, the nucleotide sequence III is A, the nucleotide sequence IV is U, the ratio of the length of the sense strand to the length of the antisense strand is 20/20, or the nucleotide sequence III and the nucleotide sequence IV are each 2 nucleotides in length, the nucleotide sequence III is GA in the 5 '-end to 3' -end direction, the nucleotide sequence IV is UC in the 5 '-end to 3' -end direction, the ratio of the length of the sense strand to the length of the antisense strand is 21/21, or the nucleotide sequence III and the nucleotide sequence IV are each 3 nucleotides in length, the nucleotide sequence III is AGA in the 5 '-end to 3' -end direction, the nucleotide sequence IV is UCU in the length ratio of the sense strand to the antisense strand, the ratio of the length of the sense strand to the antisense strand is 22/22, or the nucleotide sequence III and the nucleotide sequence IV are each 4 nucleotides in length in the 5 '-end to 3' -end direction, the nucleotide sequence III is GA and the nucleotide sequence IV is 23/23 in the length ratio of the sense strand to the UCU.
In some embodiments, nucleotide sequence III and nucleotide sequence IV are fully reverse-complementary, thus, the base of nucleotide sequence III is given, and the base of nucleotide sequence IV is also determined.
Fourth siRNA
In some embodiments, the siRNA of the present disclosure, the nucleotide sequence I is equal in length to the nucleotide sequence set forth in SEQ ID NO:97 and is not more than 3 nucleotides different, and the nucleotide sequence II is equal in length to the nucleotide sequence set forth in SEQ ID NO:98 and is not more than 3 nucleotides different:
5'-GGUACAACAGUUGAUUUAZd1-3'(SEQ ID NO:97);
5'-Zd2UAAAUCAACUGUUGUACC-3'(SEQ ID NO:98),
Wherein Zd1 is A and Zd2 is U, nucleotide sequence I comprises nucleotide Zd3 corresponding in position to Zd1, nucleotide sequence II comprises nucleotide Zd4 corresponding in position to Zd2, and Zd4 is the first nucleotide at the 5' -end of the antisense strand.
In some embodiments, the nucleotide sequence I differs from the nucleotide sequence set forth in SEQ ID NO. 97 by NO more than 1 nucleotide and/or the nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO. 98 by NO more than 1 nucleotide.
In some embodiments, the difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO. 98 includes a difference at position Zd4, and Zd4 is selected from A, G or C.
In some embodiments the Zd3 is a nucleotide complementary to Zd4. In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary, wherein substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences, wherein substantially reverse complementary means that there are no more than 1 base mismatch between the two nucleotide sequences, and wherein fully reverse complementary means that there are no base mismatches between the two nucleotide sequences.
In some embodiments, the sense strand of the siRNA comprises a nucleotide sequence as set forth in SEQ ID NO. 99 and the antisense strand of the siRNA comprises a nucleotide sequence as set forth in SEQ ID NO. 100:
5'-GGUACAACAGUUGAUUUAZd3 -3'(SEQ ID NO:99);
5'-Zd4UAAAUCAACUGUUGUACC-3'(SEQ ID NO:100),
Wherein, Zd4 is the first nucleotide at the 5' end of the antisense strand, Zd3 is selected from A, U, G or C, and Zd4 is a nucleotide complementary to Zd3.
In some embodiments, Zd3 is a and Zd4 is U.
In some embodiments, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, the nucleotide sequence III and the nucleotide sequence IV are each 1-4 nucleotides in length, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is attached to the 5 'end of the nucleotide sequence I, and the nucleotide sequence IV is attached to the 3' end of the nucleotide sequence II.
In some embodiments, the nucleotide sequence IV is substantially reverse-complementary or fully reverse-complementary to a second nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO. 97 and is the same length as the nucleotide sequence IV in the HAV viral RNA. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are each 1 nucleotide in length in the 5'-3' direction, the base of nucleotide sequence III is U and the base of nucleotide sequence IV is a; at this time, the length ratio of the sense strand and the antisense strand was 20/20, or the length of each of the nucleotide sequences III and IV was 2 nucleotides, the base composition of the nucleotide sequence III was AU in the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence IV was AU, at this time, the length ratio of the sense strand and the antisense strand was 21/21, or the length of each of the nucleotide sequences III and IV was 3 nucleotides, the base composition of the nucleotide sequence III was GAU, the base composition of the nucleotide sequence IV was AUC in the direction from the 5 'end to the 3' end, the length ratio of the sense strand and the antisense strand was 22/22, or the length of each of the nucleotide sequences III and IV was 4 nucleotides, the base composition of the nucleotide sequence III was UGAU, the base composition of the nucleotide sequence IV was AUCA, and the length ratio of the sense strand and the antisense strand was 23/23.
In some embodiments, nucleotide sequence III and nucleotide sequence IV are fully reverse-complementary, thus, the base of nucleotide sequence III is given, and the base of nucleotide sequence IV is also determined. In some embodiments, each U or T in the nucleotide sequence of the siRNA of the disclosure can be arbitrarily replaced with each other. These nucleotide differences do not significantly reduce the HAV viral RNA inhibition ability of the siRNA or increase the off-target effect of the siRNA. These siRNAs comprising nucleotide differences are also within the scope of the present disclosure.
The following description of nucleotide modifications in nucleotide sequence V, siRNA and modified sequences applies to the siRNAs of the present disclosure described above, e.g., first, second, third, and fourth siRNAs. That is, the following description of sirnas shall be regarded as referring to the four sirnas of the present disclosure described above, for example, unless a specific siRNA is specifically indicated, "the sirnas further contain a nucleotide sequence V" means "the sirnas of the present disclosure, for example, the first, second, third, or fourth sirnas further contain a nucleotide sequence V" unless otherwise specified. In some embodiments, the sense strand and the antisense strand are different in length, the antisense strand further comprising a nucleotide sequence V of 1 to 3 nucleotides in length attached to the 3 'end of the antisense strand, constituting a 3' overhang of the antisense strand.
In some embodiments, the sense strand further comprises a nucleotide sequence VI, 1 to 3 nucleotides in length, attached to the 3 'end of the sense strand, constituting a 3' overhang of the sense strand.
In some embodiments, the siRNA provided by the present disclosure includes nucleotide sequence V, but does not include nucleotide sequence VI. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure can be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23, 21/24, 22/23, 22/24, 22/25, 23/24, 23/25, or 23/26. In some embodiments, the siRNA provided by the present disclosure comprises nucleotide sequences V and VI. In some embodiments, the length of nucleotide sequence V is the same as or different from the length of nucleotide sequence VI. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure can be (19-26): (19-26). In some embodiments, the nucleotide sequences V and/or VI are 2 nucleotides in length, and thus, the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure may be 19/21, 21/23, 23/25 or 25/25.
Each of the nucleotides in the nucleotide sequence V may be any nucleotide, in some embodiments, the nucleotide sequence V is a contiguous 2 thymidylate (dTdT) or a contiguous 2 uracil ribonucleotide (UU) for ease of synthesis and cost effectiveness, or the nucleotide sequence V is complementary to a nucleotide at a corresponding position of the HAV viral RNA for improved affinity of the siRNA antisense strand to the HAV viral RNA. In some embodiments, the corresponding position refers to the position of the third nucleotide sequence or the fourth nucleotide sequence. Thus, in some embodiments, the ratio of the length of the sense strand to the length of the antisense strand of the siRNA of the present disclosure is 19/21 or 21/23, at which time the siRNA of the present disclosure has better mRNA silencing activity.
The third nucleotide sequence refers to a nucleotide sequence adjacent to the 5' end of SEQ ID NO. 1 or SEQ ID NO. 33 or SEQ ID NO. 65 or SEQ ID NO. 97 or the second nucleotide sequence and of equal length as the nucleotide sequence V.
Each of the nucleotides in the nucleotide sequence VI may be any nucleotide, in some embodiments, two consecutive thymidylate nucleotides (dTdT) or two consecutive uracil ribonucleotides (UU) for ease of synthesis and cost-effectiveness of synthesis, or the nucleotide sequence VI may be identical to a nucleotide at a corresponding position of the HAV viral RNA for improved affinity of the siRNA sense strand to the antisense strand. In some embodiments, the corresponding position refers to the position of the fourth nucleotide sequence. Thus, in some embodiments, the sirnas of the present disclosure comprise nucleotide sequences V and VI, and the ratio of the lengths of the sense strand and the antisense strand of the sirnas is 21/21 or 23/23, at which point the sirnas of the present disclosure have better mRMA silencing activity.
The fourth nucleotide sequence is a nucleotide sequence adjacent to the 3' end of SEQ ID NO.1 or SEQ ID NO. 33 or SEQ ID NO. 65 or SEQ ID NO. 97 and of equal length to nucleotide sequence VI.
In some embodiments, for the first siRNA, the sense strand of the siRNA comprises a nucleotide as set forth in SEQ ID NO. 5 and the base composition of the third nucleotide sequence is AC, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO. 6:
5'-CCAGUUUGGGAAUUGACAZa3-3'(SEQ ID NO:5);
5'-Za4UGUCAAUUCCCAAACUGGGU-3'(SEQ ID NO:6);
Wherein, Za4 is the first nucleotide at the 5' end of the antisense strand, Za3 is selected from A, U, G or C, and Za4 is a nucleotide complementary to Za3.
Or the sense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 7, and the antisense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 8:
5'-ACCCAGUUUGGGAAUUGACAZa3-3'(SEQ ID NO:7);
5'-Za4UGUCAAUUCCCAAACUGGGUAU-3'(SEQ ID NO:8);
Wherein, Za4 is the first nucleotide at the 5' end of the antisense strand, Za3 is selected from A, U, G or C, and Za4 is a nucleotide complementary to Za3.
In some embodiments, for the second siRNA, the sense strand of the siRNA comprises a nucleotide as set forth in SEQ ID NO. 37 and the base composition of the third nucleotide sequence is AA, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO. 38:
5'-GAUAUGUCUUGUGUUGAUZb3-3'(SEQ ID NO:37);
5'-Zb4AUCAACACAAGACAUAUCUU-3'(SEQ ID NO:38);
Wherein, Zb4 is the first nucleotide at the 5' end of the antisense strand, Zb3 is selected from A, U, G or C, and Zb4 is a nucleotide complementary to Zb3;
or the sense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO:39, and the antisense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO: 40:
5'-AAGAUAUGUCUUGUGUUGAUZb3-3'(SEQ ID NO:39);
5'-Zb4AUCAACACAAGACAUAUCUUUG-3'(SEQ ID NO:40);
Wherein, Zb4 is the first nucleotide at the 5' end of the antisense strand, Zb3 is selected from A, U, G or C, and Zb4 is a nucleotide complementary to Zb3.
In some embodiments, for the third siRNA, the sense strand of the siRNA comprises a nucleotide as set forth in SEQ ID NO:69, the base composition of the third nucleotide sequence is GA, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 70:
5'-GGUGGAACUUACUAUUCAZc3-3'(SEQ ID NO:69);
5'-Zc4UGAAUAGUAAGUUCCACCUC-3'(SEQ ID NO:70);
Wherein, Zc4 is the first nucleotide at the 5' end of the antisense strand, Zc3 is selected from A, U, G or C, and Zc4 is a nucleotide complementary to Zc3;
Or the sense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO:71, and the antisense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO: 72:
5'-GAGGUGGAACUUACUAUUCAZc3-3'(SEQ ID NO:71);
5'-Zc4 UGAAUAGUAAGUUCCACCUCUA-3'(SEQ ID NO:72);
Wherein, Zc4 is the first nucleotide at the 5' end of the antisense strand, Zc3 is selected from A, U, G or C, and Zc4 is a nucleotide complementary to Zc3.
In some embodiments, for the fourth siRNA, the sense strand of the siRNA comprises a nucleotide as set forth in SEQ ID NO:101 and the base composition of the third nucleotide sequence is UA, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 102:
5'-GGUACAACAGUUGAUUUAZd3-3'(SEQ ID NO:101);
5'-Zd4UAAAUCAACUGUUGUACCAU-3'(SEQ ID NO:102);
Wherein, Zd4 is the first nucleotide at the 5' end of the antisense strand, Zd3 is selected from A, U, G or C, and Zc4 is a nucleotide complementary to Zd3;
Or the sense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO. 103, and the antisense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO. 104:
5'-AUGGUACAACAGUUGAUUUAZd3-3'(SEQ ID NO:103);
5'-Zd4UAAAUCAACUGUUGUACCAUCA-3'(SEQ ID NO:104);
Wherein, Zd4 is the first nucleotide at the 5' end of the antisense strand, Zd3 is selected from A, U, G or C, and Zd4 is a nucleotide complementary to Zd3.
In the present disclosure, unless otherwise specified, the nucleotide sequences appearing are all arranged in the 5'-3' direction.
In some embodiments, the siRNA of the present disclosure is siHAVa, siHAVa2, siHAVa3, siHAVb1, siHAVb2, siHAVb3, siHAVc1, siHAVc2, siHAVc3, siHAVd1, siHAVd2, and siHAVd3 listed in table 1.
As previously described, the nucleotides in the sirnas of the present disclosure are each independently a modified or unmodified nucleotide. In some embodiments, some or all of the nucleotides in the siRNAs of the present disclosure are modified nucleotides, and in some embodiments, such modifications on the nucleotide groups do not result in a significant impairment or loss of the function of the siRNAs of the present disclosure in inhibiting HAV viral RNA.
In some embodiments, the siRNA of the present disclosure contains at least 1 modified nucleotide. In the context of the present disclosure, the term "modified nucleotide" is used to refer to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is replaced with another group, or a nucleotide in which the base on the nucleotide is a modified base. The modified nucleotide does not result in a significant impairment or loss of function of the siRNA in inhibiting HAV viral RNA. For example, the modified nucleotide disclosed in J.K.Watts,G.F.Deleavey,and M.J.Damha,Chemically modified siRNA:tools and applications.Drug Discov Today,2008,13(19-20):842-55 can be selected.
In some embodiments, the present disclosure provides that at least one nucleotide in the sense strand or the antisense strand of the siRNA is a modified nucleotide and/or at least one phosphate group is a phosphate group having a modifying group, in other words, at least a portion of the phosphate groups and/or ribose groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand are phosphate groups and/or ribose groups having a modifying group.
In some embodiments, all of the nucleotides in the sense strand and/or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand of the siRNA provided by the present disclosure is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.
In some embodiments, the fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, the fluoro-modified nucleotides in nucleotide sequence I are not more than 5, and the nucleotides at positions 7, 8 and 9 of the nucleotide sequence I are fluoro-modified nucleotides in the direction from the 5 'end to the 3' end, the fluoro-modified nucleotides in nucleotide sequence II are not more than 7, and the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence II are fluoro-modified nucleotides.
In some embodiments, the nucleotides at positions 7, 8, 9 or 5, 7, 8, 9 of the nucleotide sequence I are fluoro-modified nucleotides in the sense strand in a 5 'to 3' end orientation, the nucleotides at the remaining positions in the sense strand are non-fluoro-modified nucleotides, and the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluoro-modified nucleotides in the antisense strand in a 5 'to 3' end orientation.
In the context of the present disclosure, a "fluoro-modified nucleotide" refers to a nucleotide formed by substitution of the hydroxyl group at the 2' -position of the ribosyl of the nucleotide with fluorine, which has a structure represented by the following formula (7). "non-fluoro modified nucleotide" refers to a nucleotide, or nucleotide analogue, in which the hydroxyl group at the 2' -position of the ribosyl of the nucleotide is substituted with a non-fluoro group. In some embodiments, each non-fluoro modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by substitution of the hydroxyl group at the 2' position of the ribosyl of the nucleotide with a non-fluoro group.
Nucleotides in which the hydroxyl group at the 2 '-position of the ribosyl group is substituted with a non-fluorine group are well known to those skilled in the art and may be selected from one of 2' -alkoxy-modified nucleotides, 2 '-substituted alkoxy-modified nucleotides, 2' -alkyl-modified nucleotides, 2 '-substituted alkyl-modified nucleotides, 2' -amino-modified nucleotides, 2 '-substituted amino-modified nucleotides, 2' -deoxynucleotides.
In some embodiments, the 2 '-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2' -OMe), as shown in formula (8). In some embodiments, the 2' -substituted alkoxy-modified nucleotide may be, for example, a 2' -O-methoxyethyl-modified nucleotide (2 ' -MOE), as shown in formula (9). In some embodiments, the 2 '-amino modified nucleotide (2' -NH2) is represented by formula (10). In some embodiments, the 2' -Deoxynucleotide (DNA) is represented by formula (11):
Nucleotide analogs refer to groups that are capable of replacing nucleotides in a nucleic acid, but that differ in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, the nucleotide analog may be an iso-nucleotide, a bridged nucleotide (bridged nucleic acid, abbreviated BNA), or an acyclic nucleotide.
BNA refers to a constrained or inaccessible nucleotide. BNA may contain a five-, six-, or seven-membered ring bridging structure with "fixed" C3' -endo-saccharides tucked. The bridge is typically incorporated at the 2'-, 4' -position of the ribose to provide a 2',4' -BNA nucleotide. In some embodiments, the BNA may be LNA, ENA, cET BNA, etc., wherein LNA is shown as formula (12), ENA is shown as formula (13), cET BNA is shown as formula (14):
Acyclic nucleotides are a class of nucleotides in which the sugar ring of a nucleotide is opened. In some embodiments, the acyclic nucleotide can be an Unlocking Nucleic Acid (UNA) or a Glycerol Nucleic Acid (GNA), wherein UNA is represented by formula (15), and GNA is represented by formula (16):
in the above formula (15) and formula (16), R is selected from H, OH or alkoxy (O-alkyl).
An isopucleotide refers to a compound in which the position of a base on the ribose ring is changed in a nucleotide. In some embodiments, the isonucleotide may be a compound formed by shifting a base from the 1' -position to the 2' -position or the 3' -position of the ribose ring, as shown in formula (17) or (18).
In the compounds of the formulae (17) to (18) described above, base represents a nucleobase, for example A, U, G, C or T, R is selected from H, OH, F or a non-fluoro group as described above.
In some embodiments, the nucleotide analog is selected from one of an iso-nucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluoro modified nucleotide is a methoxy modified nucleotide, which in the foregoing and below refers to a nucleotide formed by substitution of the 2' -hydroxy group of the ribosyl group with a methoxy group.
In the above and below, "fluoro-modified nucleotide" refers to a compound having a structure shown in formula (7) formed by substitution of 2 '-hydroxyl group of nucleotide with fluoro, and "methoxy-modified nucleotide" refers to a compound having a structure shown in formula (8) formed by substitution of 2' -hydroxyl group of ribose group of nucleotide with methoxy.
In some embodiments, the siRNA of the present disclosure is an siRNA having modifications such that in the sense strand, the nucleotides at positions 7,8,9 or 5, 7,8,9 of the nucleotide sequence I are fluoro modified nucleotides, the nucleotides at the remaining positions in the sense strand are methoxy modified nucleotides, and in the antisense strand, the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8,9, 14, 16 of the nucleotide sequence II are fluoro modified nucleotides, the nucleotides at the remaining positions in the antisense strand are methoxy modified nucleotides.
In some embodiments, the siRNA of the present disclosure is an siRNA having modifications in which the nucleotides at positions 5, 7,8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides, and the nucleotides at positions 2,6, 8,9, 14 and 16 of the nucleotide sequence II in the antisense strand of the siRNA in the 5 'end to 3' end direction are fluoro-modified nucleotides, the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides;
Or in the direction from the 5 'end to the 3' end, the nucleotides at positions 5, 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, the nucleotides at the rest of the sense strand of the siRNA are methoxy-modified nucleotides, and in the direction from the 5 'end to the 3' end, the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides, the nucleotides at the rest of the antisense strand of the siRNA are methoxy-modified nucleotides;
Or the nucleotides at positions 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, the nucleotides at the rest of the sense strand of the siRNA are methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides, the nucleotides at the rest of the antisense strand of the siRNA are methoxy-modified nucleotides, in the direction from the 5 'end to the 3' end.
In some embodiments, the sirnas provided by the present disclosure are arbitrarily selected from one of the following sirnas ：siHAVa1-M1、siHAVa2-M1、siHAVa3-M1、siHAVb1-M1、siHAVb2-M1、siHAVb3-M1、siHAVc1-M1、siHAVc2-M1、siHAVc3-M1、siHAVd1-M1、siHAVd2-M1 and siHAVd-M1.
The siRNA with the modification has low cost, and can ensure that ribonuclease in blood is not easy to cut nucleic acid, thereby increasing the stability of the nucleic acid and ensuring that the nucleic acid has stronger performance of resisting nuclease hydrolysis. Meanwhile, the modified siRNA has higher activity of inhibiting HAV virus RNA.
In some embodiments, at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA provided by the present disclosure are phosphate groups having a modifying group. In some embodiments, the phosphate group having a modifying group is a phosphorothioate subunit formed by substitution of at least one oxygen atom of a phosphodiester bond in the phosphate group with a sulfur atom, and in some embodiments, the phosphate group having a modifying group is a phosphorothioate subunit having a structure as shown in formula (1):
This modification stabilizes the double-stranded structure of the siRNA, maintaining high specificity and high affinity for base pairing.
In some embodiments, the present disclosure provides that the phosphorothioate subunit linkage is present in at least one of the group consisting of between the first and second nucleotides at either end of the sense strand or the antisense strand, between the second and third nucleotides at either end of the sense strand or the antisense strand, or any combination thereof. In some embodiments, phosphorothioate subunit linkages are present at all of the above positions except the 5' end of the sense strand. In some embodiments, phosphorothioate subunit linkages are present at all of the above positions except at the 3' terminus of the sense strand. In some embodiments, the phosphorothioate subunit linkage is present in at least one of the following positions:
Between nucleotide 1 and nucleotide 2 of the 5' end of the sense strand;
between nucleotide 2 and nucleotide 3 of the 5' end of the sense strand;
the 3' end of the sense strand is between nucleotide 1 and nucleotide 2;
the 3' end of the sense strand is between nucleotide 2 and nucleotide 3;
the 5' end of the antisense strand is between nucleotide 1 and nucleotide 2;
The 5' end of the antisense strand is between nucleotide 2 and nucleotide 3;
Between nucleotide 1 and nucleotide 2 of the 3' -end of the antisense strand, and
The 3' -end of the antisense strand is between nucleotide 2 and nucleotide 3.
In some embodiments, the sirnas provided by the present disclosure are arbitrarily selected from one of the following sirnas ：siHAVa1-M1S、siHAVa1-M1X、siHAVa2-M1S、siHAVa2-M1X、siHAVa3-M1S、siHAVa3-M1X、siHAVb1-M1S、siHAVb1-M1X、siHAVb2-M1S、siHAVb2-M1X、siHAVb3-M1S、siHAVb3-M1X、siHAVc1-M1S、siHAVc1-M1X、siHAVc2-M1S、siHAVc2-M1X、siHAVc3-M1S、siHAVc3-M1X、siHAVd1-M1S、siHAVd1-M1X、siHAVd2-M1S、siHAVd2-M1X、siHAVd3-M1S and siHAVd3-M1X.
In some embodiments, the 5' -terminal nucleotide of the siRNA antisense strand is a 5' -phosphonucleotide or a 5' -phosphoanalog modified nucleotide.
Commonly used nucleotides modified with such 5' -phosphonucleotides or 5' -phosphoanalogs are well known to those skilled in the art, e.g., a 5' -phosphonucleotide may have the following structure:
As further disclosed in ,Anastasia Khvorova and Jonathan K.Watts,The chemical evolution of oligonucleotide therapies of clinical utility.Nature Biotechnology,2017,35(3):238-48 are the following 4 5' -phosphate analog modified nucleotides:
Wherein R is H, OH, methoxy and fluorine, and Base represents a nucleobase and is A, U, C, G or T.
In some embodiments, the 5 '-phosphate nucleotide is a nucleotide comprising a 5' -phosphate modification shown in formula (2), the 5 '-phosphate analogue modified nucleotide is a nucleotide comprising a vinyl phosphate (5' - (E) -vinylphosphonate, E-VP) modification shown in formula (3), or is a phosphorothioate modified nucleotide shown in formula (5).
In some embodiments, the siRNA provided by the present disclosure is arbitrarily selected from one of the following groups ：siHAVa1-M1P1、siHAVa2-M1P1、siHAVa3-M1P1、siHAVa1-M1SP1、siHAVa2-M1SP1、siHAVa3-M1SP1、siHAVa1-M1XP1、siHAVa2-M1XP1、siHAVa3-M1XP1、siHAVb1-M1P1、siHAVb2-M1P1、siHAVb3-M1P1、siHAVb1-M1SP1、siHAVb2-M1SP1、siHAVb3-M1SP1、siHAVb1-M1XP1、siHAVb2-M1XP1、siHAVb3-M1XP1、siHAVc1-M1P1、siHAVc2-M1P1、siHAVc3-M1P1、siHAVc1-M1SP1、siHAVc2-M1SP1、siHAVc3-M1SP1、siHAVc1-M1XP1、siHAVc2-M1XP1、siHAVc3-M1XP1、siHAVd1-M1P1、siHAVd2-M1P1、siHAVd3-M1P1、siHAVd1-M1SP1、siHAVd2-M1SP1、siHAVd3-M1SP1、siHAVd1-M1XP1、siHAVd2-M1XP1 and siHAVd3-M1XP1.
The inventors of the present disclosure have surprisingly found that the siRNA provided by the present disclosure achieves a high balance of stability in plasma and RNAi efficiency in animal experiments.
The inventors of the present disclosure have unexpectedly found that the siRNA provided by the present disclosure not only has significantly enhanced plasma and lysosomal stability, but also retains very high viral RNA inhibitory activity.
The siRNA provided by the present disclosure can be obtained by methods of siRNA preparation conventional in the art (e.g., methods of solid phase synthesis and liquid phase synthesis). Among them, solid-phase synthesis already has commercial subscription services. Methods of preparing nucleoside monomers having corresponding modifications and methods of introducing modified nucleotide groups into siRNA can also be known to those of skill in the art by introducing modified nucleotide groups into siRNA described in the present disclosure using nucleoside monomers having corresponding modifications.
SiRNA conjugates
In another aspect, the present disclosure provides an siRNA conjugate comprising the above siRNA and a conjugate group conjugated to the siRNA.
Generally, the conjugate group comprises at least one targeting group and/or delivery assistance group that is pharmaceutically acceptable. In some embodiments, the conjugate group further comprises a linker (linker), and the linker and/or the targeting group or the delivery assisting group are sequentially linked.
In some embodiments, the conjugate group comprises a linker and a pharmaceutically acceptable targeting group and/or a delivery assisting group, and the siRNA, the linker and the targeting group or the delivery assisting group are sequentially covalently or non-covalently linked, each of the targeting groups being selected from a ligand capable of binding to a cell surface receptor, each delivery assisting group being selected from a group capable of increasing the biocompatibility of the siRNA conjugate in a delivery target organ or tissue.
In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The siRNA molecule may be non-covalently or covalently conjugated to the conjugate group, e.g., may be covalently conjugated to the conjugate group. The conjugation site of the siRNA to the conjugation group may be at the 3' end or 5' end of the sense strand of the siRNA, at the 5' end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA to the conjugation group is at the 3' end of the sense strand of the siRNA.
In some embodiments, the conjugate group may be attached to the phosphate group, the 2' -hydroxyl group, or the base of the nucleotide. In some embodiments, the conjugate group may also be attached to the 3' -hydroxyl group, in which case the nucleotides are linked using a 2' -5' phosphodiester linkage. When the conjugate group is attached to the end of the siRNA strand, the conjugate group is typically attached to a phosphate group of a nucleotide, and when the conjugate group is attached to the internal sequence of the siRNA, the conjugate group is typically attached to a ribose sugar ring or base. Various connection modes can be referred to in the literature ：Muthiah Manoharan et.al.siRNAconjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.ACS Chemical biology,2015,10(5):1181-7.
The targeting group can be attached to the siRNA molecule via a suitable linker, which can be selected by one skilled in the art depending on the particular type of targeting group. The types of these linkers, targeting groups and attachment to siRNA can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference. In some embodiments, the siRNA and the conjugate group may be linked by acid labile, or reducible, chemical bonds that degrade in the acidic environment of the intracellular inclusion bodies, thereby allowing the siRNA to be in a free state. For non-degradable conjugation, the conjugation group can be attached to the sense strand of the siRNA, thereby minimizing the effect of conjugation on siRNA activity.
In some embodiments, the targeting group can be a ligand conventionally used in the art of siRNA administration, such as the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.
In some embodiments, at least one or each of the targeting groups is selected from a ligand capable of binding to a cell surface receptor comprising the HAV viral RNA.
In some embodiments, at least one or each of the targeting groups is selected from a ligand capable of binding to mammalian liver parenchymal cell surface receptor (ASGPR). In some embodiments, each of the targeting groups is independently a ligand that is affinity to an asialoglycoprotein receptor on the surface of mammalian hepatocytes. In some embodiments, each of the targeting groups is independently an asialoglycoprotein or a sugar. In some embodiments, each of the targeting groups is independently an asialoglycoprotein, such as an asialoglycoprotein of the serotypes (asialoorosomucoid, ASOR) or an asialoglycoprotein of the onset (asialofetuin, ASF). In some embodiments of the present invention, in some embodiments, each of the targeting groups is independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylose furanose, L-xylose furanose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-mannopyranose, beta-D-mannopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-fructofuranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-galactofuranose, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-isobutyrylgalactosamine, 2-amino-3-O) -1- [ (R ] -2-ethyl ] -2-carboxymethyl ] -2-D-galactopyranose, 4-dimethyl-6-D-amino-6-D-deoxyglucopyranose, 4-dimethyl-4-D-galactopyranose, 2-deoxy-2-sulphonamino-D-glucopyranose, N-glycolyl- α -neuraminic acid, 5-thio- β -D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl- α -D-glucopyranoside methyl ester, 4-thio- β -D-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio- α -D-glucoheptopyranoside ethyl ester, 2, 5-anhydro-D-psicosonitrile, ribose, D-4-thioribose, L-ribose, L-4-thioribose. In some embodiments, at least one or each of the targeting groups is galactose or N-acetylgalactosamine.
In some embodiments, the linker in the siRNA conjugates of the present disclosure has a structure as shown in formula (301):
wherein k is an integer of 1 to 3;
LA has a structure containing an amide bond as shown in formula (302), LB has a structure containing N-acyl pyrrolidine as shown in formula (303), contains a carbonyl group and an oxygen atom, and LC is a linking group based on hydroxymethyl aminomethane, dihydroxymethyl aminomethane or trimethylol aminomethane;
Wherein n302、q302 and p302 are each independently integers from 2 to 6, alternatively n302、q302 and p302 are each independently integers from 2 or 3, n303 is an integer from 4 to 16, alternatively n303 is an integer from 8 to 12,Indicating the site of covalent attachment of the group.
In the linker, each LA is connected with one targeting group through an ether bond and is connected with the LC part through an oxygen atom of a hydroxyl group in the LC part, and LB is connected with the nitrogen atom of an amino group in the LC part through an amide bond formed by a carbonyl group in the formula (303) and is connected with the siRNA through an oxygen atom in the formula (303) through a phosphate bond or a phosphorothioate bond.
In some embodiments, the siRNA conjugates provided by the present disclosure have a structure as shown in formula (305):
where Nu represents the siRNA provided by the present disclosure.
In some embodiments, the linker in the siRNA conjugates of the present disclosure has a structure represented by formula (306):
wherein n306 is an integer from 0 to 3, each p306 is independently an integer from 1 to 6,The linking group is linked by at least one of the oxygen atoms marked with # forming a phosphate bond or a phosphorothioate bond with the siRNA, and the rest of the oxygen atoms marked with # are linked with a hydrogen atom to form a hydroxyl group or are linked with a C1-C3 alkyl to form a C1-C3 alkoxy group;
in some embodiments, the siRNA conjugates of the present disclosure have a structure as shown in formula (307):
where Nu represents the siRNA provided by the present disclosure.
In some embodiments, the siRNA conjugates of the present disclosure have a structure represented by formula (308):
Wherein, the
N1 is an integer selected from 1-3, n3 is an integer selected from 0-4;
Each m1, m2 or m3 is independently an integer selected from 2 to 10;
R10、R11、R12、R13、R14 or R15 are each independently H, or selected from the group consisting of C1-C10 alkyl, C1-C10 haloalkyl, and C1-C10 alkoxy;
r3 has the structure shown in formula A59:
Wherein E1 is OH, SH, or BH2, nu represents the siRNA provided by the present disclosure;
r2 is a linear alkylene of 1 to 20 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of C (O), NH, O, S, CH = N, S (O)2、C2-C10 alkenylene, C2-C10 alkynylene, C6-C10 arylene group, C3-C18 heterocyclylene and C5-C10 heteroarylene, and wherein R2 may optionally have any one or more substituents from the group consisting of C1-C10 alkyl, C6-C10 aryl, C5-C10 heteroaryl, C1-C10 haloalkyl, -OC1-C10 alkyl, -OC1-C10 alkylphenyl, -C1-C10 alkyl-OH, -OC1-C10 haloalkyl, -SC1-C10 alkyl, -SC1-C10 alkylphenyl, -C1-C10 alkyl-SH, -SC1-C10 haloalkyl, halogen substituent, -OH, -SH, -NH2、-C1-C10 alkyl-NH2、-N(C1-C10 alkyl) (C1-C10 alkyl), -NH (C1-C10 alkyl), -N (C1-C10 alkyl) (C1-C10 alkylphenyl), -NH (C1-C10 alkylphenyl), -, Cyano, nitro, -CO2H、-C(O)O(C1-C10 alkyl), -CON (C1-C10 alkyl) (C1-C10 alkyl), -CONH (C1-C10 alkyl), -CON (C1-C10 alkyl) (C1-C10 alkylphenyl), -CONH (C1-C10 alkylphenyl), -CONH2、-NHC(O)(C1-C10 alkyl), -NHC (O) (phenyl), -N (C1-C10 alkyl) C (O) (C1-C10 alkyl), -N (C1-C10 alkyl) C (O) (phenyl), -C (O) C1-C10 alkyl, -C (O) C1-C10 alkylphenyl, -C (O) C1-C10 haloalkyl, -OC (O) C1-C10 alkyl, -SO2(C1-C10 alkyl), -SO2 (phenyl), -SO2(C1-C10 haloalkyl), -SO2NH2、-SO2NH(C1-C10 alkyl), -SO2 NH (phenyl), -NHSO2(C1-C10 alkyl), -NHSO2 (phenyl), and-NHSO2(C1-C10 haloalkyl);
Each L1 is independently a linear alkylene group of 1 to 70 carbon atoms in length, wherein one or more carbon atoms may optionally be replaced by any one or more selected from the group consisting of C (O), NH, O, S, CH = N, S (O)2、C2-C10 alkenylene, C2-C10 alkynylene, C6-C10 arylene group, C3-C18 heterocyclylene and C5-C10 heteroarylene, and wherein L1 optionally has any one or more substituents from the group consisting of C1-C10 alkyl, C6-C10 aryl, C5-C10 heteroaryl, C1-C10 haloalkyl, -OC1-C10 alkyl, -OC1-C10 alkylphenyl, -C1-C10 alkyl-OH, -OC1-C10 haloalkyl, -SC1-C10 alkyl, -SC1-C10 alkylphenyl, -C1-C10 alkyl-SH, -SC1-C10 haloalkyl, halogen substituent, -OH, -SH, -NH2、-C1-C10 alkyl-NH2、-N(C1-C10 alkyl) (C1-C10 alkyl), -NH (C1-C10 alkyl), -N (C1-C10 alkyl) (C1-C10 alkylphenyl), -NH (C1-C10 alkylphenyl), -, Cyano, nitro, -CO2H、-C(O)O(C1-C10 alkyl), -CON (C1-C10 alkyl) (C1-C10 alkyl), -CONH (C1-C10 alkyl), -CONH2,-NHC(O)(C1-C10 alkyl), -NHC (O) (phenyl), -N (C1-C10 alkyl) C (O) (C1-C10 alkyl), -N (C1-C10 alkyl) C (O) (phenyl), -C (O) C1-C10 alkyl, -C (O) C1-C10 alkylphenyl, -C (O) C1-C10 haloalkyl, -OC (O) C1-C10 alkyl, -SO2(C1-C10 alkyl), -SO2 (phenyl), -SO2(C1-C10 haloalkyl), -SO2NH2、-SO2NH(C1-C10 alkyl), -SO2 NH (phenyl), -NHSO2(C1-C10 alkyl), -NHSO2 (phenyl), and-NHSO2(C1-C10 haloalkyl);
represents the site of covalent attachment of the group;
M1 represents a targeting group, the definition and optional scope of which are the same as described above. In some embodiments, each M1 is independently selected from one of the ligands having an affinity for an asialoglycoprotein receptor on the surface of a mammalian liver cell.
The skilled artisan will appreciate that although L1 is defined as a linear alkylene group for convenience, it may not be a linear group or be named differently, such as an amine or alkenyl group resulting from the substitutions and/or substitutions described above. For the purposes of this disclosure, the length of L1 is the number of atoms in the chain connecting the two attachment points. For this purpose, the ring (e.g., heterocyclylene or heteroarylene) resulting from substitution of the carbon atom of the linear alkylene group is counted as one atom.
When M1 is a ligand having an affinity for an asialoglycoprotein receptor on the surface of a mammalian liver cell, n1 may be an integer from 1 to 3 in some embodiments, n3 may be an integer from 0 to 4, ensuring that the number of M1 ligands in the conjugate is at least 2, and in some embodiments, n1+n3≥2, such that the number of M1 ligands is at least 3, allows the M1 ligand to bind more readily to the liver surface asialoglycoprotein receptor, thereby facilitating entry of the conjugate into the cell by endocytosis. Experiments have shown that when the number of M1 ligands is greater than 3, the increase in ease of binding of the M1 ligand to the hepatic surface asialoglycoprotein receptor is not significant, and thus, in some embodiments, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1+n3=2 to 3, from a comprehensive view of ease of synthesis, cost of structure/process, and delivery efficiency.
In some embodiments, where M1, M2, and M3 are independently selected from integers from 2-10, the spatial position between the plurality of M1 ligands may be tailored for binding of the M1 ligand to the hepatic surface asialoglycoprotein receptor, in order to make the conjugates provided by the present disclosure simpler, easier to synthesize, and/or lower cost, in some embodiments, each of M1, M2, and M3 is independently an integer from 2-5, in some embodiments m1=m2=m3.
Those skilled in the art will appreciate that when R10、R11、R12、R13、R14 and R15 are each independently selected from one of H, C1-C10 alkyl, C1-C10 haloalkyl, and C1-C10 alkoxy, the objectives of the present disclosure can be achieved without altering the properties of the conjugates disclosed herein. In some embodiments, R10、R11、R12、R13、R14 and R15 are each independently selected from H, methyl, and ethyl. In some embodiments, R10、R11、R12、R13、R14 and R15 are both H.
According to the siRNA conjugates provided herein, R3 is a group of the structure shown in formula a59, wherein E1 is OH, SH, or BH2, in some embodiments E1 is OH or SH, based on ease of availability of the preparation starting materials.
In some embodiments, R2 is selected to achieve a linkage to the N atom on the nitrogen-containing backbone to a 59. In the context of the present disclosure, a "nitrogen-containing backbone" refers to a chain structure in which the carbon atoms to which R10、R11、R12、R13、R14 and R15 are attached are interconnected with an N atom. Thus, R2 can be any linking group capable of linking the a59 group to the N atom on the nitrogen-containing backbone in a suitable manner. In some embodiments, where the siRNA conjugates of the present disclosure are prepared by a process of solid phase synthesis, it is desirable to have both a linking site in the R2 group that is linked to the N atom on the nitrogen-containing backbone and a linking site that is linked to the P atom in R3. In some embodiments, the site in R2 attached to the N atom on the nitrogen-containing backbone forms an amide bond with the N atom and the site attached to the P atom on R3 forms a phosphate bond with the P atom. In some embodiments, R2 is 2 to 20 atoms, or 4 to 15 atoms in length. In some embodiments, R2 is B5, B6, B5', or B6':
Wherein, theIndicating the site of covalent attachment of the group.
Q2 may be an integer in the range of 1 to 10, and in some embodiments q2 is an integer in the range of 1 to 5.
The role of L1 is to link the M1 ligand to N on a nitrogen-containing backbone, providing a targeting function for the siRNA conjugates of the present disclosure. In some embodiments, L1 is selected from a linked combination of one or more of the groups of formulas A1-A26. In some embodiments, L1 is selected from the group consisting of A1, A4, A5, A6, A8, A10, A11, and A13, in some embodiments, L1 is selected from the group consisting of A1, A4, A8, A10, and A11, and in some embodiments, L1 is selected from the group consisting of A1, A8, and A10, and at least 2.
In some embodiments, L1 may be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms in length. In some embodiments, L1 is 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 atoms in length.
In some embodiments, j1 is an integer from 2 to 10, and in some embodiments, j1 is an integer from 3 to 5. In some embodiments, j2 is an integer from 2 to 10, and in some embodiments, j2 is an integer from 3 to 5. R 'is a C1-C4 alkyl group, and in some embodiments, R' is one of methyl, ethyl, and isopropyl. Ra is one of a27, a28, a29, a30, and a31, and in some embodiments Ra is a27 or a28.Rb is a C1-C5 alkyl group, and in some embodiments Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, j1, j2, R', ra, rb are each selected in formulas A1-a26 to achieve N-linking of the M1 ligand to the nitrogen-containing backbone and to make the spatial position between the M1 ligand more suitable for binding of the M1 ligand to the hepatic surface asialoglycoprotein receptor.
In some embodiments, the siRNA conjugates of the present disclosure have a structure represented by formula (403)、(404)、(405)、(406)、(407)、(408)、(409)、(410)、(411)、(412)、(413)、(414)、(415)、(416)、(417)、(418)、(419)、(420)、(421) or (422):
where Nu represents the siRNA of the present disclosure.
In some embodiments, the P atom in formula A59 can be attached to any possible position in the siRNA sequence, e.g., the P atom in formula A59 can be attached to any one of the nucleotides of the sense strand or the antisense strand of the siRNA, and in some embodiments, the P atom in formula A59 is attached to any one of the nucleotides of the sense strand of the siRNA. In some embodiments, the P atom in formula A59 is attached to the end of the sense strand or the antisense strand of the siRNA, and in some embodiments, the P atom in formula A59 is attached to the end of the sense strand of the siRNA. The end refers to the first 4 nucleotides from one end of the sense strand or the antisense strand. In some embodiments, the P atom in formula A59 is attached to the end of the sense strand or the antisense strand of the siRNA, and in some embodiments, the P atom in formula A59 is attached to the 3' end of the sense strand of the siRNA. In the case of the above-described position of the sense strand linked to the siRNA, the conjugates provided by the present disclosure, upon entry into a cell, upon unwinding, can release the separate siRNA antisense strand to inhibit HAV viral RNA via the RNAi machinery.
The P atom in formula A59 can be attached to any possible position on the nucleotide in the siRNA, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide, or the base of the nucleotide. In some embodiments, the P atom in formula a59 can be linked to the 2', 3', or 5' position of a nucleotide in the siRNA by formation of a phosphodiester linkage. In some embodiments, the P atom in formula a59 is attached to an oxygen atom formed upon dehydrogenation of the 3' hydroxyl group of the 3' terminal nucleotide of the siRNA sense strand, or the P atom in formula a59 is attached to a nucleotide by substitution of hydrogen in the 2' -hydroxyl group of one nucleotide in the siRNA sense strand, or the P atom in formula a59 is attached to a nucleotide by substitution of hydrogen in the 5' hydroxyl group of the 5' terminal nucleotide of the siRNA sense strand.
The inventors of the present disclosure unexpectedly found that the siRNA conjugates of the present disclosure have significantly improved stability in plasma and higher HAV viral RNA silencing activity. In some embodiments, the siRNA of the present disclosure may be one of the sirnas shown in table 1.
TABLE 1siRNA of the present disclosure
Wherein, uppercase letter C, G, U, A represents the base composition of the nucleotide, lowercase letter m represents that one nucleotide adjacent to the left side of the letter m is a methoxy modified nucleotide, lowercase letter f represents that one nucleotide adjacent to the left side of the letter f is a fluoro modified nucleotide, lowercase letter s represents that phosphorothioate subunit connection is formed between the two nucleotides at the left and right sides of the letter, and P1 represents that one nucleotide adjacent to the right side of P1 is a 5 '-phosphate nucleotide or a 5' -phosphate analogue modified nucleotide. In some embodiments, P1 is VP, ps, or P representing a particular modification, wherein the letter combination VP represents that one nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate (5 '- (E) -vinylphosphonate, E-VP) modified nucleotide, the letter combination Ps represents that one nucleotide adjacent to the right of the letter combination Ps is a phosphorothioate modified nucleotide, and the capital letter P represents that one nucleotide adjacent to the right of the letter P is a 5' -phosphate nucleotide.
It is clear to those skilled in the art that modified nucleotide groups can be introduced into the siRNAs described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods of preparing nucleoside monomers with corresponding modifications and methods of introducing modified nucleotide groups into siRNA are also well known to those of skill in the art. All modified nucleoside monomers are commercially available or can be prepared using known methods.
Any reasonable synthetic route can be used to prepare the siRNA conjugates of the present disclosure. For example, for a conjugate molecule comprising a targeting group and an active reactive group that can react with phosphoramidite to form a covalent linkage, the active group in the conjugate molecule can be first protected with a protecting agent and then attached to a solid support, followed by phosphoramidite solid phase synthesis, by attaching nucleoside monomers one by one in the 3 'to 5' direction according to the nucleotide type and sequence of the sense strand and the antisense strand of the siRNA, the attachment of each nucleoside monomer comprising a deprotection, coupling, capping, oxidation or sulfidation four-step reaction, isolating the sense strand and the antisense strand of the siRNA, and annealing to obtain the siRNA conjugates of the present disclosure.
Further, the preparation of siRNA conjugates can also be performed with reference to the disclosures of the prior literature. For example, WO2019010274A1 describes in example 1a method of sequentially attaching a linking group having a specific structure and a targeting ligand to an siRNA via a reaction. The entire contents of which are incorporated herein by reference.
Pharmaceutical composition
In one aspect, the present disclosure provides a pharmaceutical composition comprising an siRNA and/or an siRNA conjugate as described above as an active ingredient and a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier may be a carrier conventionally used in the siRNA administration field, for example, but not limited to, magnetic nanoparticles (magnetic nanoparticles such as Fe3O4 or Fe2O3 -based nanoparticles), carbon nanotubes (carbon nanotubes), mesoporous silicon (mesoporous silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), polyethylenimine (PEI), polyamide dendrimers (polyamidoamine (PAMAM) dendrimers), polylysine (L-lysine), PLL), chitosan (chitosan), 1,2-dioleoyl-3-trimethylammonium propane (1, 2-dioleoyl-3-trimethylammonium-propane, DOTAP), poly D-or L-lactic/glycolic acid copolymer (PLGA), poly (aminoethylphosphate) (2-aminoethyl ethylene phosphate), PPEEA) and poly (methylethyl methacrylate) (2-dimethylaminoethyl methacrylate), and one or more of them.
In some embodiments, the amount of siRNA and/or siRNA conjugate and pharmaceutically acceptable carrier in the pharmaceutical composition is not particularly required, and in some embodiments, the weight ratio of siRNA and/or siRNA conjugate to pharmaceutically acceptable carrier may be 1 (1-500), and in some embodiments, the weight ratio is 1 (1-50).
In some embodiments, the pharmaceutical composition may further comprise other pharmaceutically acceptable excipients, which may be one or more of various formulations or compounds conventionally employed in the art. For example, the pharmaceutically acceptable additional excipients may include at least one of a pH buffer, a protectant, and an osmolality adjusting agent.
The pH buffer solution can be a tris hydrochloride buffer solution with the pH value of 7.5-8.5 and/or a phosphate buffer solution with the pH value of 5.5-8.5, for example, the pH value of 5.5-8.5.
The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The protective agent may be present in an amount of 0.01 to 30% by weight, based on the total weight of the pharmaceutical composition.
The osmolality adjusting agent may be, for example, sodium chloride and/or potassium chloride. The osmolality adjusting agent is present in an amount such that the osmolality of the pharmaceutical composition is 200-700 milliosmoles per kilogram (mOsm/kg). The amount of osmolality adjusting agent can be readily determined by one skilled in the art based on the desired osmolality. In some embodiments, the dosage of the formulation of the pharmaceutical composition may be adjusted during the administration process according to the mode of administration.
In some embodiments, the pharmaceutical composition can be a liquid preparation, such as injection, or can be freeze-dried powder injection, and is mixed with liquid auxiliary materials to prepare a liquid preparation when administration is carried out. The liquid formulation may be used for, but is not limited to, subcutaneous, intramuscular, injectable administration, and the pharmaceutical composition may be delivered by, but is not limited to, nasal administration, oropharyngeal inhalation, spray administration, and the like. In some embodiments, the pharmaceutical composition is administered by subcutaneous injection.
In some embodiments, the pharmaceutical composition may be in the form of a liposomal formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposomal formulation comprises an amine-containing transfection compound (which may also be referred to hereinafter as an organic amine), a helper lipid, and/or a pegylated lipid. Wherein the organic amine, the helper lipid and the pegylated lipid may be selected from one or more of the amine-containing transfection compounds described in chinese patent application CN103380113a (which is incorporated herein by reference in its entirety) or pharmaceutically acceptable salts or derivatives thereof, the helper lipid and the pegylated lipid, respectively.
In some embodiments, the organic amine may be a compound as depicted in formula (201) described in chinese patent application CN103380113a, or a pharmaceutically acceptable salt thereof:
Wherein:
X101 and X102 are each independently O, S, N-A or C-A, wherein A is hydrogen or A C1-C20 hydrocarbon chain;
Y101 and Z101 are each independently c= O, C = S, S = O, CH-OH or SO2;
R101、R102、R103、R104、R105、R106 and R107 are each independently hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight chain heteroaliphatic group, a substituted or unsubstituted, branched or straight chain acyl group, a substituted or unsubstituted, branched or straight chain aryl group, a substituted or unsubstituted, branched or straight chain heteroaryl group;
x is an integer from 1 to 10;
n is an integer from 1 to 3, m is an integer from 0 to 20, and p is 0 or 1, wherein, if m=p=0, then R102 is hydrogen;
And, if at least one of n or m is 2, then R103 and the nitrogen in formula (201) form a structure as shown in formula (202) or formula (203):
wherein g, e and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each of N represents a nitrogen atom in formula (201).
In some embodiments, R103 is a polyamine. In other embodiments, R103 is a ketal. In some embodiments, each of R101 and R102 in formula (201) is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl group having from 3 to about 20 carbon atoms, such as from 8 to about 18 carbon atoms, and from 0 to 4 double bonds, such as from 0 to 2 double bonds.
In some embodiments, if each of n and m independently has a value of 1 or 3, then R103 can be any of the following formulas (204) - (213):
Wherein in formulae (204) - (213), g, e and f are each independently integers from 1 to 6, each "HCC" represents a hydrocarbon chain, and each shows a possible point of attachment of R103 to the nitrogen atom in formula (201), wherein each H at any of the positions may be replaced to effect attachment to the nitrogen atom in formula (201).
Wherein the compound of formula (201) may be prepared as described in chinese patent application CN103380113 a.
In some embodiments, the organic amine is an organic amine as shown in formula (214) and/or an organic amine as shown in formula (215):
the auxiliary lipid is cholesterol, cholesterol analogues and/or cholesterol derivatives;
the polyethylene glycol lipid is 1, 2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine-N- [ methoxy (polyethylene glycol) ] -2000.
In some embodiments, the molar ratio between the organic amine, the helper lipid, and the pegylated lipid in the pharmaceutical composition is (19.7-80): (19.7-80): (0.3-50), for example, may be (50-70): (20-40): (3-20).
In some embodiments, the particles of the pharmaceutical composition formed from the siRNA of the present disclosure and the amine-containing transfection reagent described above have an average diameter of about 30nm to about 200nm, typically about 40nm to about 135nm, more typically the average diameter of the liposome particles is about 50nm to about 120nm, about 50nm to about 100nm, about 60nm to about 90nm, or about 70nm to about 90nm, e.g., the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160nm.
In some embodiments, the weight ratio (weight/weight ratio) of siRNA to total lipid (e.g., organic amine, helper lipid, and/or pegylated lipid) in a pharmaceutical composition formed from an siRNA of the present disclosure and an amine-containing transfection reagent as described above is in the range of from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10, e.g., the weight ratio of siRNA of the present disclosure to total lipid is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or 1:18.
In some embodiments, the components of the pharmaceutical composition may be present independently at the point of sale and may be present in liquid formulations at the point of use. In some embodiments, the pharmaceutical compositions formed by the siRNAs provided by the present disclosure and the pharmaceutically acceptable carriers described above can be prepared according to known methods, except that the existing siRNAs are replaced by the siRNAs provided by the present disclosure, and in some embodiments, can be prepared according to the following methods:
Suspending organic amine, auxiliary lipid and polyethylene glycol lipid in the above molar ratio in alcohol, and mixing to obtain lipid solution, wherein the amount of alcohol is such that the total mass concentration of the obtained lipid solution is 2-25mg/mL, for example 8-18mg/mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, e.g., one or more of ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, e.g., may be ethanol.
The siRNA provided by the present disclosure is dissolved in a buffer salt solution to obtain an siRNA aqueous solution. The concentration of the buffer salt solution is 0.05-0.5M, for example, may be 0.1-0.2M, the pH of the buffer salt solution is adjusted to 4.0-5.5, for example, may be 5.0-5.2, and the amount of the buffer salt solution is such that the concentration of siRNA does not exceed 0.6mg/mL, for example, may be 0.2-0.4mg/mL. The buffer salt is selected from one or more of soluble acetate and soluble citrate, and can be sodium acetate and/or potassium acetate.
Mixing the lipid solution and siRNA aqueous solution, and incubating the mixed product at 40-60 ℃ for at least 2 minutes, for example, 5-30 minutes, to obtain the incubated liposome preparation. The volume ratio of the lipid solution to the siRNA aqueous solution is 1 (2-5), and can be 1:4, for example.
Concentrating or diluting the incubated liposome preparation, removing impurities, and sterilizing to obtain the pharmaceutical composition provided by the disclosure, wherein the physical and chemical parameters are that the pH value is 6.5-8, the encapsulation efficiency is not lower than 80%, the particle size is 40-200nm, the polydispersity index is not higher than 0.30, the osmotic pressure is 250-400mOsm/kg, for example, the physical and chemical parameters can be that the pH value is 7.2-7.6, the encapsulation efficiency is not lower than 90%, the particle size is 60-100nm, the polydispersity index is not higher than 0.20, and the osmotic pressure is 300-400mOsm/kg.
Wherein concentration or dilution may be performed before, after, or simultaneously with removal of impurities. As a method for removing impurities, various methods are available, for example, a tangential flow system, a hollow fiber column, ultrafiltration at 100kDa, and Phosphate Buffer (PBS) of pH7.4 as an ultrafiltration exchange solution can be used. As a method of sterilization, various methods are available, and for example, filtration sterilization on a 0.22 μm filter can be used.
SiRNA and siRNA conjugate of the disclosure and application of pharmaceutical composition containing siRNA
In some embodiments, the present disclosure provides the use of an siRNA and/or siRNA conjugate and/or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a disease or condition associated with modulation of HAV function.
In some embodiments, the present disclosure provides a method of treating a disease or condition associated with modulation of HAV function, the method comprising administering to a subject in need thereof an effective amount of an siRNA/or siRNA conjugate and/or pharmaceutical composition of the present disclosure. By administering the siRNA active ingredients of the present disclosure to a subject in need thereof, the goal of treating the caused disease can be achieved by the mechanism of RNA interference. Thus, the siRNA and/or siRNA conjugates and/or pharmaceutical compositions of the present disclosure are useful for treating, or for the preparation of a medicament for treating, a disease or symptom associated with modulation of HAV function.
The term "administration" as used herein refers to placing an siRNA, siRNA conjugate and/or pharmaceutical composition of the present disclosure into a subject by a method or route that results in, at least in part, positioning the siRNA, siRNA conjugate and/or pharmaceutical composition of the present disclosure at a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include topical and systemic administration. In general, local administration results in more siRNA conjugate being delivered to a specific site than the subject's systemic circulation, while systemic administration results in the siRNA, siRNA conjugate and/or pharmaceutical composition of the present disclosure being delivered to the subject's basal systemic circulation. It is contemplated that the present disclosure is directed to providing means for treating hepatitis a disease, in some embodiments employing a mode of administration that delivers a drug to liver tissue.
The administration to the subject may be by any suitable route known in the art, including but not limited to, oral or parenteral routes such as intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal and topical (including buccal and sublingual). The frequency of administration may be 1 or more times daily, weekly, biweekly, tricyclically, monthly or yearly.
The dosages of the siRNA, siRNA conjugate or pharmaceutical composition described in the present disclosure may be conventional dosages in the art, which may be determined according to various parameters, particularly the age, weight and sex of the subject. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining LD50 (the lethal dose to death of 50% of the population) and ED50 (the dose that causes 50% of the maximal response intensity in the dose response, and the dose that causes 50% of the subjects to develop a positive response in the mass response). The range of doses for human use can be derived based on data obtained from cell culture assays and animal studies. In some embodiments, the dosage of the formulation of the siRNA, siRNA conjugate or pharmaceutical composition is adjusted during the administration process according to the mode of administration.
In administering the sirnas, siRNA conjugates, and/or pharmaceutical compositions described herein, e.g., for male or female, 6-12 weeks old, 18-25g of C57BL/6J or 30-45g of ob/ob mice, the amount of siRNA can be (i) 0.001-100mg/kg of body weight, in some embodiments 0.01-50mg/kg of body weight, in some embodiments 0.05-20mg/kg of body weight, in other embodiments 0.1-15mg/kg of body weight, and in other embodiments 0.1-10mg/kg of body weight, based on the amount of siRNA, (ii) 0.001-50mg/kg of body weight, in some embodiments 0.01-10mg/kg of body weight, in some embodiments 0.05-5mg/kg of body weight, and in some embodiments 0.1-3mg/kg of body weight, based on the amount of siRNA in a pharmaceutical composition formed by the siRNA and a pharmaceutically acceptable carrier.
In some embodiments, the present disclosure provides a method of inhibiting HAV viral RNA in a cell, the method comprising contacting an effective amount of an siRNA and/or siRNA conjugate and/or pharmaceutical composition of the present disclosure with the cell, introducing the siRNA and/or siRNA conjugate and/or pharmaceutical composition of the present disclosure into the cell, and inhibiting HAV viral RNA in the cell by a mechanism of RNA interference.
The amount of siRNA in the modified siRNA, siRNA conjugates, and/or pharmaceutical compositions provided using the methods provided by the present disclosure is generally an amount sufficient to reduce viral RNA and result in an extracellular concentration of 1pM to 1 μM, or 0.01nM to 100nM, or 0.05nM to 50nM, or 0.05nM to about 5nM at the surface of target cells comprising viral RNA. The amount required to achieve this local concentration will vary depending on a variety of factors including the method of delivery, the site of delivery, the number of cell layers between the site of delivery and the target cell or tissue, the route of delivery (local or systemic), and the like. The concentration at the delivery site may be significantly higher than the concentration at the surface of the target cell or tissue.
Kit for detecting a substance in a sample
In one aspect, the present disclosure provides a kit comprising an effective amount of at least one of the siRNA, siRNA conjugate and pharmaceutical composition of the present disclosure.
In some embodiments, the kits described herein can provide siRNA in one container. In some embodiments, the kits described herein can comprise a container that provides a pharmaceutically acceptable excipient. In some embodiments, other ingredients, such as stabilizers or preservatives, and the like, may also be included in the kit. In some embodiments, the kits described herein can comprise at least one additional therapeutic agent in a container other than the container in which the siRNA described herein is provided. In some embodiments, the kit may comprise instructions for mixing the siRNA with a pharmaceutically acceptable carrier and/or adjuvant or other ingredients, if any.
In the kits of the present disclosure, the siRNA and pharmaceutically acceptable carrier and/or adjuvant, as well as the modified siRNA, siRNA conjugate and/or pharmaceutical composition, and/or pharmaceutically acceptable carrier and/or adjuvant, may be provided in any form, such as liquid form, dry form or lyophilized form. In some embodiments of the present invention, in some embodiments, the siRNA and pharmaceutically acceptable carrier and/or adjuvant are substantially pure and/or sterile and the pharmaceutical composition and/or pharmaceutically acceptable carrier and/or adjuvant of the siRNA conjugate. In some embodiments, sterile water may be provided in a kit of the present disclosure.
The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereby.
Examples
Unless otherwise specified, reagents and media used in the following examples are commercially available, and the procedures for nucleic acid electrophoresis, real-time PCR, and the like used are carried out by the method described in Molecular Cloning (Cold Spring Harbor LBboratory Press (1989)).
Unless otherwise indicated, the reagent ratios provided below are all calculated as volume ratios (v/v). Data analysis employed GRAPHPAD PRISM 8.0.0 statistical analysis software.
Unless otherwise specified, reagents and media used in the following examples are commercially available, and the procedures for nucleic acid electrophoresis, real-time PCR, and the like used are carried out by the method described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).
PREPARATION EXAMPLES 1-4 Synthesis of conjugates 1-4 provided by the present disclosure
Conjugates 1 to 4 in Table 2A below were prepared and obtained according to the preparation method described in preparation example 13 of CN110959011A, except that the sense strand and the antisense strand of siRNA were synthesized according to the nucleotide sequences of siRNA of conjugates 1 to 4 in Table 2A below, respectively.
Conjugates 1-4 have a structure represented by formula (403)
Wherein Nu in formula (403) represents the siRNA of the present disclosure, table 2A lists the siRNA nucleotide sequences of conjugates 1-4, the attachment position of the conjugate group is the ribose 3 'position of the 3' terminal nucleotide of the sense strand in the siRNA, and the conjugates 1-4 are in sodium salt form.
Table 2A siRNA sequences in siRNA conjugates
Wherein, the capital letter C, G, U, A represents the base composition of the nucleotide, the lowercase letter m represents that one nucleotide adjacent to the left side of the letter m is methoxy modified nucleotide, the lowercase letter f represents that one nucleotide adjacent to the left side of the letter f is fluoro modified nucleotide, and the lowercase letter s represents that phosphorothioate subunit is connected between the two nucleotides around the letter s.
Molecular weight measurements were performed using a liquid chromatography Mass spectrometer (LC-MS, liquid Chromatography-Mass SP1ectrometry, available from Waters, model number LCT PREMIER) after diluting each siRNA conjugate to a concentration of 0.2mg/mL (as siRNA) using ultra-pure water (Milli-Q ultra-pure water meter, resistivity 18.2MΩ. Cm (25 ℃)). The results of the measured and theoretical molecular weight measurements of the sense and antisense strands of conjugates 1-4 are detailed in Table 2B below. The actual measurement value is consistent with the theoretical value, which indicates that the synthesized conjugate 1-4 is the double-stranded nucleic acid sequence of the target design.
TABLE 2 molecular weight determination of conjugates 1-4
Comparative preparation example 1 Synthesis of reference siRNA conjugates
A reference siRNA conjugate was prepared according to the method of preparation example 13 in CN110959011A, except that the nucleotide sequence used was the nucleotide sequence corresponding to NC in Table 2A, giving a reference siRNA conjugate, the NC conjugate was represented by the structural formula (403), and the NC conjugate was an siRNA conjugate as a negative control, which did not have sequence homology to HAV viral RNA.
Experimental example 1 this experiment demonstrates the inhibitory activity of siRNA and its conjugate in human hepatoma cells in vitro (in vitro) Huh 7.5.1.
Huh7.5.1 human hepatoma cells of this experiment were purchased from Changsha Ai Bi vitamin technology Co., ltd.; HM175/18f hepatitis A virus (obtained from ATCC), and the virus solution was diluted and cultured in DMEM medium supplemented with 2% FBS until the virus copy number was 1X 109 GE/ml and the virus titer was 3X 106 pfu/ml, to obtain P2 generation virus seed solution.
Mu.L of the HM175/18f hepatitis A virus P2 generation virus seed solution obtained above was taken, and 90. Mu.L of a DMEM medium containing 2% FBS was added thereto, followed by mixing uniformly to obtain 100. Mu.L of a DMEM medium containing HAV virus.
Huh7.5.1 human hepatoma cells in logarithmic growth phase were selected, inoculated into 96-well plates at 4.0X105 cells/well, 100. Mu.L of DMEM medium was added to each well, after culturing in air containing 5% CO2 at 37℃for 24 hours, the medium in the well was removed, 100. Mu.L of the DMEM medium containing HAV virus obtained above was added to each well, and after further culturing for 2 hours, the medium in the well was removed, 100. Mu.L of fresh 2% FBS in the DMEM medium was added to each well.
Each of the siRNA conjugates to be tested was prepared as a 2. Mu.M stock solution using opti-MEM medium (GIBCO Co.), and each of the siRNA conjugates to be tested was conjugate 1, conjugate 2, conjugate 3, conjugate 4 or conjugate NC, respectively.
Each siRNA conjugate to be tested was formulated in an opti-MEM medium at a concentration (based on siRNA in the conjugate) of 2. Mu.M, 200nM, 20nM, 2nM, 0.2nM, 0.02nM, 0.002nM and 0.0002nM for 8 different concentrations of siRNA conjugate working fluid, respectively.
For each siRNA conjugate to be tested, a 1A1-1A8 solution was prepared, and each 1A1-1A8 solution contained 10. Mu.L of conjugate working solution and 10. Mu.L of Opti-MEM medium, respectively, in order.
A1B solution was prepared, and each 1B solution contained 0.6. Mu.L of LipofectamineTM (Invitrogen corporation) and 19.4. Mu.L of Opti-MEM medium.
For each conjugate, 1 part of 1B solution was mixed with 1a1-1A8 solution of each siRNA conjugate, respectively, and incubated at room temperature for 10min to give transfection complex 1X1-1X8 of each siRNA conjugate.
100. Mu.L of fresh 2% FBS DMEM medium was added to each of the culture wells containing the virus-infected cell fluid described above in 48-well plates, and the transfection complexes of each siRNA conjugate 1X1-1X8 were added, mixed uniformly in an amount of 20. Mu.L/well to give the transfection complexes of each siRNA conjugate at a final concentration of about 50nM, 5nM, 0.5nM, 0.05nM, 0.005nM, 0.0005nM, 0.00005nM and 0.000005nM (based on the amount of siRNA in the siRNA conjugate), and3 culture wells were transfected with the transfection complexes of each siRNA conjugate 1X1-1X8, respectively, to give the transfection mixture containing the siRNA conjugate, which was recorded as a test group. For each siRNA conjugate, the transfection mixture in 3 culture wells with the lowest final concentration of conjugate (0.000005 nM) was scored as control.
The transfection mixture containing the siRNA conjugate was transfected into the culture wells for 6 hours, after which the medium in the culture wells was drained, and 200. Mu.L of fresh 2% FBS in DMEM medium was added to each well, and the culture was continued for 72 hours.
Total RNA was extracted from each well of cells using a fully automated nucleic acid extractor (model: NP-GeneRotex, 96) according to the instructions provided by the supplier.
For each well of cells, 1. Mu.g of total RNA was taken, and the total RNA of each well of cells was reverse transcribed using a reverse transcription kit GoldenstarTM RT6 CDNA SYNTHESIS KIT (Biotechnology Co., ltd., beijing, optimum) in which GoldenstarTMOligo(dT)17 was selected as a primer, and 20. Mu.L of a reverse transcription reaction system was prepared according to the procedure of reverse transcription in the kit specification. The condition of reverse transcription is that for each reverse transcription reaction system, the reverse transcription reaction system is placed at 50 ℃ for 50min, then at 85 ℃ for 5min, finally at 4 ℃ for 30s, and 80 mu L of DEPC water is added into the reverse transcription reaction system after the reaction is finished, so as to obtain a solution containing cDNA.
For each reverse transcription reaction system, 5. Mu.L of the cDNA-containing solution was used as a template, respectivelySYBR qPCR SuperMix Plus kit (available from offshore protein technologies Co., ltd., cat. No. E096-01B) was used to prepare 20. Mu.L of a qPCR reaction system, wherein the PCR primer sequences for amplifying the target HAV reverse transcription DNA and the reference gene GAPDH were as shown in Table 3, and the final concentration of each primer was 0.25. Mu.M. The qPCR reaction systems are placed on a ABI StepOnePlus Real-Time PCR instrument, and amplified by a three-step method, wherein the amplification procedure is that the denaturation is carried out for 10min at 95 ℃, then the denaturation is carried out for 30s at 95 ℃, the annealing is carried out for 30s at 60 ℃, and the elongation is carried out for 30s at 72 ℃, and the denaturation, annealing and elongation processes are repeated for 40 times, so that a product W1 containing amplified target HAV reverse transcription DNA and internal reference gene GAPDH is obtained. The product W1 is then incubated at 95 ℃ for 15s, at 60 ℃ for 1min and at 95 ℃ for 15s, and a real-time fluorescent quantitative PCR instrument is used for respectively collecting the dissolution curves of the target HAV reverse transcription DNA and the internal reference gene GAPDH in the product W1 to obtain Ct values of the target HAV reverse transcription DNA and the internal reference gene GAPDH.
TABLE 3 primer information for HAV reverse transcribed DNA and internal reference gene GAPDH
By using the comparative Ct (delta Ct) method, relative quantitative calculations were performed on the target HAV reverse transcribed DNA in each test group as follows:
delta Ct (test group) =ct (test group target reverse transcription DNA) -Ct (test group reference gene)
Delta Ct (control) =ct (control target reverse transcription DNA) -Ct (control reference gene)
ΔΔct (test group) =Δct (test group) - Δct (control group average)
ΔΔct (control) =Δct (control) - Δct (control average)
Wherein, Δct (control group average) is the arithmetic average of Δct (control group) for each of the 3 culture wells of the control group. Thus, each culture well of the test and control groups corresponds to one ΔΔct value.
The relative amounts of HAV copy number under the action of siRNA conjugates at different concentrations were calculated according to the following formula, with Ct value in each group of the lowest concentration siRNA conjugates as a control.
Test group HAV virus relative copy number = 2-ΔΔCt( test set );
Relative inhibition of HAV virus in test group = (relative copy number of HAV virus in test group 1) ×100%.
Based on the relative copy number of HAV virus in the test group in Huh7.5.1 human hepatoma cells after transfection of different concentrations of siRNA conjugate to be tested, a log (inhibitor) vs. Variable slope (four parameters) dose-response curve was fitted using the nonlinear regression analysis function of Graphpad 5.0 software.
Calculating the IC50 value of the target sequence of the siRNA to be detected according to the function corresponding to the fitted dose-effect curve, wherein the function is as follows,
Wherein:
y is the relative copy number of the HAV virus,
X is the logarithmic value of the transfected siRNA concentration,
Bot is the Y value at the bottom of the steady state period,
Top is the Y value at the Top of the steady state period,
X 'is the corresponding X value when Y is halfway between the bottom and top, and HillSlope is the slope of the curve at X'.
From this dose-response curve and the corresponding function, the corresponding X50 value when y=50% is determined, and the IC50 value=10≡x50 (nM) for each siRNA is calculated.
FIGS. 1A-1D are dotted graphs showing relative copy numbers of HAV viral RNA after transfection of different concentrations of siRNA conjugates in Huh7.5.1 human hepatoma cells, and IC50 values calculated using fitted curves.
The results of FIGS. 1A-1D show that in vitro Huh7.5.1 human hepatoma cells, conjugate 1, conjugate 2, conjugate 3 and conjugate 4 exhibited very high HAV viral RNA inhibitory activity with IC50 values of 1.1550nM, 0.0110nM, 0.0087nM and 0.4299nM, respectively. Further, at a concentration of 5nM, the relative inhibition ratio of conjugate 2 and conjugate 3 was greater than 90%, and the relative inhibition ratio of conjugate 1 and conjugate 4 was also greater than 75%, both exhibiting excellent HAV viral RNA inhibition effects.
Experimental example 2 this experiment demonstrates the inhibitory activity of siRNA and siRNA conjugate thereof in vitro monkey primary hepatocytes.
The monkey primary hepatocytes of this experiment (cat#LV-PmonH 001), plating medium (cat#LV-WEP 007) and maintenance medium (cat#LV-WEM 007) were purchased from Wo Biotech Co.
Mu.L of the HM175/18f hepatitis A virus P2 generation virus solution prepared in experimental example 1 was taken and added to 90. Mu.L of the maintenance medium, and mixed uniformly to obtain 100. Mu.L of the maintenance medium containing HAV virus.
Monkey primary hepatocytes were inoculated into 96-well plates at a plating density of 1.8x5 cells/cm2, 100 μl of plating medium was added to each well, after culturing for 24 hours, the medium in the culture well was drained, 100 μl of the above-obtained HAV virus-containing maintenance medium was added to each well, and culturing was continued for 24 hours. The medium in the culture wells was drained and 100. Mu.L of fresh maintenance medium supplemented with 2% FBS was added to each well.
Each siRNA conjugate to be tested was formulated separately into 2 μm conjugate working fluid using maintenance medium. The siRNA conjugates to be tested used were conjugate 1, conjugate 2, conjugate 3, conjugate 4 or conjugate NC, respectively.
For each siRNA conjugate to be tested, 2A solutions were prepared, each 2A solution containing 10. Mu.L of conjugate working solution and 10. Mu.L of maintenance medium, respectively.
In a 48-well plate, 100. Mu.L of a maintenance medium was added, and each siRNA conjugate was added with a 2A solution, and mixed uniformly in an amount of 10. Mu.L/well to obtain a transfection mixture 2X with a final concentration of each siRNA conjugate of about 50nM (based on the amount of siRNA in the siRNA conjugate), and 3 culture wells were transfected with each siRNA conjugate 2A solution, and for conjugate 1-conjugate 4, a transfection mixture containing the siRNA conjugate was obtained and was designated as a test group. For conjugate NC, a transfection mixture containing conjugate NC was obtained and was noted as control.
For each transfection mixture 2X, after 6 hours of incubation at 37℃in an incubator with 5% CO2 in air, the medium in the wells was drained and 100. Mu.L of fresh maintenance medium supplemented with 2% FBS was added to each well and incubation was continued for 48 hours.
Total RNA was extracted from each well of cells using a fully automatic nucleic acid extractor (model: NP-GeneRotex 96). The relative copy number of HAV virus was tested according to the method of experimental example 1, except that the test results of the conjugate NC group were calculated as a control group. The results are shown in FIG. 2.
FIG. 2 is a bar graph showing relative copy numbers of HAV virus after transfection of 50nM concentration of siRNA conjugates of the present disclosure or control conjugates, respectively, in vitro monkey primary cells. As shown in the results of fig. 2, in vitro, the relative copy number of HAV virus of conjugate 3 was 0.18, i.e., the relative inhibition rate was 82%, the relative copy number of viruses of conjugates 1 and 2 was 0.28, i.e., the relative inhibition rate was 72%, and the relative copy number of virus of conjugate 4 was 0.43, i.e., the relative inhibition rate was 57% in the primary monkey cells compared to the control group to which conjugate NC was administered. It can be seen that the conjugates of the present disclosure are able to efficiently enter monkey primary hepatocytes and exert HAV virus inhibitory effects without the use of transfection reagents, and that conjugates 1-4 all show good inhibitory activity against HAV viral RNA.
Experimental example 3 this experimental example demonstrates the inhibitory activity of the siRNA and conjugates of the present disclosure following infection with different strains in huh7.5.1 human hepatoma cells in vitro.
(3-1) Inhibitory Activity against 18F Strain
Mu.L of the HM175/18F hepatitis A virus solution cultured in Experimental example 1 was taken, 90. Mu.L of fresh DMEM medium supplemented with 2% FBS was added thereto, and mixed well to obtain 100. Mu.L of DMEM medium containing HAV virus 18F.
Logarithmic growth Huh7.5.1 human liver cancer cells were selected, inoculated into 96-well plates at 4.0x105 cells/well, cultured in 100. Mu.L of DMEM medium per well at 37℃under an air atmosphere containing 5% CO2 for 24 hours, and after the medium in the culture wells was removed, 100. Mu.L of DMEM medium containing HAV virus 18F was additionally added to each culture well, and the culture was continued for 24 hours.
Each siRNA conjugate to be tested was formulated as 2. Mu.M conjugate working fluid using Opti-MEM medium (GIBCO Co.), and each siRNA conjugate to be tested was conjugate 1, conjugate 2, conjugate 3, conjugate 4 or conjugate NC, respectively.
For each siRNA conjugate to be tested, a 3A solution was prepared, and each 3A solution contained 10. Mu.L of conjugate working solution and 10. Mu.L of Opti-MEM medium, respectively, in sequence.
A3B solution was prepared, and each 3B solution contained 0.6. Mu.L of LipofectamineTM (Invitrogen corporation) and 19.4. Mu.L of Opti-MEM medium.
For each conjugate, 1 part of 3B solution was mixed with 3A solution of each siRNA conjugate, and incubated at room temperature for 10min to give transfection complex 3Xa of each siRNA conjugate.
In the culture wells, 100. Mu.L of the DMEM medium containing the above 2% FBS (Huh 7.5.1 cells and hepatitis A virus) was added to each of the siRNA conjugates, respectively, the transfected complex 3Xa was added to each of the siRNA conjugates, and mixed uniformly in an amount of 20. Mu.L/well to give transfected complexes each having a final concentration of about 50nM (based on the amount of siRNA in the siRNA conjugates), each of the transfected complex 3Xa of the siRNA conjugates was transfected into 3 culture wells, and the transfected mixture containing the siRNA conjugates was obtained for conjugate 1-conjugate 4 and was recorded as a test group. For conjugate NC, a transfection mixture containing conjugate NC was obtained and was noted as control.
After the transfection mixture was transfected into the culture wells for 6 hours, the medium in the culture wells was drained, and 100. Mu.L of fresh DMEM medium supplemented with 2% FBS was added to each well, and the culture was continued for 72 hours.
Total RNA was extracted from each well of cells using a fully automatic nucleic acid extractor (model: NP-GeneRotex 96). HAV virus relative copy number was tested according to the method of Experimental example 1. The difference was that the relative copy number was calculated by normalizing the transfection mixture containing conjugate NC as a control group.
(3-2) Inhibitory Activity against H2 Strain
The relative copy number of HAV virus in Huh7.5.1 human hepatoma cells was tested for the live attenuated hepatitis A vaccine H2 strain according to the method described in (3-1) above. The difference is that the 18F strain is replaced by a hepatitis A attenuated live vaccine H2 strain, the HAV strain is preserved by the national institute of released army and military science, 2BS cells are used for passage, and qPCR (quantitative polymerase chain reaction) is used for measuring the copy number of the virus to be 1 multiplied by 108 GE/ml. mu.L of a hepatitis A virus H2 strain P2 generation virus solution is taken, 90 mu.L of a DMEM medium containing 2% FBS is added, and the mixture is uniformly mixed to obtain 100 mu.L of a DMEM medium containing HAV virus H2 strain.
FIG. 3 is a bar graph showing the relative copy numbers of HAV viruses of different strains after transfection of 50nM concentration of siRNA conjugates of the present disclosure or control conjugates, respectively, in Huh7.5.1 human hepatoma cells. From the results of FIG. 3, it can be seen that at a concentration of 50nM, the inhibition rate of each of conjugate 1, conjugate 2, conjugate 3 or conjugate 4 on the relative RNA copy number of 18F strain in Huh7.5.1 human hepatoma cells was greater than 84%, and the relative inhibition rate on H2 strain was greater than 60%. Each siRNA conjugate showed good inhibitory activity against both strains compared to NC group.
Experimental example 4 this experiment demonstrates the inhibitory activity of siRNA and its conjugates in huh7.5.1 human hepatoma cells in vitro at different time points.
Huh7.5.1 cells were transfected using Lipofectamine RNAiMAX (Invitrogen). 4.0X105.1 human liver cancer cells were inoculated into 96-well plates, cultured overnight and transfected. The final concentration of siRNA conjugate was 5nM, which was conjugate 1, conjugate 2, conjugate 3, conjugate 4, or conjugate NC, respectively.
Mu.L of HM175/18f-Nluc hepatitis A reporter virus (1X 108 GE/ml) solution was added to 90. Mu.L of 2% FBS DMEM medium and mixed well. Wherein the HM175/18f-Nluc (Nluc-tagged recombinant hepatitis A Virus) was obtained according to the method described in Krishnamurthy Konduru et al, HEPATITIS A Virus (HAV) PACKAGING SIZE limit virology Journal volume 6,Article number:204 (2009), except that the luciferase Nluc gene was usedLuciferase) replaces Blasticidin (Bsd) gene.
Culture was continued by the method of Experimental example 1 by adding an additional 100. Mu.L of 2% FBS-containing DMEM medium (containing 10. Mu.L of HM175/18f-Nluc hepatitis A reporter virus) to each well. Samples were taken at-80 ℃ for storage (samples were taken every 24 hours) on day 1, day 2, day 3 and day 4 after transfection, respectively.
For each conjugate, the co-transfected cells described above were washed 1 time with PBS, according to the followingThe instructions of the fluorogenic enzyme detection kit are operated in the following steps ofThe 96-well plate luminescence detector detects to obtain the luminescence value (RLU value) of each group of cells. The growth curve is shown in fig. 4.
Fig. 4 is a line graph showing the degree of proliferation of HAV virus in huh7.5.1 human hepatoma cells, at day 0 to day 4, after transfection of the siRNA conjugates or the control conjugates of the present disclosure, respectively. Further, relative inhibition of each group of conjugates relative to NC group on days 1 to 4 was calculated based on the extent of proliferation of HAV virus in each group of cells and is recorded in table 4:
table 4 relative inhibition ratio of conjugates 1-4
| Days (days) | Conjugate 1 | Conjugate 2 | Conjugate 3 | Conjugate 4 |
| Day 1 | 18.70% | 39.80% | 29.40% | 32.70% |
| Day 2 | 80.90% | 77.80% | 79.90% | 70.60% |
| Day 3 | 77.50% | 73.80% | 78.30% | 72.00% |
| Day 4 | 86.40% | 88.80% | 86.10% | 89.90% |
Wherein the relative inhibition rate of the conjugate = (RLU value (NC) -RLU value (test conjugate))/RLU value (NC) ×100%.
From the results of Table 4, it was found that, compared with the negative control NC, from day 2, each of the conjugate 1, the conjugate 2, the conjugate 3 and the conjugate 4 showed a relative inhibition ratio of more than 70%, and the relative inhibition ratio on day 4 reached 86% or more, showing a good and stable inhibition effect against HAV virus. Further, on day 4, the conjugates of the present disclosure were able to reduce HAV fluorescence values in cells to pre-transfection (i.e., day 0) levels, exhibiting excellent therapeutic effects.
Experimental example 5 this experiment illustrates the in vivo (in vivo) activity test of siRNA and its siRNA conjugate in mice
This experimental example examined the activity of conjugate 3 in mice at the same concentration and with different modes of administration. Conjugate 3 prepared in preparation example 3 was dissolved in PBS to a solution of 1mg/ml (calculated as siRNA). The following treatments were then carried out:
(1) Type I interferon receptor deficient C57BL/6 mice (females, approximately 25g heavy, 6-8 weeks old, purchased from Siro Biotech Co., ltd.) were randomly divided into 3 groups of 9 mice each, numbered separately. All mice were infected with the HM175/18f strain of 2X107 GE by tail vein injection.
(2) The above siRNA conjugate 3 solution was administered to the first group of 9 mice by intravenous injection at the tail, weighed before administration and the body weight was recorded, and the single dose was calculated as 9mg/kg by weight administration, with a single dose volume of 9. Mu.L/g of the body weight of the mice, as test group 1.
The above siRNA conjugate 3 solution was administered to a second group of 9 mice by subcutaneous injection from the back of the neck, and the weight was weighed and recorded before administration, and the single dose was calculated as 9mg/kg by weight administration, with a single dose volume of 9. Mu.L/g of the mouse weight, as test group 2.
The third group of 9 mice were given PBS by intravenous injection at a single dose volume of 9. Mu.L/g as a blank group.
(3) Mice faeces were collected on day 7 and day 14 after dosing, calculated as dosing time point as day 1. The fecal sample and PBS were stirred well in a ratio of 1g:4ml, and the treated fecal sample was centrifuged at 20000 for 20 minutes using a centrifuge. After centrifugation, 200 μl of supernatant was transferred to a new centrifuge tube.
Total RNA in the supernatant virus particles was extracted using a fully automatic nucleic acid extractor (model: NP-GeneRotex 96).
The Ct value of the target HAV reverse transcribed DNA in each test group was obtained by reverse transcription in the same manner as in experimental example 1, and the copy number of HAV viral RNA in fecal samples in each test group was quantitatively calculated. The results are shown in Table 5.
TABLE 5 average copy number of HAV viral RNA in mouse faeces
| Days (days) | Blank control | Intravenous injection | Subcutaneous injection |
| Day 7 | 1×104.77GE/ml | 1×104.58GE/ml | 1×104.05GE/ml |
| Day 14 | 1×105.47GE/ml | 1×103.94GE/ml | 1×104.26GE/ml |
As can be seen from the results in table 5, on day 7 after administration of siRNA conjugate 3 of the present disclosure, the average copy number of HAV virus in fecal sample of the blank group was 1×104.77 GE/ml, the average copy number of HAV virus in fecal sample of the intravenous group was 1×104.58 GE/ml, and the average copy number of HAV virus in fecal sample of the subcutaneous group was 1×104.05 GE/ml, showing that conjugate 3 has inhibitory effect on HAV virus compared to the blank group. Further, on day 14 after administration of siRNA conjugate 3 of the present disclosure, the average copy number of the fecal sample HAV virus was 1×105.47 GE/ml for the placebo group, 1×103.94 GE/ml for the intravenous 1 fecal sample, and 1×104.26 GE/ml for the subcutaneous group.
The inhibition of HAV virus by conjugate 3 was calculated for the different modes of administration according to the following formula, taking the average copy number of the blank control group as a control.
Relative inhibition of HAV virus in test group = (average copy number of HAV virus in test group 1/average copy number of HAV virus in blank group) ×100%.
On day 14 after administration of siRNA conjugate 3 of the present disclosure, the average inhibition of HAV virus by conjugate 3 was 95.5% in intravenous groups and 93.5% in subcutaneous groups. Conjugate 3 showed significant inhibition of HAV virus, both intravenous and subcutaneous injection, compared to the blank.
The liver tissue of each mouse was taken on day 21 after administration and stored with RNAlater, calculated as the administration time point on day 1. Total RNA was extracted from hepatocytes using a fully automatic nucleic acid extractor (model: NP-GeneRotex 96). The relative copy numbers of the viral RNAs of mice in each group were calculated by using uninfected mice as a control, the Ct values of viral RNAs and GADPH were determined by the same method as in experimental example 1, and the relative copy numbers of the murine hepaviruses of mice in each group were calculated by using comparative Ct (ΔΔct) by the method of experimental example 1, and the relative copy numbers of the blank control group were used as a control, and the inhibition ratios of HAV viruses by different administration methods were calculated according to the following formulas.
Relative inhibition of HAV virus in test group = (relative copy number of HAV virus in test group 1/relative copy number of HAV virus in blank group) ×100%.
The results are shown in Table 7.
The primer information used is shown in Table 6.
TABLE 6 HAV reverse transcription DNA and internal control mouse Gene GAPDH primer information
TABLE 7 inhibition of HAV Virus in liver cells of mice on day 21 by different modes of administration
| Days (days) | Intravenous injection | Subcutaneous injection |
| Day 21 | 61.5% | 55.1% |
As can be seen from the results of table 7, the HAV virus inhibition rate of the intravenous injection group was 61.5% and the HAV virus inhibition rate of the subcutaneous injection group was 55.1% relative to the blank group on day 21 after administration of the siRNA conjugate 3 of the present disclosure, which indicates that the conjugate 3 of the present disclosure has a remarkable and long-term inhibitory effect on HAV virus.
The preferred embodiments of the present disclosure have been described in detail above, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.
In addition, the specific features described in the above embodiments may be combined in any suitable manner without contradiction. The various possible combinations are not described further in this disclosure in order to avoid unnecessary repetition. Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.