Nucleotide analogs and oligonucleotides comprising the same

Abstract

The present application provides nucleotide analogs with reduced off-target effects, oligonucleotides comprising the same, and uses thereof. In particular, the present application provides compounds of Formula (I), pharmaceutically acceptable salts, stereoisomers, deuterated forms, solvates, or prodrugs thereof, which are useful in the preparation of oligonucleotide agents (e.g., small interfering RNA (siRNA) and antisense oligonucleotides) with reduced off-target effects or enhanced gene expression inhibition activity. The present application also provides oligonucleotides comprising the nucleotide analogs. Formula (I)

Description

Technical Field

[0001] This application relates to nucleotide analogs that reduce off-target effects, oligonucleotides containing the same, and applications thereof, wherein the nucleotide analogs can be used to prepare oligonucleotide reagents (e.g., small interfering RNA (siRNA) and antisense oligonucleotides) that reduce off-target effects or enhance gene expression repression activity. Background Technology

[0002] In recent years, oligonucleotide reagents (such as small interfering RNA (siRNA) and antisense oligonucleotides) have shown great application potential in the biomedical field as tools for precise regulation of gene expression. These molecules target specific RNA sequences through base pairing principles, achieving gene silencing or functional regulation, providing new strategies for treating hereditary diseases, viral infections, and cancer. For example, siRNA mediates the degradation of target mRNA through RNA interference mechanisms, while antisense oligonucleotides can enhance or inhibit target gene expression through steric hindrance or RNase H-dependent pathways. Currently, several drugs based on oligonucleotide technology have received FDA approval, validating their clinical translational feasibility.

[0003] siRNA (small interfering RNA) drugs, as a revolutionary gene silencing tool, have shown great potential in treating genetic diseases, viral infections, and cancer. However, off-target effects are one of the major challenges limiting their clinical application. Off-target effects refer to the non-specific binding of siRNA to non-target gene sequences, leading to unintended gene silencing, which may trigger cytotoxicity, immune responses, or other adverse reactions. This phenomenon mainly stems from the partial complementarity between siRNA and target mRNA sequences, especially the matching between the seed region (nucleotides 2-8 of the siRNA) and non-target genes.

[0004] Studies have shown that off-target effects not only interfere with the accuracy of experimental results but can also lead to serious safety issues in clinical treatment. For example, off-target effects may affect normal cellular function and even induce apoptosis or tumorigenesis. Therefore, reducing the off-target effects of siRNA is crucial for improving its specificity and safety. Currently, researchers are employing various strategies to reduce off-target effects, including chemically modifying siRNA (such as 2'-O-methylation), optimizing siRNA design (such as adjusting the seed region sequence), using asymmetric siRNA structures, and developing efficient delivery systems. These methods have made significant progress in improving siRNA targeting and reducing off-target risks, but further optimization is still needed to meet the needs of clinical applications. Summary of the Invention

[0005] The problem the invention aims to solve

[0006] This application aims to provide nucleotide analogs for use in the preparation of oligonucleotide reagents (e.g., small interfering RNA (siRNA) and antisense oligonucleotides) that reduce off-target effects.

[0007] Solution for solving the problem

[0008] Compounds represented by formula (I), their pharmaceutically acceptable salts, stereoisomers, deuterated derivatives, solvates, or prodrugs

[0009] This application provides compounds of formula (I), their pharmaceutically acceptable salts, stereoisomers, deuterated derivatives, solvates, or prodrugs.

[0010] Formula (I),

[0011] Where X is selected from O or S;

[0012] R1 is selected from hydrogen, hydroxyl protecting groups, or reactive phosphorus groups;

[0013] R2 or R3 are each independently selected from hydrogen, deuterium, and C. 1-3 Alkyl, amino protecting groups, or reactive phosphorus groups;

[0014] Wherein, Base represents an unmodified or modified nucleobase, the unmodified or modified nucleobase having a structure selected from any of the following:

[0015] , , ,or ,

[0016] Among them, R4, R5, R6, R7, R8, R9, R 10 Each is independently selected from hydrogen, deuterium, amino protecting groups, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, 3-6 membered cycloalkyl, or -(C=O)C 1-6 alkyl.

[0017] In some embodiments, the reactive phosphorus group may be selected from phosphate ester groups, thiophosphate ester groups, or phosphoramide groups.

[0018] In some embodiments, the reactive phosphorus group may be and .

[0019] In a specific embodiment, the amino protecting group is selected from any one of the following: benzoyl (Bz), acetyl (Ac), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), p-methoxybenzyl (PMB), benzyl (Bn), triphenylmethyl (Tr), p-toluenesulfonyl (Tos), phthaloyl (Pht), allyloxycarbonyl (Alloc), trifluoroacetyl (Tfa), isobutyryl (iBu), or N-[(dimethylamino)methylene]; preferably benzoyl (Bz), acetyl (Ac), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), isobutyryl (iBu), or N-[(dimethylamino)methylene].

[0020] In some embodiments, the hydroxyl protecting group is selected from any of the following: methoxymethyl ether (MOM), 2-tetrahydropyran (THP), tert-butyldimethylsilyl (TBS), trimethylsilyl (TMS), triethylsilyl (TES), triphenylmethyl (Tr), 4,4′-bismethoxytriphenylmethyl (DMTr), 4,4′,4′′-trimethoxytriphenylmethyl (TMTr), benzyl (Bn), benzoyl (Bz), isobutyryl (iBu), acetyl (Ac), p-methoxybenzyl (PMB), or tert-butyldiphenylsilyl (TBDPS); preferably from triphenylmethyl (Tr), 4,4′-bismethoxytriphenylmethyl (DMTr), 4,4′,4′′-trimethoxytriphenylmethyl (TMTr), benzyl (Bn), benzoyl (Bz), isobutyryl (iBu), acetyl (Ac), or tert-butyldiphenylsilyl (TBDPS).

[0021] In some embodiments, the hydroxyl protecting group is selected from any of the following: methoxymethyl ether (MOM), 2-tetrahydropyran (THP), tert-butyldimethylsilyl (TBS), trimethylsilyl (TMS), triethylsilyl (TES), triphenylmethyl (Tr), 4,4′-bismethoxytriphenylmethyl (DMTr), 4,4′,4′′-trimethoxytriphenylmethyl (TMTr), benzyl (Bn), benzoyl (Bz), acetyl (Ac), or p-methoxybenzyl (PMB); preferably from triphenylmethyl (Tr), 4,4′-bismethoxytriphenylmethyl (DMTr), 4,4′,4′′-trimethoxytriphenylmethyl (TMTr), benzyl (Bn), benzoyl (Bz), or acetyl (Ac).

[0022] In some implementations, X is preferably O.

[0023] In some embodiments, R2 or R3 is independently selected from hydrogen, deuterium, methyl, ethyl, amino protecting groups or reactive phosphorus groups.

[0024] In some embodiments, R2 is selected from hydrogen, deuterium, methyl, or ethyl, and R3 is selected from hydrogen, deuterium, an amino protecting group, or a reactive phosphorus group.

[0025] In some embodiments, one of R2 or R3 is an amino protecting group, and the other is hydrogen or deuterium.

[0026] In a preferred embodiment, R2 is hydrogen, R3 is an amino protecting group (e.g., Boc, Bz, or Ac), and R1 is selected from reactive phosphorus groups (e.g., ).

[0027] In some implementations, R 10 Selected from hydrogen, deuterium, and C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, 3-6 membered cycloalkyl, or -(C=O)C 1-6 alkyl.

[0028] In some implementations, R4, R5, R6, R7, R8, R9, R 10 Each is independently selected from hydrogen, deuterium, amino protecting groups, and C. 1-3 Alkyl, C 1-3 Alkoxy, C 1-3 Halogenated alkyl, 3-6 membered cycloalkyl, or -(C=O)C 1-3 alkyl.

[0029] In some implementations, R4, R5, R6, R7, R8, R9, R 10 Each group is independently selected from hydrogen, amino protecting group, methyl, ethyl, propyl, isopropyl, methoxyethyl, cyclopropyl, fluoromethyl, difluoromethyl, or acetyl.

[0030] In some embodiments, R4 or R5 is independently selected from hydrogen, amino protecting groups, or C. 1-3 alkyl.

[0031] In some embodiments, R6 or R7 is independently selected from hydrogen or amino protecting groups.

[0032] In some embodiments, R8 or R9 is independently selected from hydrogen or amino protecting groups.

[0033] In a preferred embodiment, R 10 It is hydrogen or C 1-3Alkyl, preferably hydrogen or methyl.

[0034] In some implementations, the Base is selected from any of the following:

[0035] , , or ,

[0036] R5, R7, or R9 are each independently selected from hydrogen, amino protecting groups, or C. 1-3 Alkyl, R 10 Selected from hydrogen or C 1-6 Alkyl; preferably, R 10 Selected from hydrogen or C 1-3 Alkyl (e.g., methyl).

[0037] In some embodiments, the compound represented by formula (I) of this application, its pharmaceutically acceptable salt, stereoisomer, deuterated derivative, solvate, or prodrug may be selected from the following:

[0038]

[0039] Or, a pharmaceutically acceptable salt, stereoisomer, deuterated product, solvate, or prodrug.

[0040] In some embodiments, the compound represented by formula (I) of this application, its pharmaceutically acceptable salt, deuterated derivative, solvate, or prodrug may be selected from the following:

[0041] Or, a pharmaceutically acceptable salt, deuterated form, solvate, or prodrug thereof.

[0042] The compound represented by formula (i), its pharmaceutically acceptable salt, stereoisomer, deuterated derivative, solvate, or prodrug.

[0043] This application provides the compound represented by formula (i), its pharmaceutically acceptable salt, stereoisomer, deuterated derivative, solvate, or prodrug.

[0044]

[0045] Where X is selected from O or S;

[0046] R1 and R ib Each is independently selected from hydrogen, hydroxyl protecting groups, or reactive phosphorus groups;

[0047] R ia Selected from hydroxyl, hydrogen, fluorine, optionally substituted amino, optionally substituted C 1-6 Alkyl, optionally substituted C 1-6 Alkoxy, C 1-6 alkenyl or C 1-6alkynyl group;

[0048] Wherein, Base represents an unmodified or modified nucleobase, and the unmodified or modified nucleobase has a structure selected from any of the following:

[0049] , , ,or ,

[0050] Among them, R4, R5, R6, R7, R8, R9, R 10 Each is independently selected from hydrogen, deuterium, amino protecting groups, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, 3-6 membered cycloalkyl, or -(C=O)C 1-6 alkyl.

[0051] In some embodiments, the reactive phosphorus group may be selected from phosphate ester groups, thiophosphate ester groups, or phosphoramide groups.

[0052] In some embodiments, the reactive phosphorus group may be and .

[0053] In a specific embodiment, the amino protecting group is selected from any one of the following: benzoyl (Bz), acetyl (Ac), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), p-methoxybenzyl (PMB), benzyl (Bn), triphenylmethyl (Tr), p-toluenesulfonyl (Tos), phthaloyl (Pht), allyloxycarbonyl (Alloc), trifluoroacetyl (Tfa), isobutyryl (iBu), or N-[(dimethylamino)methylene]; preferably benzoyl (Bz), acetyl (Ac), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), isobutyryl (iBu), or N-[(dimethylamino)methylene].

[0054] In some embodiments, the hydroxyl protecting group is selected from any of the following: methoxymethyl ether (MOM), 2-tetrahydropyran (THP), tert-butyldimethylsilyl (TBS), trimethylsilyl (TMS), triethylsilyl (TES), triphenylmethyl (Tr), 4,4′-bismethoxytriphenylmethyl (DMTr), 4,4′,4′′-trimethoxytriphenylmethyl (TMTr), benzyl (Bn), benzoyl (Bz), acetyl (Ac), p-methoxybenzyl (PMB), or tert-butyldiphenylsilyl (TBDPS); preferably from triphenylmethyl (Tr), 4,4′-bismethoxytriphenylmethyl (DMTr), 4,4′,4′′-trimethoxytriphenylmethyl (TMTr), benzyl (Bn), benzoyl (Bz), acetyl (Ac), or tert-butyldiphenylsilyl (TBDPS).

[0055] In some embodiments, the hydroxyl protecting group is selected from any of the following: methoxymethyl ether (MOM), 2-tetrahydropyran (THP), tert-butyldimethylsilyl (TBS), trimethylsilyl (TMS), triethylsilyl (TES), triphenylmethyl (Tr), 4,4′-bismethoxytriphenylmethyl (DMTr), 4,4′,4′′-trimethoxytriphenylmethyl (TMTr), benzyl (Bn), benzoyl (Bz), isobutyryl (iBu), acetyl (Ac), p-methoxybenzyl (PMB); preferably from triphenylmethyl (Tr), 4,4′-bismethoxytriphenylmethyl (DMTr), 4,4′,4′′-trimethoxytriphenylmethyl (TMTr), benzyl (Bn), benzoyl (Bz), isobutyryl (iBu), or acetyl (Ac).

[0056] In some implementations, X is preferably O.

[0057] In a preferred embodiment, R1 is selected from reactive phosphorus groups (e.g., ...). ), R ib Selected from hydrogen or hydroxyl protecting groups.

[0058] R ia Selected from hydrogen, hydroxyl, F, methoxy, -O-CH2-CH2-O-CH3 or -CH2-CH2-CH=CH2.

[0059] In some implementations, R 10 Selected from hydrogen, deuterium, and C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, 3-6 membered cycloalkyl, or -(C=O)C 1-6 alkyl.

[0060] In some implementations, R4, R5, R6, R7, R8, R9, R10 Each is independently selected from hydrogen, deuterium, amino protecting groups, and C. 1-3 Alkyl, C 1-3 Alkoxy, C 1-3 Halogenated alkyl, 3-6 membered cycloalkyl, or -(C=O)C 1-3 alkyl.

[0061] In some implementations, R4, R5, R6, R7, R8, R9, R 10 Each group is independently selected from hydrogen, amino protecting group, methyl, ethyl, propyl, isopropyl, methoxyethyl, cyclopropyl, fluoromethyl, difluoromethyl, or acetyl.

[0062] In some embodiments, R4 or R5 is independently selected from hydrogen, amino protecting groups, or C. 1-3 alkyl.

[0063] In some embodiments, R6 or R7 is independently selected from hydrogen or amino protecting groups.

[0064] In some embodiments, R8 or R9 is independently selected from hydrogen or amino protecting groups.

[0065] In a preferred embodiment, R 10 It is hydrogen or C 1-3 Alkyl, preferably hydrogen or methyl.

[0066] In some implementations, the Base is selected from any of the following:

[0067] , , or ,

[0068] R5, R7, or R9 are each independently selected from hydrogen, amino protecting groups, or C. 1-3 Alkyl, R 10 Selected from hydrogen or C 1-6 Alkyl; preferably, R 10 Selected from hydrogen or C 1-3 Alkyl (e.g., methyl).

[0069] In some embodiments, the two Bases of the compound represented by Formula (i), its pharmaceutically acceptable salt, stereoisomer, deuterated derivative, solvate, or prodrug may be the same or different. For example, when one Base is selected from modified or unmodified U, the other Base may be selected from modified or unmodified A, C, G, or T. Or, for example, when one Base is selected from modified or unmodified U, the other Base may be selected from modified or unmodified U. Exemplarily, the compound represented by Formula (i), its pharmaceutically acceptable salt, stereoisomer, deuterated derivative, solvate, or prodrug may be

[0070] .

[0071] In some respects, this application also provides the following compounds, their pharmaceutically acceptable salts, stereoisomers, deuterated derivatives, or solvates:

[0072] , , , , , , , , , , , , , , , , , .

[0073] Oligonucleotides

[0074] On one hand, this application relates to oligonucleotides, particularly double-stranded ribonucleic acid (dsRNA).

[0075] In some embodiments, one strand of the double-stranded ribonucleic acid (BRNA) is an antisense strand, which is complementary to the mRNA sequence formed during the expression of the target gene and is used to guide the cleavage of the target mRNA (i.e., the transcription product of the target gene). The other sense strand of the BRNA includes a sequence that is at least partially complementary (including completely complementary) to the antisense strand to form a double-stranded region of the BRNA.

[0076] In some embodiments, the double-stranded ribonucleic acid (SSRNA) serves as a substrate for the endonuclease Dicer, and is cleaved into small fragments of dsRNA, i.e., siRNA. In some embodiments, the SSRNA is siRNA. siRNA assembles to form an RNA-induced silencing complex (RISC), which cleaves the target mRNA and inhibits the expression of the target gene.

[0077] The double-stranded RNA comprises a sense strand and an antisense strand, wherein the antisense strand is complementary to the target gene; and the sense strand is complementary to the antisense strand.

[0078] In some embodiments, the antisense strand comprises a sequence B that differs from the inverse complementary sequence of at least 15 consecutive nucleotides in the target sequence by no more than 3 nucleotides. Specifically, a start nucleotide is selected in the target sequence along the direction from the 5' end to the 3' end, and at least 15 nucleotides extending in the 3' direction, including the start nucleotide, serve as the binding region of the siRNA. The antisense strand comprises the inverse complementary sequence of the nucleotide sequence corresponding to the binding region. It should be noted that the start nucleotide can be a nucleotide at any position in the target sequence, as long as extending in the 3' direction from the start nucleotide yields at least 15 consecutive nucleotides (including the nucleotide at the start position).

[0079] In this invention, the nucleotide sequence of the antisense strand can be completely complementary or substantially complementary to the target sequence. When the nucleotide sequence of the antisense strand is substantially complementary to the target sequence, the nucleotide sequence of the antisense strand contains no more than three mismatched bases with the target sequence. For example, the number of mismatched bases is one, two, or three. When the nucleotide sequence of the antisense strand is completely complementary to the target sequence, the nucleotide sequence of the antisense strand does not contain any mismatched bases with the target sequence.

[0080] Furthermore, the antisense strand consists of at least 15 nucleotides. In some embodiments, the antisense strand consists of 15-28 nucleotides. For example, the length of the antisense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides. Preferably, the antisense strand consists of 18-25 nucleotides, more preferably 18-23 nucleotides, and most preferably 19, 21, or 23 nucleotides.

[0081] In some embodiments, the sense strand comprises sequence A, which differs from at least 15 consecutive nucleotides in the target sequence by no more than 3 nucleotides. The sense strand includes regions complementary to the antisense strand, and the nucleotide sequence of the sense strand is identical or substantially identical to the sequence of the antisense strand binding region on the target sequence. Therefore, the nucleotide sequence of the sense strand comprises or is at least 15 consecutive nucleotides in the target sequence that bind the antisense strand; or, the nucleotide sequence of the sense strand differs from at least 15 consecutive nucleotides in the target sequence that bind the antisense strand by 1, 2, or 3 bases.

[0082] Furthermore, the positive sense strand consists of at least 15 nucleotides. In some embodiments, the positive sense strand consists of 15-28 nucleotides. For example, the length of the positive sense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides. Preferably, the positive sense strand consists of 18-25 nucleotides, more preferably 18-23 nucleotides, and most preferably 19, 21, or 23 nucleotides.

[0083] In some specific embodiments, the nucleotide that differs from the target sequence is preferably located at the end of the sense strand. For example, in some specific embodiments, the differing nucleotide is located at the 3' end of the sense strand. In other specific embodiments, the differing nucleotide is located at the 5' end of the sense strand.

[0084] In this disclosure, the length of the justice chain and the length of the antisense chain may be the same or different.

[0085] In some implementations, the lengths of the justice chain and the antisense chain are the same. Specifically, the length ratio of the justice chain to the antisense chain is 15 / 15, 16 / 16, 17 / 17, 18 / 18, 19 / 19, 20 / 20, 21 / 21, 22 / 22, 23 / 23, 24 / 24, 25 / 25, 26 / 26, 27 / 27, or 28 / 28. Preferably, the length ratio of the justice chain to the antisense chain is 18 / 18, 19 / 19, 20 / 20, 21 / 21, 22 / 22, 23 / 23, 24 / 24, or 25 / 25; more preferably, it is 19 / 19, 20 / 20, 21 / 21, 22 / 22, or 23 / 23; and most preferably, it is 19 / 19, 21 / 21, or 23 / 23.

[0086] In some implementations, the lengths of the justice chain and the antisense chain are different. For example, the justice chain / antisense chain length ratio is 18 / 19, 18 / 20, 18 / 21, 18 / 22, 18 / 23, 18 / 24, 18 / 25, 18 / 26, 19 / 18, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 19, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25. Examples of possible lengths include 20 / 26, 21 / 18, 21 / 19, 21 / 20, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 18, 22 / 19, 22 / 20, 22 / 21, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 19, 23 / 20, 23 / 21, 23 / 22, 23 / 24, 23 / 25, or 23 / 26. In some preferred embodiments, the length ratio of the justice chain to the antisense chain is 19 / 21 or 21 / 23.

[0087] In this disclosure, the sense strand and the antisense strand can be fully complementary or substantially complementary. When they are substantially complementary, there are no more than 3 mismatched bases in the double-stranded region formed by the sense strand and the antisense strand.

[0088] In some embodiments, the nucleotide sequence of the sense strand of the double-stranded ribonucleic acid comprises sequence A and optional sequence D, wherein the 3' end of sequence A in the sense strand is linked to sequence D, which consists of 1-2 nucleotides, preferably 1-2 thymine deoxyribonucleotides; and / or, the nucleotide sequence of the antisense strand of the double-stranded ribonucleic acid comprises sequence B and optional sequence E, wherein the 3' end of sequence B in the antisense strand is linked to sequence E, which consists of 1-2 nucleotides, preferably 1-2 thymine deoxyribonucleotides.

[0089] In some embodiments, after the sense strand and antisense strand are at least partially complementary to form a double-stranded region, the sense strand, antisense strand, or both have protruding nucleotides extending out of the double-stranded region. The number of protruding nucleotides can be one or more, for example, one or two. Furthermore, the one or two protruding nucleotides can be located at the 5' end, 3' end, or both ends of any antisense strand or sense strand, and each protruding nucleotide can be of any type.

[0090] For example, when the sense strand consists of a 21-unit sequence A plus sequence D, and the antisense strand consists of a 21-unit sequence B plus sequence E, the nucleotides 1-19 of sequence A are completely anticomplementary to the nucleotides 1-19 of sequence B in the 5'-to-3' direction, resulting in protruding nucleotides at the 3' end of sequences D and E, respectively. Similarly, when the sense strand consists of a 19-unit sequence A, and the antisense strand consists of a 21-unit sequence B plus E, the nucleotides 1-19 of sequence A are completely anticomplementary to the nucleotides 1-19 of sequence B in the 5'-to-3' direction, resulting in protruding nucleotides at the 3' end of sequence E on the antisense strand.

[0091] In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and the 3' end of the sense strand has 1-2 protruding nucleotides extending out of the double-stranded region, while the 3' end of the antisense strand is blunt. In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region, while the 3' end of the sense strand is blunt. In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and both the sense strand and the antisense strand have 1-2 protruding nucleotides extending out of the double-stranded region at their 3' ends. In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and both the sense strand and the antisense strand have blunt 3' ends.

[0092] In some embodiments, each nucleotide of the sense strand is independently a modified or unmodified nucleotide. In some embodiments, each nucleotide of the antisense strand is independently a modified or unmodified nucleotide.

[0093] In some embodiments, each nucleotide of the sense strand is independently a modified or unmodified nucleotide. In some embodiments, each nucleotide of the antisense strand is independently a modified or unmodified nucleotide.

[0094] In some embodiments, the sense and / or antisense strands contain phosphodiester bonds and modifications thereof. Modifications to the phosphodiester bonds include phosphothiophosphate bonds, methylphosphonate bonds, borane phosphate bonds, phosphoramidide bonds, or amide bonds linking any two linked nucleotides. For example, the sense strand may contain phosphodiester bonds, phosphothiophosphate bonds, methylphosphonate bonds, borane phosphate bonds, phosphoramidide bonds, or amide bonds linking any two linked nucleotides. In some embodiments, the sense strand may contain phosphodiester bonds, phosphothiophosphate bonds, methylphosphonate bonds, borane phosphate bonds, phosphoramidide bonds, or amide bonds linking any two linked nucleotides.

[0095] In some embodiments, any two nucleotides linked in the sense strand are connected by a phosphodiester bond, a phosphothiodiester bond, or an amide bond. In some embodiments, any two nucleotides linked in the antisense strand are connected by a phosphodiester bond, a phosphothiodiester bond, or an amide bond.

[0096] In some embodiments, any two nucleotides linked in the sense strand are connected by a phosphodiester bond or a phosphothiodiester bond. In some embodiments, any two nucleotides linked in the antisense strand are connected by a phosphodiester bond or a phosphothiodiester bond.

[0097] In some embodiments, the ribosome group of the 5' terminal nucleotide of the positive strand is a 5' hydroxyl group.

[0098] In some embodiments, the ribosome of the 5' terminal nucleotide of the antisense strand has a 5' hydroxyl group, a 5' phosphate ester group, or a 5' phosphate derivative group. The type of group at the 5' position of the ribosome depends on the preparation method used, which is known to those skilled in the art based on the corresponding preparation method.

[0099] When the ribose group of the 5' terminal nucleotide in the antisense strand has a 5' phosphate ester group or a 5' phosphate derivative group, the following structure is formed:

[0100] , , Wherein, Base represents a nucleobase, such as A, U, G, C, or T; R' is a hydroxyl group or hydrogen, or is substituted by various groups known to those skilled in the art, such as 2'-fluoro (2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, and 2'-deoxyribonucleotides.

[0101] In this paper, when the ribonucleotide at the 5' end of the sense strand and / or antisense strand has a hydroxyl group instead of a 5' phosphate ester group or a 5' phosphate-derived group, the structure of the 5' end nucleotide is as follows:

[0102] ,

[0103] Wherein, Base represents a nucleobase, such as A, U, G, C or T, and R is a hydroxyl or hydrogen or is substituted by various groups known to those skilled in the art. For example, R can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, or 2'-substituted amino.

[0104] Oligonucleotide modifications and conjugates

[0105] A second aspect of this disclosure provides a modification of a double-stranded ribonucleic acid (SSRNA). Further, the SSRNA modification is a siRNA modification.

[0106] In some implementations, the double-stranded ribonucleic acid modification includes modification of at least one nucleotide.

[0107] In this disclosure, the modifications to the double-stranded ribonucleic acid include one or more modifications selected from nucleotide modifications, nucleotide derivative modifications, phosphodiester bond modifications, and 5'-terminal modifications.

[0108] In this document, "nucleotide modification" refers to a nucleotide or nucleotide derivative formed by replacing the 2' hydroxyl group of the ribose group with another group, or a nucleotide whose nucleobase is a modified nucleobase. Such nucleotide modification does not result in a significant weakening or loss of the siRNA's ability to repress gene expression. For example, modified nucleotides disclosed in JKWatts, GF Deleavey, and MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20): 842-55 can be selected. Nucleotide modification can improve the stability of siRNA and maintain its high repressive efficiency against target genes.

[0109] For example, the modified nucleosides have the following structures:

[0110] Wherein, Base represents a nucleobase, such as A, U, G, C, or T, and the hydroxyl group at the 2' position of the ribose group is replaced by R. These hydroxyl groups at the 2' position can be replaced by various groups known to those skilled in the art; for example, R can be selected from halogens, alkyl groups, alkoxy groups, substituted alkyl groups, and substituted alkoxy groups. For example, in some specific embodiments, the modified nucleotides include, but are not limited to, 2'-fluoro(2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, and 2'-deoxyribonucleotides.

[0111] In some embodiments, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe, 2'-O-CH3) modified nucleotide, etc.

[0112] In some embodiments, the 2'-substituted alkoxy modified nucleotide is a nucleotide modified with 2'-methoxyethoxy (2'-O-CH2-CH2-O-CH3) or a nucleotide modified with 2'-O-CH2-CH=CH2, etc.

[0113] In some embodiments, the 2'-substituted alkyl-modified nucleotide is a nucleotide modified with 2'-CH2-CH2-CH=CH2, etc.

[0114] In some embodiments, the modification of the nucleotide is a modification of the nucleobase. Nucleobase modification can be of various types known to those skilled in the art. Exemplary examples include, but are not limited to, Ψ, Inosine (I), Cm, Um, Gm, or derivatives thereof.

[0115] In this document, nucleotide derivatives can refer to compounds that can replace nucleotides in nucleic acids but whose structure differs from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. In some embodiments, nucleotide derivatives can be isonucleotides, debased residues, bridged nucleic acids (BNAs), or acyclic nucleotides. BNAs refer to restricted or inaccessible nucleotides. BNAs can contain a bridging structure with a "fixed" C3'-endoglucan condensation, such as a five-membered, six-membered, or seven-membered ring. This bridge is typically incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET, etc.

[0116] For example, the nucleoside of LNA is shown in formula (1), the nucleoside of ENA is shown in formula (2), and the nucleoside of cET is shown in formula (3):

[0117]

[0118] Equation (1) Equation (2) Equation (3)

[0119] Base refers to nucleobases, such as A, U, G, C, or T.

[0120] Acyclic nucleotides are a class of nucleotides formed by the opening of the sugar ring, such as unopened nucleic acids (UNA) or glycerol nucleic acids (GNA). UNA is shown in formula (4), and GNA is shown in formula (5).

[0121]

[0122] Equation (4) Equation (5)

[0123] In equations (4) and (5) above, Base refers to a nucleobase, and Ra is independently selected from H, OH, or alkoxy (-OC). 1-6 alkyl).

[0124] In some embodiments, the nucleotide derivative may include nucleotide analogs of compounds of formula (I) described above in this application, i.e., nucleotide analogs of compounds of formula (I), pharmaceutically acceptable salts, stereoisomers or deuterated derivatives thereof.

[0125] In some embodiments, nucleotide derivative modification refers to the replacement of nucleotides in nucleic acids with nucleotide derivatives. Exemplarily, the nucleotide derivatives are selected from isonucleotides, debased residues, LNA, ENA, cET, UNA, GNA, or nucleotide analogs described in this application. In this application, when a nucleotide analog from a compound represented by formula (I) is included, preferably, the nucleobases therein are identical to the nucleobases of the substituted nucleotide. Alternatively, in this application, when a nucleotide analog from a compound represented by formula (I) is included, preferably, the nucleobases therein conform to the Watson-Crick base pairing principle with the nucleobases on the corresponding nucleotides of the other strand of the double-stranded ribonucleic acid.

[0126] In some embodiments, the nucleotides in the double-stranded RNA are replaced with isonucleotides, also referred to as isonucleotide modification in the context of this disclosure. In some embodiments, isonucleotide modification includes incorporating an isonucleotide at one or more sites on the sense and / or antisense strands of the double-stranded RNA (e.g., siRNA) to replace the natural nucleoside for coupling at the corresponding positions.

[0127] In some embodiments, the isonucleoside modification is D-isonucleoside modification. In other embodiments, the isonucleoside modification is L-isonucleoside modification. In still other embodiments, the isonucleoside modification is a combination of D-isonucleoside and L-isonucleoside modification.

[0128] In some embodiments, the positive strand may contain one or more capping residues or portions, sometimes referred to in the art as a “cap,” “terminal cap,” or “capping residue.” As used herein, a “capping residue” refers to a nonnucleotide compound or other structural unit that can be incorporated into one or more ends of the nucleotide sequence of a double-stranded ribonucleic acid (e.g., siRNA) disclosed herein. In some cases, capping residues may provide certain beneficial properties to double-stranded ribonucleic acid (e.g., siRNA), such as protection against exonuclease degradation. In some embodiments, an inverse debased deoxyribose residue (invAb) is added as a capping residue. In some embodiments, the capping residue appears at the 3' end of the positive strand or both the 5' and 3' ends.

[0129] In some embodiments, one or more inverse debased deoxyribose residues (invAb) are added to the 3' end of the positive strand. In some embodiments, one or more inverse debased deoxyribose residues (invAb) are added to both the 5' end and the 3' end of the positive strand. In some embodiments, the inverse debased deoxyribose residues may be linked via phosphate ester bonds, thiophosphate ester bonds, or other nucleoside internucleotide bonds. When describing modification sites in the modification method, (invAb) is not counted as the first site of the nucleotide sequence. (invAb) may have the following structure:

[0130] When located at the 3' end, it is When located at the 5' end, it is .

[0131] In some embodiments, the modified double-stranded ribonucleic acid (BRNA) comprises a phosphodiester bond modification at at least one position, such as a thiophosphate, methylphosphonate, methoxypropylphosphonate, or amide bond modification. In some embodiments, the modified BRNA comprises a phosphodiester bond, methylphosphonate bond, borane phosphate bond, phosphoramidite bond, or amide bond linking any two linked nucleotides.

[0132] In some embodiments, modification of the phosphodiester bond refers to the substitution of an oxygen atom in the phosphodiester bond by a sulfur atom to form a phosphothiodiester bond. Phosphothiodiester bonds can stabilize the double-stranded structure of double-stranded ribonucleic acid (e.g., siRNA) and maintain the specificity of base pairing. An exemplary phosphothiodiester bond structure is shown below:

[0133] .

[0134] In some embodiments, the 5'-terminal modification includes a 5'-terminal phosphate ester modification, for example, the nucleotide at the 5' end of the sense strand and / or antisense strand has a 5' phosphate-derived group, including 5'-(E)-vinylphosphonate (5'-(E)-VP, also known as EVP), 5'-methylphosphonate (5'-MP), and (S)-5'-C-methyl analogues.

[0135] In some embodiments, the ribosome of the 5' terminal nucleotide of the antisense strand and / or the sense strand may have a 5' hydroxyl group. As an example, when the ribosome of the 5' terminal nucleotide of the antisense strand has a 5' phosphate ester group or a 5' phosphate-derived group, the following structure is formed:

[0136] , , Wherein, Base represents a nucleobase, such as A, U, G, C or T; R' is independently selected from hydroxyl or hydrogen or substituted by various groups known to those skilled in the art, for example, R' can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino.

[0137] In this paper, when the ribonucleotide at the 5' end of the sense strand and / or antisense strand has a hydroxyl group instead of a 5' phosphate ester group or a 5' phosphate-derived group, the structure of the 5' end nucleotide is as follows:

[0138] ,

[0139] Wherein, Base represents a nucleobase, such as A, U, G, C or T, and R is a hydroxyl or hydrogen or is substituted by various groups known to those skilled in the art. For example, R can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, or 2'-substituted amino.

[0140] In some embodiments, the double-stranded ribonucleic acid modifier further comprises at least one of the following chemical modifications:

[0141] (1) Modification of at least one nucleotide in the positive strand,

[0142] (2) Modification of the phosphodiester bond at at least one position in the positive chain.

[0143] (3) Modification of at least one nucleotide in the antisense strand,

[0144] (4) Modification of phosphodiester bonds at at least one position in the antisense chain.

[0145] Furthermore, the double-stranded ribonucleic acid modifier is an siRNA modifier containing at least one of the chemical modifications in (1)-(4).

[0146] In some embodiments, the positive strand of the double-stranded ribonucleic acid (BRNA) modifier, along the 5'-to-3' direction, includes the following modification: at least three (e.g., at least three to six, such as 3, 4, 5, or 6) of the ribonucleotides at positions 3-11 of sequence A in the positive strand are 2'-fluorinated ribonucleotides. For example, along the 5'-to-3' direction, the positive strand of the BRNA modifier includes the following modification: at least three (e.g., 3, 4, 5, or 6) of the ribonucleotides at positions 3, 5, 7, 8, 9, 10, and 11 of sequence A in the positive strand are 2'-fluorinated ribonucleotides. For example, along the 5' end to the 3' end, the positive strand of the double-stranded ribonucleic acid modification includes the following modification: at least three (e.g., 3, 4, 5, or 6) of the ribonucleotides at positions 3, 5, 7, 8, 9, and 11 of the sequence A of the positive strand are 2'-fluoro modified ribonucleotides.

[0147] In a further preferred embodiment, the ribonucleotides at other positions in sequence A of the sense strand may be 2'-methoxy-modified ribonucleotides. In some embodiments, the sense strand may also be modified with one or more nucleotide derivatives.

[0148] In some embodiments, the positive strand of the double-stranded ribonucleic acid modification includes the following modification along the 5' end to the 3' end direction: the ribonucleotides at positions 5, 7, 8 and 9 of sequence A of the positive strand are 2'-fluoro-modified ribonucleotides; optionally, the ribonucleotides at other positions of sequence A of the positive strand are 2'-methoxy-modified ribonucleotides.

[0149] Alternatively, in some embodiments, the positive strand of the double-stranded ribonucleic acid modification includes the following modification along the 5' end to the 3' end direction: the ribonucleotides at positions 3, 7, 8 and 9 of sequence A of the positive strand are 2'-fluoro-modified ribonucleotides; optionally, the ribonucleotides at other positions of sequence A of the positive strand are 2'-methoxy-modified ribonucleotides.

[0150] Alternatively, in some embodiments, the positive strand of the double-stranded ribonucleic acid modification includes the following modification along the 5' end to the 3' end direction: the ribonucleotides at positions 3, 7, 9 and 11 of sequence A of the positive strand are 2'-fluoro-modified ribonucleotides; optionally, the ribonucleotides at other positions of sequence A of the positive strand are 2'-methoxy-modified ribonucleotides.

[0151] Alternatively, in some embodiments, the positive strand of the double-stranded ribonucleic acid modification includes the following modification along the 5' end to the 3' end: the ribonucleotides at positions 7, 9, 10, and 11 of sequence A of the positive strand are 2'-fluoro-modified ribonucleotides; optionally, the ribonucleotides at other positions of sequence A of the positive strand are 2'-methoxy-modified ribonucleotides.

[0152] In some embodiments, when neither the 5' nor the 3' end of the positive chain is connected (invAb), the positive chain contains a phosphothioester bond located at the following positions:

[0153] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0154] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0155] Between the first nucleotide and the second nucleotide starting at the 3' end of the positive strand; and / or

[0156] Between the second and third nucleotides starting at the 3' end of the positive strand;

[0157] or,

[0158] The positive chain contains phosphothiophosphate diester bonds located at the following positions:

[0159] Between the first nucleotide and the second nucleotide starting at the 5' end of the positive strand; and / or

[0160] Between the second and third nucleotides starting at the 5' end of the positive strand.

[0161] In some embodiments, when only one (invAb) is attached to the 5' end of the positive chain, the positive chain contains a phosphothioester bond located at the following positions:

[0162] Between (invAb) at the 5' end of the positive strand and the first nucleotide starting at the 5' end;

[0163] Between the first and second nucleotides starting at the 5' end of the positive strand.

[0164] In some embodiments, when only one (invAb) is attached to the 3' end of the positive chain, the positive chain contains a phosphothioester bond located at the following positions:

[0165] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0166] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0167] Between (invAb) at the 3' end of the positive strand and the first nucleotide starting at the 3' end.

[0168] In some embodiments, when the 5' end and 3' end of the positive chain are each connected to an (invAb), the positive chain contains a phosphothioester bond located at the following positions:

[0169] Between (invAb) at the 5' end of the positive strand and the first nucleotide starting at the 5' end;

[0170] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0171] Between (invAb) at the 3' end of the positive strand and the first nucleotide starting at the 3' end.

[0172] In some implementations, the ribosome of the 5' terminal nucleotide of the positive strand is a 5' hydroxyl group.

[0173] In some embodiments, the nucleotide structure of the ribosome of the 5' terminal nucleotide of the positive strand having a 5' hydroxyl group is shown in Formula X:

[0174] (Formula X)

[0175] Wherein, Base represents a nucleobase, such as A, U, G, C or T; R is a hydroxyl group or is substituted by various groups known to those skilled in the art, for example, R can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino, 2'-deoxynucleotide.

[0176] In some embodiments, the positive strand of the siRNA modifier includes phosphothioester bonds at the following positions along the 5' end to the 3' end: between the first and second nucleotides starting at the 5' end, between the second and third nucleotides starting at the 5' end, between the first and second nucleotides starting at the 3' end, and between the second and third nucleotides starting at the 3' end.

[0177] In some embodiments, the positive strand of the siRNA modifier includes phosphothioester bonds at the following positions along the 5' end to the 3' end: between the first and second nucleotides starting at the 5' end, and between the second and third nucleotides starting at the 5' end.

[0178] In some embodiments, the antisense strand of the double-stranded ribonucleic acid (BRNA) modifier includes the following modification along the 5'-to-3' direction: at least three (e.g., at least three to seven, such as 3, 4, 5, 6, or 7) of the ribonucleotides at positions 2-16 of sequence B of the antisense strand are 2'-fluorinated ribonucleotides. For example, along the 5'-to-3' direction, the antisense strand of the BRNA modifier includes the following modification: at least three (e.g., 3, 4, 5, 6, or 7) of the ribonucleotides at positions 2, 6, 7, 8, 9, 10, 14, and 16 of sequence B of the antisense strand are 2'-fluorinated ribonucleotides. For example, along the 5' end to the 3' end, the antisense strand of the double-stranded ribonucleic acid modification includes the following modification: at least three (e.g., 3, 4, 5, 6, or 7) of the ribonucleotides at positions 2, 6, 7, 10, 14, and 16 of sequence B of the antisense strand are 2'-fluoro modified ribonucleotides.

[0179] In a further preferred embodiment, the ribonucleotides at other positions in sequence B of the antisense strand may be 2'-methoxy modified ribonucleotides.

[0180] In some embodiments, the antisense strand may also have one or more nucleotide derivative modifications, such as GNA or nucleotide analogs from compounds represented by formula (I) above in this application.

[0181] In some embodiments, the antisense strand of the double-stranded ribonucleic acid (BRNA) modifier, along the 5'-to-3' direction, comprises the following modification: the ribonucleotides at positions 2, 6, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; optionally, sequence B of the antisense strand may have one or more nucleotide derivative modifications, such as GNA or nucleotide analogs from compounds of formula (I) described above in this application, and the remaining ribonucleotides in sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides. For example, along the 5'-to-3' direction, the antisense strand of the BRNA modifier comprises the following modification: the ribonucleotides at positions 2, 6, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; and the remaining ribonucleotides in sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides.

[0182] In some embodiments, the antisense strand of the double-stranded ribonucleic acid (BRNA) modifier, along the 5'-to-3' direction, includes the following modification: the ribonucleotides at positions 2, 7, 10, and 14 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; optionally, sequence B of the antisense strand may have one or more nucleotide derivative modifications, such as GNA or nucleotide analogs from the compound represented by formula (I) above in this application, and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides. For example, along the 5'-to-3' direction, the antisense strand of the BRNA modifier includes the following modification: the ribonucleotides at positions 2, 7, 10, and 14 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides.

[0183] In some embodiments, the antisense strand of the double-stranded ribonucleic acid modification includes the following modification along the 5' end to the 3' end: the ribonucleotides at positions 2, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; optionally, sequence B of the antisense strand may have one or more nucleotide derivative modifications, such as GNA or nucleotide analogs from the compound represented by formula (I) above in this application, and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides. In a specific embodiment, the antisense strand of the double-stranded ribonucleic acid modification includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 14, and 16 of sequence B in the antisense strand are 2'-fluoro-modified ribonucleotides; the ribonucleotide at position 6 in the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA or a nucleotide analogue from the compound represented by formula (I) above in this application; and the ribonucleotides at the remaining positions in the antisense strand are 2'-methoxy-modified ribonucleotides.

[0184] In some embodiments, the antisense strand of the double-stranded ribonucleic acid modification includes the following modification along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; optionally, sequence B of the antisense strand may have one or more nucleotide derivative modifications, such as GNA or nucleotide analogs from the compound of formula (I) above described in this application, and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides. For example, along the 5' end to the 3' end, the antisense strand of the double-stranded ribonucleic acid modification includes the following modifications: the ribonucleotides at positions 2, 6, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; the ribonucleotide at position 7 of sequence B of the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA or a nucleotide analogue from the compound shown in formula (I) above in this application; and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides.

[0185] In some embodiments, the antisense strand of the double-stranded ribonucleic acid modification includes the following modification along the 5' end to the 3' end: the ribonucleotides at positions 2, 7, 10, and 14 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; optionally, sequence B of the antisense strand may have one or more nucleotide derivative modifications, such as GNA or nucleotide analogs from the compound of formula (I) above described in this application, and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides. For example, the antisense strand of the double-stranded ribonucleic acid modification includes the following modifications: the ribonucleotides at positions 2, 7, 10, and 14 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; the ribonucleotide at position 6 of sequence B of the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA or a nucleotide analogue from the compound shown in formula (I) above in this application; and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides.

[0186] In some embodiments, the antisense strand of the double-stranded ribonucleic acid modification includes the following modification along the 5' end to the 3' end: the ribonucleotides at positions 2, 10, and 14 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; optionally, sequence B of the antisense strand may have one or more nucleotide derivative modifications, such as GNA or nucleotide analogs from the compound of formula (I) above described in this application, and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides. For example, along the 5' end to the 3' end, the antisense strand of the double-stranded ribonucleic acid modification includes the following modifications: the ribonucleotides at positions 2, 10, and 14 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; the ribonucleotide at position 7 of sequence B of the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA or a nucleotide analogue from the compound shown in formula (I) above in this application; and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides.

[0187] In some embodiments, the antisense strand of the double-stranded ribonucleic acid modification includes the following modification along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 8, 9, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; optionally, sequence B of the antisense strand may have one or more nucleotide derivative modifications, such as GNA or nucleotide analogs from the compound of formula (I) above described in this application, and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides. For example, along the 5' end to the 3' end, the antisense strand of the double-stranded ribonucleic acid modification includes the following modifications: the ribonucleotides at positions 2, 6, 8, 9, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; the ribonucleotide at position 7 of sequence B of the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA or a nucleotide analogue from the compound shown in formula (I) above in this application; and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides.

[0188] In some embodiments, the antisense strand of the double-stranded ribonucleic acid modification includes the following modification along the direction from the 5' end to the 3' end: the ribonucleotides at positions 2, 8, 9, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; optionally, sequence B of the antisense strand may have one or more nucleotide derivative modifications, such as GNA or nucleotide analogs from the compound of formula (I) above described in this application, and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides. For example, along the 5' end to the 3' end, the antisense strand of the double-stranded ribonucleic acid modification includes the following modifications: the ribonucleotides at positions 2, 8, 9, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; the ribonucleotide at position 6 or 7 of sequence B of the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA or a nucleotide analogue from the compound shown in formula (I) above in this application; and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides.

[0189] In some embodiments, the antisense strand of the double-stranded ribonucleic acid modification includes phosphothioester bonds at the following positions along the 5' end to the 3' end: between the first and second nucleotides starting at the 5' end, between the second and third nucleotides starting at the 5' end, between the first and second nucleotides starting at the 3' end, and / or between the second and third nucleotides starting at the 3' end.

[0190] In some embodiments, the antisense strand of the double-stranded ribonucleic acid modification includes phosphothioester bonds at the following positions along the 5' end to the 3' end: between the first and second nucleotides starting at the 5' end, between the second and third nucleotides starting at the 5' end, between the first and second nucleotides starting at the 3' end, and between the second and third nucleotides starting at the 3' end.

[0191] In some embodiments, the antisense strand may also be modified with one or more nucleotide derivatives, such as GNA or a nucleotide analog from the compound represented by formula (I) above in this application, and the nucleotide derivative-modified nucleoside has a phosphodiester bond, phosphothioester bond, or amide bond with one or both of its adjacent nucleosides. For example, the ribonucleotide at position 6 or 7 of sequence B of the antisense strand may be a ribonucleotide modified with the nucleotide derivative GNA or a nucleotide analog from the compound represented by formula (I) above in this application, and may have a phosphothioester bond or amide bond with the nucleotide at position 5 or 6.

[0192] In some embodiments, the ribosome of the 5' terminal nucleotide of the antisense strand has a 5' hydroxyl group, a 5' phosphate ester group, or a 5' phosphate derivative group. The type of group at the 5' position of the ribosome depends on the preparation method used, which is known to those skilled in the art based on the corresponding preparation method.

[0193] In some embodiments, when the ribonucleotide at the 5' end of the antisense strand has a 5' phosphate ester group or a 5' phosphate-derived group, the following structure is formed:

[0194] , , Wherein, Base represents a nucleobase, such as A, U, G, C, or T; R' is independently a hydroxyl or hydrogen group or is substituted by various groups known to those skilled in the art, such as 2'-fluoro (2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, and 2'-deoxyribonucleotides.

[0195] Oligonucleotide modifications containing the nucleoside monomer of this application

[0196] In one aspect, this application provides modifications of oligonucleotides in which one or more nucleosides are replaced by nucleoside monomers represented by formula (II).

[0197] Equation (II),

[0198] Where X is selected from O or S;

[0199] R2 is selected from hydrogen, deuterium, or C. 1-3 alkyl;

[0200] Wherein, Base represents an unmodified or modified nucleobase, the unmodified or modified nucleobase having a structure selected from any of the following:

[0201] , , ,or ,

[0202] Among them, R4, R5, R6, R7, R8, R9, R 10 Each is independently selected from hydrogen, deuterium, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, 3-6 membered cycloalkyl, or -(C=O)C 1-6 alkyl.

[0203] In some embodiments, the oligonucleotide modification is a modification of double-stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand, each strand having 15-40 nucleotides; at least one nucleotide at positions 2-8 of the antisense strand is replaced by a nucleotide monomer of formula (II) in the 5'-3' direction. In some embodiments, nucleotides 1, 2, 3, or 4 at positions 2-8 of sequence B in the antisense strand are replaced by a nucleotide monomer of formula (II) in the 5'-3' direction.

[0204] In some embodiments, the oligonucleotide modification is a modification of double-stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand, each strand having 15-40 nucleotides; wherein, from the 5' end to the 3' end, at least one nucleotide at position 2-8 of the antisense strand is replaced by a nucleotide monomer of formula (II):

[0205] Equation (II),

[0206] Where X is selected from O or S;

[0207] R2 is selected from hydrogen, deuterium, or C. 1-3 alkyl;

[0208] Wherein, Base represents an unmodified or modified nucleobase, the unmodified or modified nucleobase having a structure selected from any of the following:

[0209] , , ,or ,

[0210] Among them, R4, R5, R6, R7, R8, R9, R 10 Each is independently selected from hydrogen, deuterium, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, 3-6 membered cycloalkyl, or -(C=O)C 1-6 alkyl.

[0211] In some implementations, X is preferably O.

[0212] In some embodiments, R2 is selected from hydrogen, deuterium, or methyl.

[0213] In some implementations, R2 is hydrogen.

[0214] In some implementations, R 10 Selected from hydrogen, deuterium, and C 1-6 Alkyl, C1-6 Alkoxy, C 1-6 Halogenated alkyl, 3-6 membered cycloalkyl, or -(C=O)C 1-6 alkyl.

[0215] In some implementations, R4, R5, R6, R7, R8, R9, R 10 Each is independently selected from hydrogen, deuterium, amino protecting groups, and C. 1-3 Alkyl, C 1-3 Alkoxy, C 1-3 Halogenated alkyl, 3-6 membered cycloalkyl, or -(C=O)C 1-3 alkyl.

[0216] In some implementations, R4, R5, R6, R7, R8, R9, R 10 Each is independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, methoxyethyl, cyclopropyl, fluoromethyl, difluoromethyl, or acetyl.

[0217] In some embodiments, R4 or R5 are each independently selected from hydrogen or C. 1-3 Alkyl group, preferably hydrogen or methyl. For example, both R4 and R5 are hydrogen; or R4 is hydrogen and R5 is methyl.

[0218] In some implementations, R6 and R7 are hydrogen.

[0219] In some implementations, R8 and R9 are hydrogen.

[0220] In a preferred embodiment, R 10 It can be hydrogen or methyl.

[0221] In some implementations, the Base is selected from any of the following:

[0222] , , or ,

[0223] R5, R7, or R9 are each independently selected from hydrogen or C. 1-3 Alkyl, R 10 Selected from hydrogen or C 1-6 Alkyl; preferably, R 10 Selected from hydrogen or C 1-3 Alkyl (e.g., methyl).

[0224] In some embodiments, the nucleoside monomer represented by formula (II) is selected from one or more of the following:

[0225] ;

[0226] Preferably, the nucleoside monomer represented by formula (II) is selected from one or more of the following:

[0227] .

[0228] In some embodiments, one, two, three, four, or five nucleoside residues at positions 2, 3, 4, 5, 6, 7, or 8 of the antisense strand are replaced by a nucleoside monomer of formula (II) in the direction from the 5' end to the 3' end. In some embodiments, one, two, three, four, or five nucleoside residues at positions 2, 3, 4, 5, 6, 7, or 8 of sequence B in the antisense strand are replaced by a nucleoside monomer of formula (II) in the direction from the 5' end to the 3' end.

[0229] In a specific implementation, the Base in the nucleoside monomer shown in formula (II) conforms to the Watson-Crick base complementary pairing principle with the nucleobases on the corresponding nucleotides of the sense strand.

[0230] In a specific embodiment, the nucleoside monomer represented by formula (II) has a phosphodiester bond or modification thereof with its adjacent nucleoside. The modification includes, but is not limited to, thiophosphate diester bonds, methylphosphonate bonds, borane phosphate bonds, phosphoramide diester bonds or amide bonds, such as amide bonds.

[0231] In some embodiments, the antisense strand of the double-stranded ribonucleic acid modification may also contain other modifications. In a preferred embodiment, all ribonucleotides in the antisense strand are modified ribonucleotides.

[0232] In some embodiments, the positive strand of the double-stranded ribonucleic acid modification also contains modifications. In a preferred embodiment, all ribonucleotides in the positive strand are modified ribonucleotides.

[0233] For information on oligonucleotides, double-stranded ribonucleotides, and their modifications, please refer to the description above.

[0234] In some embodiments, the oligonucleotides, double-stranded ribonucleotides, and their modifications or conjugates described herein are in the form of pharmaceutically acceptable salts. In specific embodiments, the salts described above are selected from base addition salts, acid addition salts, and combinations thereof.

[0235] In some embodiments, the base addition salt is selected from sodium, potassium, calcium, ammonium, organic amine, magnesium salts and combinations thereof, and the acid addition salt is selected from inorganic acid salts, organic acid salts and combinations thereof.

[0236] In some embodiments, the inorganic acid is selected from hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and combinations thereof, and the organic acid is selected from acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and combinations thereof.

[0237] In some embodiments, the pharmaceutically acceptable salt is selected from sodium salts.

[0238] For details regarding modifications to the justice chain and antithesis chain, please refer to the description above.

[0239] In some embodiments, the positive strand contains or is selected from 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides from the nucleotide sequences shown in any one of SEQ ID NOs: 1, 7, 16, 33, and 39 of this application. In some embodiments, the positive strand contains or is selected from 17, 18, 19, 20, or 21 consecutive nucleotides from the nucleotide sequences shown in any one of SEQ ID NOs: 1, 7, 16, 33, and 39 of this application. In some embodiments, the positive strand contains or is selected from 17, 18, or 19 consecutive nucleotides from the nucleotide sequences shown in any one of SEQ ID NOs: 1, 7, 16, 33, and 39 of this application.

[0240] In some embodiments, the antisense strand contains or is selected from 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides from the nucleotide sequences shown in any one of SEQ ID NOs: 2, 8, 17, 34, and 40 as described in this application. In some embodiments, the antisense strand contains or is selected from 19, 20, 21, 22, or 23 consecutive nucleotides from the nucleotide sequences shown in any one of SEQ ID NOs: 2, 8, 17, 34, and 40 as described in this application. In some embodiments, the antisense strand contains or is selected from 19, 20, or 21 consecutive nucleotides from the nucleotide sequences shown in any one of SEQ ID NOs: 2, 8, 17, 34, and 40 as described in this application.

[0241] In some embodiments, the positive strand or its contained nucleotide sequence differs from the sequence shown in any one of SEQ ID NO: 1, 7, 16, 33, and 39 of this application by 0, 1, 2, or 3 nucleotides. In some embodiments, the positive strand or its contained nucleotide sequence differs from the sequence shown in any one of SEQ ID NO: 1, 7, 16, 33, and 39 of this application by 0, 1, or 2 nucleotides. In some embodiments, the positive strand or its contained nucleotide sequence differs from the sequence shown in any one of SEQ ID NO: 1, 7, 16, 33, and 39 of this application by 0 or 1 nucleotide.

[0242] In some embodiments, the antisense strand or its contained nucleotide sequence differs from the sequence shown in any one of SEQ ID NO: 2, 8, 17, 34, and 40 of this application by 0, 1, 2, or 3 nucleotides. In some embodiments, the antisense strand or its contained nucleotide sequence differs from the sequence shown in any one of SEQ ID NO: 2, 8, 17, 34, and 40 of this application by 0, 1, or 2 nucleotides. In some embodiments, the antisense strand or its contained nucleotide sequence differs from the sequence shown in any one of SEQ ID NO: 2, 8, 17, 34, and 40 of this application by 0 or 1 nucleotide.

[0243] Double-stranded ribonucleic acid and its modifications for inhibiting CFB gene expression

[0244] A further aspect of this disclosure provides a double-stranded RNA (dsRNA) for repressing the expression of the CFB gene. One strand of this dsRNA is an antisense strand, which is complementary to the mRNA sequence formed during the expression of the target gene (i.e., the CFB gene) to guide the cleavage of the target mRNA (i.e., the transcript of the CFB gene). The sense strand of the dsRNA includes a sequence that is at least partially complementary (including completely complementary) to the antisense strand to form a double-stranded region of the dsRNA.

[0245] In some embodiments, the double-stranded RNA serves as a substrate for the endonuclease Dicer, and is cleaved into small fragments of dsRNA, i.e., siRNA. In some embodiments, the double-stranded RNA is siRNA. The siRNA assembles to form an RNA-induced silencing complex (RISC), which cleaves the target mRNA and inhibits the expression of the CFB gene.

[0246] Based on the target sequence derived from human CFB mRNA (NM_001710.6), design double-stranded ribonucleic acid (e.g., siRNA) that binds to the target mRNA.

[0247] In some embodiments, the double-stranded ribonucleic acid for inhibiting CFB comprises a sense strand and an antisense strand, wherein the sense strand comprises a sequence A that differs from the nucleotide sequence shown in SEQ ID NO: 1 by no more than 3 (e.g., no more than 2 or 1) nucleotides, and the antisense strand comprises a sequence B that differs from the nucleotide sequence shown in SEQ ID NO: 2 by no more than 3 (e.g., no more than 2 or 1) nucleotides; the sequences A and B are at least partially reverse complementary to form the double-stranded region of the modified double-stranded ribonucleic acid;

[0248] 5'-AUGGUGUGAAGCCAAGAUA-3' (SEQ ID NO: 1),

[0249] 5'-UAUCUUGGCUUCACACCAU-3' (SEQ ID NO: 2).

[0250] In a specific implementation, the sense strand contains a nucleotide sequence as shown in SEQ ID NO: 1 as sequence A, and the antisense strand contains a nucleotide sequence as shown in SEQ ID NO: 2 as sequence B.

[0251] In some embodiments, at least one nucleoside residue at positions 2-8 of sequence B in the antisense strand is replaced by a nucleoside monomer of formula (II). In some embodiments, one, two, or three nucleoside residues at positions 4, 5, 6, or 7 of sequence B in the antisense strand are replaced by a nucleoside monomer of formula (II). In some embodiments, a nucleoside residue at position 6 of sequence B in the antisense strand is replaced by a nucleoside monomer of formula (II).

[0252] In some embodiments, the nucleoside residue at position 6 or 7 of sequence B in the antisense strand is replaced by a nucleoside monomer as shown in formula (II).

[0253] In a preferred embodiment, the nucleotides of sequence A and sequence B in the antisense and sense strands of the double-stranded ribonucleic acid used to suppress CFB are both modified nucleotides.

[0254] In some embodiments, the nucleotides at positions 2, 7, 10, and 14 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, following the direction from the 5' end to the 3' end; optionally, each nucleotide at other positions of sequence B of the antisense strand is independently one of non-2'-fluoro-modified nucleotides.

[0255] In some embodiments, the nucleotides at positions 3, 7, 8, and 9 of sequence A of the positive strand are 2'-fluorinated nucleotides, following the direction from the 5' end to the 3' end; optionally, each nucleotide at other positions of sequence A of the positive strand is independently one of non-2'-fluorinated nucleotides.

[0256] In another specific embodiment, the antisense strand may optionally contain the nucleotide sequence E.

[0257] Preferably, the antisense chain includes EVP modification, etc.

[0258] In a specific embodiment, following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 7, 10, and 14 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy-modified nucleotide; or,

[0259] Following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 7, 10, and 14 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, the nucleotide at position 6 of sequence B of the antisense strand is GNA-modified, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy-modified nucleotide; or

[0260] In the direction from the 5' end to the 3' end, the nucleotides at positions 2, 7, 10, and 14 of sequence B of the antisense strand are 2'-fluorinated nucleotides, the nucleoside residue at position 6 of sequence B of the antisense strand is replaced by a nucleoside monomer as shown in formula (II), and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxylated nucleotide. Optionally, the nucleosides at positions 5 and 6 of sequence B of the antisense strand are linked by an amide bond.

[0261] In a specific embodiment, the antisense strand in the double-stranded ribonucleic acid modification for inhibiting CFB may have a structure selected from any of the following sequences:

[0262] 1) 5'-EVPusAfsucuuGfgcUfucaCfaccausTsT-3' (SEQ ID NO: 3);

[0263] 2) 5'-EVPusAfsucu U GfgcUfucaCfaccausTsT-3' (SEQ ID NO: 4); or

[0264] 3) 5'-EVPusAfsucuU NP1 GfgcUfucaCfaccausTsT-3' (SEQ ID NO: 5);

[0265] In this context, lowercase letters a, u, c, and g represent nucleotides modified with 2'-methoxy groups; Af, Uf, Cf, and Gf represent nucleotides modified with 2'-fluoride groups; and EVP indicates that the nucleotide adjacent to the right of this letter combination is a 5'-trans-vinylphosphonate nucleotide. U (Bold, italic, underlined): indicates uracil nucleotides modified with GNA; 's' indicates that two adjacent nucleotides are linked by a phosphothioester bond; 'T' indicates: thymine deoxyribonucleotide; U NP1 express: .

[0266] In a specific implementation, the nucleotides at positions 3, 7, 8, and 9 of sequence A of the positive strand are 2'-fluorinated nucleotides, following the direction from the 5' end to the 3' end.

[0267] In a specific embodiment, the nucleotides at positions 3, 7, 8, and 9 of sequence A of the positive strand are 2'-fluoro-modified nucleotides, and each nucleotide at other positions of sequence A of the positive strand is a 2'-methoxy-modified nucleotide.

[0268] In some embodiments, the justice chain includes one or more (invAbs), preferably one or two. In some embodiments, (invAbs) are located at the 5' end and / or the 3' end of the justice chain. For example, (invAbs) are located at the 5' end of the justice chain; or, (invAbs) are located at the 3' end of the justice chain; or, (invAbs) are located at both the 5' end and the 3' end of the justice chain, wherein each of the (invAbs) is independently one or two (invAbs), preferably one (invAb).

[0269] In a preferred embodiment, one or more (invAb) are added to the 5' end and / or 3' end of the justice chain, for example, one or two. In some embodiments, one (invAb) is added to the 5' end of the justice chain. In some embodiments, one (invAb) is added to the 3' end of the justice chain. In some embodiments, one (invAb) is added to both the 5' end and the 3' end of the justice chain.

[0270] In some embodiments, the positive chain contains phosphothioester bonds located at the positions described below, along the direction from the 5' end to the 3' end:

[0271] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0272] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0273] Between the first and second nucleotides starting at the 3' end of the positive strand;

[0274] Between the second and third nucleotides starting at the 3' end of the positive strand;

[0275] Between the reverse debased deoxyribose residue starting at the 5' end of the positive strand and the first nucleotide at the 5' end of the positive strand; and / or

[0276] The reverse debased deoxyribose residue starting at the 3' end of the positive strand is between the first nucleotide at the 3' end of the positive strand.

[0277] Preferably, the positive chain comprises a phosphothioester bond located at the following positions: between the reverse debased deoxyribose residue starting at the 5' end of the positive chain and the first nucleotide, between the first nucleotide and the second nucleotide, and between the reverse debased deoxyribose residue starting at the 3' end and the first nucleotide.

[0278] Preferably, the positive chain comprises a phosphothioester bond located at the following positions: between the first and second nucleotides starting at the 5' end of the positive chain, between the second and third nucleotides, and between the reverse debased deoxyribose residue starting at the 3' end and the first nucleotide.

[0279] Preferably, the positive chain comprises a phosphothioester bond located at the following positions: between the reverse debased deoxyribose residue starting at the 5' end of the positive chain and the first nucleotide, and between the first nucleotide and the second nucleotide.

[0280] In a specific embodiment, the sense strand in the modified double-stranded ribonucleic acid may have the following sequence:

[0281] 5'-asusGfgugUfGfAfagccaagauas(invAb)-3' (SEQ ID NO: 6);

[0282] Wherein, lowercase letters a, u, c, g represent: 2'-methoxy modified nucleotides; Af, Uf, Cf, Gf represent 2'-fluorinated modified nucleotides; s represents two adjacent nucleotides, and the nucleotide is linked to (invAb) by a phosphothioester bond; (invAb) represents a reverse debased deoxyribose residue; optionally, if the 5' end of the positive strand does not have a reverse debased deoxyribose residue, the ribosome of the nucleotide has a 5' hydroxyl group, preferably having a 5' hydroxyl group.

[0283] In some embodiments, this disclosure provides a kit comprising: a) the double-stranded ribonucleic acid, its modifier, its conjugate, or a pharmaceutical composition thereof as described in this disclosure; and b) the following instructions for use:

[0284] (1) Inhibit CFB gene expression;

[0285] (2) Used for the prevention or treatment of diseases, symptoms or disorders associated with abnormal expression of the CFB gene;

[0286] (3) For the treatment of subjects with diseases that would benefit from reduced CFB gene expression.

[0287] This disclosure provides for at least one use of the modified double-stranded ribonucleic acid, or its conjugates, or pharmaceutical compositions thereof described herein:

[0288] (1) Inhibit CFB gene expression, or prepare drugs for inhibiting CFB gene expression;

[0289] (2) To prevent or treat diseases, symptoms or disorders related to abnormal expression of the CFB gene, or to prepare drugs for the prevention or treatment of diseases, symptoms or disorders related to abnormal expression of the CFB gene.

[0290] (3) To treat subjects with diseases that would benefit from reduced CFB gene expression, or to prepare a medicine for treating subjects with diseases, conditions or disorders that would benefit from reduced CFB gene expression.

[0291] This disclosure further provides the use of siRNA molecules (including modified siRNAs, siRNA conjugates) or pharmaceutical compositions thereof in at least one of (1)-(3) above.

[0292] In this disclosure, "subject" includes humans or non-human animals, preferably vertebrates, and more preferably mammals. Subjects may include transgenic organisms. Most preferably, the subject is a human. Further, the subject has at least one of the following characteristics:

[0293] (1) Abnormal expression of CFB gene in vivo, more specifically, abnormally high expression of CFB gene;

[0294] (2) Suffering from a disease, condition or disorder associated with abnormal expression of the CFB gene;

[0295] (3) People who have a disease that would benefit from reduced CFB gene expression. Such as people who have or are prone to diseases, conditions or disorders associated with abnormal CFB gene expression.

[0296] In this article, “diseases, conditions or disorders associated with abnormal expression of the CFB gene” include, without limitation, primary membranous nephropathy, hemolytic uremic syndrome, C3 glomerulonephropathy, IgA nephropathy, paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration, and ophthalmic diseases.

[0297] Double-stranded ribonucleic acid and its modifications for inhibiting SCAP gene expression

[0298] A further aspect of this disclosure provides a double-stranded RNA (dsRNA) for repressing the expression of the SCAP gene. One strand of this dsRNA is an antisense strand, which is complementary to the mRNA sequence formed during the expression of the target gene (i.e., the SCAP gene) to guide the cleavage of the target mRNA (i.e., the transcript of the SCAP gene). The sense strand of the dsRNA includes a sequence that is at least partially complementary (including completely complementary) to the antisense strand to form a double-stranded region of the dsRNA.

[0299] In some embodiments, the double-stranded RNA serves as a substrate for the endonuclease Dicer, and is cleaved into small fragments of dsRNA, i.e., siRNA. In some embodiments, the double-stranded RNA is siRNA. The siRNA assembles to form an RNA-induced silencing complex (RISC), which cleaves the target mRNA and inhibits the expression of the SCAP gene.

[0300] Based on the target sequence derived from human SCAP mRNA (NM_012235.4), design double-stranded ribonucleic acid (e.g., siRNA) that binds to the target mRNA.

[0301] In some embodiments, the double-stranded ribonucleic acid for inhibiting SCAP comprises a sense strand and an antisense strand, wherein the sense strand comprises sequence A which differs from the nucleotide sequence shown in SEQ ID NO: 7 by no more than 3 (e.g., no more than 2 or 1) nucleotides, and the antisense strand comprises sequence B which differs from the nucleotide sequence shown in SEQ ID NO: 8 by no more than 3 (e.g., no more than 2 or 1) nucleotides; the sequences A and B are at least partially reverse complementary to form the double-stranded region of the modified double-stranded ribonucleic acid;

[0302] 5'-UCUCCUUUUGGGACCUAAA-3' (SEQ ID NO: 7),

[0303] 5'-UUUAGGUCCCAAAAGGAGA-3' (SEQ ID NO: 8).

[0304] In a specific implementation, the sense strand contains a nucleotide sequence as shown in SEQ ID NO: 7 as sequence A, and the antisense strand contains a nucleotide sequence as shown in SEQ ID NO: 8 as sequence B.

[0305] In some embodiments, at least one nucleoside residue at positions 2-8 of sequence B in the antisense strand is replaced by a nucleoside monomer of formula (II). In some embodiments, one, two, or three nucleoside residues at positions 4, 5, 6, or 7 of sequence B in the antisense strand are replaced by a nucleoside monomer of formula (II). In some embodiments, a nucleoside residue at position 6 or 7 of sequence B in the antisense strand is replaced by a nucleoside monomer of formula (II).

[0306] In some embodiments, the nucleoside residue at position 6 or 7 of sequence B in the antisense strand is replaced by a nucleoside monomer as shown in formula (II).

[0307] In a preferred embodiment, the nucleotides of sequence A and sequence B in the antisense and sense strands of the double-stranded ribonucleic acid used to suppress SCAP are both modified nucleotides.

[0308] In some embodiments, at least three sites (e.g., 3-7, such as 3, 4, 5, 6, or 7) of the nucleotides at positions 2, 6, 7, 10, 14, and 16 of sequence B of the antisense strand are 2'-fluorinated nucleotides, in the direction from the 5' end to the 3' end, and each nucleotide at the other positions of sequence B of the antisense strand is independently one of non-2'-fluorinated nucleotides.

[0309] In some embodiments, at least three sites (e.g., 3-6, such as 3, 4, 5, or 6) of the 3rd, 5th, 7th, 8th, 9th, and 11th positions of the sequence A of the positive strand are 2'-fluorinated nucleotides, in the direction from the 5' end to the 3' end, and each nucleotide at the other positions of the sequence A of the positive strand is independently one of the non-2'-fluorinated nucleotides.

[0310] In another specific embodiment, the antisense strand may optionally contain the nucleotide sequence E.

[0311] Preferably, the antisense chain includes EVP modification, etc.

[0312] In a specific embodiment, the nucleotides at positions 2, 7, 10, and 14 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy-modified nucleotide.

[0313] In a specific embodiment, following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy-modified nucleotide; or,

[0314] In the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, the nucleotide at position 7 of sequence B of the antisense strand is GNA-modified, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy-modified nucleotide. Optionally, the nucleotides at positions 6 and 7 of sequence B of the antisense strand are linked by an amide bond; or

[0315] In the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14 and 16 of sequence B of the antisense strand are 2'-fluorinated nucleotides, the nucleoside residue at position 7 of sequence B of the antisense strand is replaced by the nucleoside monomer shown in formula (II), and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxylated nucleotide.

[0316] In a specific embodiment, following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 10, and 14 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy-modified nucleotide; or,

[0317] In the direction from the 5' end to the 3' end, the nucleotides at positions 2, 10, and 14 of sequence B of the antisense strand are 2'-fluorinated nucleotides, the nucleoside residue at position 7 of sequence B of the antisense strand is replaced by a nucleoside monomer as shown in formula (II), and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxylated nucleotide. Optionally, the nucleosides at positions 6 and 7 of sequence B of the antisense strand are linked by an amide bond.

[0318] In a specific implementation, the antisense strand in the modified double-stranded ribonucleic acid used to suppress SCAP may have any of the following sequences:

[0319] 1) 5'-EVPusUfsuaggUfccCfaaaAfggagasTsT-3' (SEQ ID NO: 9);

[0320] 2) 5'-EVPusUfsuagGf U cccaaaAfgGfagasTsT-3' (SEQ ID NO: 10);

[0321] 3) 5'-EVPusUfsuagGfU NP1 cccaaaAfgGfagasTsT-3' (SEQ ID NO: 11); or

[0322] 4) 5'-EVPusUfsuaggU NP1 ccCfaaaAfggagasTsT-3' (SEQ ID NO: 12);

[0323] In this context, lowercase letters a, u, c, and g represent nucleotides modified with 2'-methoxy groups; Af, Uf, Cf, and Gf represent nucleotides modified with 2'-fluoride groups; and EVP indicates that the nucleotide adjacent to the right of this letter combination is a 5'-trans-vinylphosphonate nucleotide. U (Bold, italic, underlined): indicates uracil nucleotides modified with GNA; 's' indicates that two adjacent nucleotides are linked by a phosphothioester bond; 'T' indicates: thymine deoxyribonucleotide; U NP1 express: .

[0324] In a specific embodiment, the nucleotides at positions 3, 7, 9, and 11 of sequence A of the positive strand are 2'-fluoro-modified nucleotides, following the direction from the 5' end to the 3' end, and each nucleotide at other positions of sequence A of the positive strand is a 2'-methoxy-modified nucleotide.

[0325] In a specific embodiment, the nucleotides at positions 3, 7, 8, and 9 of sequence A of the positive strand are 2'-fluoro-modified nucleotides, and each nucleotide at other positions of sequence A of the positive strand is a 2'-methoxy-modified nucleotide.

[0326] In a specific embodiment, the nucleotides at positions 5, 7, 8, and 9 of sequence A of the positive strand are 2'-fluoro-modified nucleotides, following the direction from the 5' end to the 3' end, and each nucleotide at other positions of sequence A of the positive strand is a 2'-methoxy-modified nucleotide.

[0327] In some embodiments, the justice chain includes one or more (invAbs), preferably one or two. In some embodiments, (invAbs) are located at the 5' end and / or the 3' end of the justice chain. For example, (invAbs) are located at the 5' end of the justice chain; or, (invAbs) are located at the 3' end of the justice chain; or, (invAbs) are located at both the 5' end and the 3' end of the justice chain, wherein each of the (invAbs) is independently one or two (invAbs), preferably one (invAb).

[0328] In a preferred embodiment, one or more (invAb) are added at the 5' and / or 3' end of the justice chain, for example, one or two. In some embodiments, one (invAb) is added at the 5' end of the justice chain. In some embodiments, one (invAb) is added at the 3' end of the justice chain. In some embodiments, one (invAb) is added at both the 5' end and the 3' end of the justice chain.

[0329] In some embodiments, the positive chain contains phosphothioester bonds located at the positions described below, along the direction from the 5' end to the 3' end:

[0330] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0331] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0332] Between the first and second nucleotides starting at the 3' end of the positive strand;

[0333] Between the second and third nucleotides starting at the 3' end of the positive strand;

[0334] Between the reverse debased deoxyribose residue starting at the 5' end of the positive strand and the first nucleotide at the 5' end of the positive strand; and / or

[0335] The reverse debased deoxyribose residue starting at the 3' end of the positive strand is between the first nucleotide at the 3' end of the positive strand.

[0336] Preferably, the positive chain comprises a phosphothioester bond located at the following positions: between the reverse debased deoxyribose residue starting at the 5' end of the positive chain and the first nucleotide, between the first nucleotide and the second nucleotide, and between the reverse debased deoxyribose residue starting at the 3' end and the first nucleotide.

[0337] Preferably, the positive chain comprises a phosphothioester bond located at the following positions: between the first and second nucleotides starting at the 5' end of the positive chain, between the second and third nucleotides, and between the reverse debased deoxyribose residue starting at the 3' end and the first nucleotide.

[0338] Preferably, the positive chain comprises a phosphothioester bond located at the following positions: between the reverse debased deoxyribose residue starting at the 5' end of the positive chain and the first nucleotide, and between the first nucleotide and the second nucleotide.

[0339] In a specific embodiment, the sense strand in the modified double-stranded ribonucleic acid may have a structure selected from any of the following sequences:

[0340] 1)5'-uscsUfccuUfuUfgGfgaccuaaa-3' (SEQ ID NO: 13);

[0341] 2)5'- uscsUfccuUfUfUfgggaccuaaas(invAb)-3' (SEQ ID NO: 14);

[0342] 3)5'- uscsucCfuUfUfUfgggaccuaaa-3' (SEQ ID NO: 15);

[0343] In this context, lowercase letters a, u, c, and g represent: nucleotides modified with a 2'-methoxy group; Af, Uf, Cf, and Gf represent nucleotides modified with a 2'-fluoride group; s represents two adjacent nucleotides connected by a phosphothioester bond; and (invAb) represents a reverse debasing deoxyribose residue. Optionally, if the 5' end of the positive strand does not have a reverse debasing deoxyribose residue, the ribosome of the nucleotide has a 5' hydroxyl group, preferably a 5' hydroxyl group.

[0344] In some embodiments, this disclosure provides a kit comprising: a) the double-stranded ribonucleic acid, its modifier, its conjugate, or a pharmaceutical composition thereof as described in this disclosure; and b) the following instructions for use:

[0345] (1) Inhibit SCAP gene expression;

[0346] (2) Used for the prevention or treatment of diseases, conditions or disorders associated with abnormal expression of the SCAP gene;

[0347] (3) For the treatment of subjects with diseases that would benefit from reduced SCAP gene expression.

[0348] This disclosure provides for at least one use of the modified double-stranded ribonucleic acid, or its conjugates, or pharmaceutical compositions thereof described herein:

[0349] (1) Inhibit SCAP gene expression, or prepare drugs for inhibiting SCAP gene expression;

[0350] (2) To prevent or treat diseases, symptoms or disorders associated with abnormal expression of the SCAP gene, or to prepare drugs for the prevention or treatment of diseases, symptoms or disorders associated with abnormal expression of the SCAP gene.

[0351] (3) To treat subjects with diseases that would benefit from reduced SCAP gene expression, or to prepare a medicine for treating subjects with diseases, conditions or disorders that would benefit from reduced SCAP gene expression.

[0352] This disclosure further provides the use of siRNA molecules (including siRNA modifiers, siRNA conjugates) or pharmaceutical compositions thereof in at least one of (1)-(3) above.

[0353] In this disclosure, "subject" includes humans or non-human animals, preferably vertebrates, and more preferably mammals. Subjects may include transgenic organisms. Most preferably, the subject is a human. Further, the subject has at least one of the following characteristics:

[0354] (1) Abnormal expression of SCAP gene in vivo, more specifically, abnormally high expression of SCAP gene;

[0355] (2) Suffering from a disease, condition or disorder associated with abnormal expression of the SCAP gene;

[0356] (3) People who have a disease that would benefit from reduced SCAP gene expression. Such as people who have or are predisposed to diseases, conditions or disorders associated with abnormal SCAP gene expression.

[0357] In this article, “diseases, conditions or disorders associated with abnormal expression of the SCAP gene” include, without limitation, esophageal cancer, hyperlipidemia, obesity, fatty liver diseases such as non-alcoholic fatty liver disease (NAFLD), and liver fibrosis.

[0358] Double-stranded ribonucleic acid and its modifications for inhibiting INHBE gene expression

[0359] A further aspect of this disclosure provides a double-stranded RNA (dsRNA) for inhibiting the expression of the INHBE gene. One strand of this dsRNA is an antisense strand, which is complementary to the mRNA sequence formed during the expression of the target gene (i.e., the INHBE gene) to guide the cleavage of the target mRNA (i.e., the transcript of the INHBE gene). The sense strand of the dsRNA includes a sequence that is at least partially complementary (including completely complementary) to the antisense strand to form a double-stranded region of the dsRNA.

[0360] In some embodiments, the double-stranded ribonucleic acid serves as a substrate for the endonuclease Dicer, and is cleaved into small fragments of dsRNA, i.e., siRNA. In some embodiments, the double-stranded ribonucleic acid is siRNA. The siRNA assembles to form an RNA-induced silencing complex (RISC), which cleaves the target mRNA and inhibits the expression of the INHBE gene.

[0361] Based on the target sequence derived from human INHBE mRNA (NM_031479.5), a double-stranded ribonucleic acid (e.g., siRNA) is designed to bind to the target mRNA.

[0362] In some embodiments, the double-stranded ribonucleic acid for inhibiting INHBE comprises a sense strand and an antisense strand, wherein the sense strand comprises sequence A, which differs from the nucleotide sequence shown in SEQ ID NO: 16 by no more than 3 (e.g., no more than 2 or 1) nucleotides, and the antisense strand comprises sequence B, which differs from the nucleotide sequence shown in SEQ ID NO: 17 by no more than 3 (e.g., no more than 2 or 1) nucleotides; the sequences A and B are at least partially reverse complementary to form the double-stranded region of the modified double-stranded ribonucleic acid;

[0363] 5'-CAGCUUUGCUACUGUCACU-3' (SEQ ID NO: 16),

[0364] 5'- AGUGACAGUAGCAAAGCUGAG-3' (SEQ ID NO: 17).

[0365] In a specific implementation, the sense strand contains a nucleotide sequence as shown in SEQ ID NO: 16 as sequence A, and the antisense strand contains a nucleotide sequence as shown in SEQ ID NO: 17 as sequence B.

[0366] In some embodiments, at least one nucleoside residue at positions 2-8 of sequence B in the antisense strand is replaced by a nucleoside monomer of formula (II). In some embodiments, one, two, or three nucleoside residues at positions 4, 5, 6, or 7 of sequence B in the antisense strand are replaced by a nucleoside monomer of formula (II). In some embodiments, a nucleoside residue at position 6 or 7 of sequence B in the antisense strand is replaced by a nucleoside monomer of formula (II).

[0367] In some embodiments, the nucleoside residue at position 6 or 7 of sequence B in the antisense strand is replaced by a nucleoside monomer as shown in formula (II).

[0368] In a preferred embodiment, the nucleotides of sequence A and sequence B in the antisense and sense strands of the double-stranded ribonucleic acid used to suppress INHBE are both modified nucleotides.

[0369] In some embodiments, at least three sites (e.g., 3-7, such as 3, 4, 5, 6, or 7) of the nucleotides at positions 2, 6, 7, 10, 14, and 16 of sequence B of the antisense strand are 2'-fluorinated nucleotides, in the direction from the 5' end to the 3' end, and each nucleotide at the other positions of sequence B of the antisense strand is independently one of non-2'-fluorinated nucleotides.

[0370] In some embodiments, at least three sites (e.g., 3-6, such as 3, 4, 5, or 6) of the 3rd, 5th, 7th, 8th, and 9th positions of the sequence A of the positive strand are 2'-fluorinated nucleotides, and each nucleotide at the other positions of the sequence A of the positive strand is independently one of non-2'-fluorinated nucleotides.

[0371] In another specific embodiment, the antisense strand may optionally contain the nucleotide sequence E.

[0372] Preferably, the antisense chain includes EVP modification, etc.

[0373] In a specific embodiment, the nucleotides at positions 2, 6, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy-modified nucleotide.

[0374] In a specific embodiment, following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy-modified nucleotide; or,

[0375] Following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, the nucleotide at position 6 of sequence B of the antisense strand is GNA-modified, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy-modified nucleotide. Optionally, the nucleotides at positions 5 and 6 of sequence B of the antisense strand are linked by an amide bond; or

[0376] In the direction from the 5' end to the 3' end, the nucleotides at positions 2, 14, and 16 of sequence B of the antisense strand are 2'-fluorinated nucleotides, the nucleoside residue at position 6 of sequence B of the antisense strand is replaced by a nucleoside monomer as shown in formula (II), and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxylated nucleotide. Optionally, the nucleosides at positions 5 and 6 of sequence B of the antisense strand are linked by an amide bond.

[0377] In a specific embodiment, following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 7, 10, and 14 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy-modified nucleotide; or,

[0378] In the direction from the 5' end to the 3' end, the nucleotides at positions 2, 7, 10, and 14 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, the nucleotide at position 6 of sequence B of the antisense strand is GNA-modified, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy-modified nucleotide. Optionally, the nucleotides at positions 5 and 6 of sequence B of the antisense strand are linked by an amide bond; or

[0379] In the direction from the 5' end to the 3' end, the nucleotides at positions 2, 7, 10, and 14 of sequence B of the antisense strand are 2'-fluorinated nucleotides, the nucleoside residue at position 6 of sequence B of the antisense strand is replaced by a nucleoside monomer as shown in formula (II), and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxylated nucleotide. Optionally, the nucleosides at positions 5 and 6 of sequence B of the antisense strand are linked by an amide bond.

[0380] In a specific implementation, the antisense strand in the modified double-stranded ribonucleic acid used to suppress INHBE may have a structure selected from any of the following sequences:

[0381] 1) 5'-EVPasGfsugaCfaguagcaAfaGfcugsasg-3' (SEQ ID NO: 18);

[0382] 2) 5'-EVPasGfsuga C aguagcaAfaGfcugsasg -3' (SEQ ID NO: 19);

[0383] 3) 5'-EVPasGfsugaC NP1 aguagcaAfaGfcugsasg -3' (SEQ ID NO: 20);

[0384] 4) 5'-EVPasGfsugacAfguAfgcaAfagcugsasg-3' (SEQ ID NO: 21);

[0385] 5) 5'- EVPasGfsuga C AfguAfgcaAfagcugsasg-3' (SEQ ID NO: 22); or

[0386] 6) 5'-EVPasGfsugaC NP1 AfguAfgcaAfagcugsasg-3' (SEQ ID NO: 23);

[0387] In this context, lowercase letters a, u, c, and g represent nucleotides modified with 2'-methoxy groups; Af, Uf, Cf, and Gf represent nucleotides modified with 2'-fluoride groups; and EVP indicates that the nucleotide adjacent to the right of this letter combination is a 5'-trans-vinylphosphonate nucleotide. C (Bold, italic, underlined): indicates a cytosine nucleotide modified with GNA; 's' indicates that two adjacent nucleotides are linked by a phosphothioester bond; C NP1 express: .

[0388] In a specific embodiment, the nucleotides at positions 3, 7, 8, and 9 of sequence A of the positive strand are 2'-fluoro-modified nucleotides, and each nucleotide at other positions of sequence A of the positive strand is a 2'-methoxy-modified nucleotide.

[0389] In a specific embodiment, the nucleotides at positions 5, 7, 8, and 9 of sequence A of the positive strand are 2'-fluoro-modified nucleotides, following the direction from the 5' end to the 3' end, and each nucleotide at other positions of sequence A of the positive strand is a 2'-methoxy-modified nucleotide.

[0390] In some embodiments, the justice chain includes one or more (invAbs), preferably one or two. In some embodiments, (invAbs) are located at the 5' end and / or the 3' end of the justice chain. For example, (invAbs) are located at the 5' end of the justice chain; or, (invAbs) are located at the 3' end of the justice chain; or, (invAbs) are located at both the 5' end and the 3' end of the justice chain, wherein each of the (invAbs) is independently one or two (invAbs), preferably one (invAb).

[0391] In a preferred embodiment, one or more (invAb) are added at the 5' end and / or the 3' end of the justice chain, for example, one or two. In some embodiments, one (invAb) is added at the 5' end of the justice chain. In some embodiments, one (invAb) is added at the 3' end of the justice chain. In some embodiments, one (invAb) is added at both the 5' end and the 3' end of the justice chain.

[0392] In some embodiments, the positive chain contains phosphothioester bonds located at the positions described below, along the direction from the 5' end to the 3' end:

[0393] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0394] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0395] Between the first and second nucleotides starting at the 3' end of the positive strand;

[0396] Between the second and third nucleotides starting at the 3' end of the positive strand;

[0397] Between the reverse debased deoxyribose residue starting at the 5' end of the positive strand and the first nucleotide at the 5' end of the positive strand; and / or

[0398] The reverse debased deoxyribose residue starting at the 3' end of the positive strand is between the first nucleotide at the 3' end of the positive strand.

[0399] Preferably, the positive chain comprises a phosphothioester bond located at the following positions: between the reverse debased deoxyribose residue starting at the 5' end of the positive chain and the first nucleotide, between the first nucleotide and the second nucleotide, and between the reverse debased deoxyribose residue starting at the 3' end and the first nucleotide.

[0400] Preferably, the positive chain comprises a phosphothioester bond located at the following positions: between the first and second nucleotides starting at the 5' end of the positive chain, between the second and third nucleotides, and between the reverse debased deoxyribose residue starting at the 3' end and the first nucleotide.

[0401] Preferably, the positive chain comprises a phosphothioester bond located at the following positions: between the reverse debased deoxyribose residue starting at the 5' end of the positive chain and the first nucleotide, and between the first nucleotide and the second nucleotide.

[0402] In a specific embodiment, the sense strand in the modified double-stranded ribonucleic acid may have a structure selected from any of the following sequences:

[0403] 1)5'-csasgcUfuUfGfCfuacugucacu-3' (SEQ ID NO: 24);

[0404] 2)5'-csasGfcuuUfGfCfuacugucacus(invAb)-3' (SEQ ID NO: 25);

[0405] In this context, lowercase letters a, u, c, and g represent: nucleotides modified with a 2'-methoxy group; Af, Uf, Cf, and Gf represent nucleotides modified with a 2'-fluoride group; s represents two adjacent nucleotides connected by a phosphothioester bond; and (invAb) represents a reverse debasing deoxyribose residue. Optionally, if the 5' end of the positive strand does not have a reverse debasing deoxyribose residue, the ribosome of the nucleotide has a 5' hydroxyl group, preferably a 5' hydroxyl group.

[0406] In some embodiments, this disclosure provides a kit comprising: a) the double-stranded ribonucleic acid, its modifier, its conjugate, or a pharmaceutical composition thereof as described in this disclosure; and b) the following instructions for use:

[0407] (1) Inhibit INHBE gene expression;

[0408] (2) Used for the prevention or treatment of diseases, symptoms or disorders associated with abnormal expression of the INHBE gene;

[0409] (3) Used to treat subjects with diseases that would benefit from reduced INHBE gene expression.

[0410] This disclosure provides for at least one use of the modified double-stranded ribonucleic acid, or its conjugates, or pharmaceutical compositions thereof described herein:

[0411] (1) Inhibit INHBE gene expression, or prepare drugs for inhibiting INHBE gene expression;

[0412] (2) To prevent or treat diseases, symptoms or disorders associated with abnormal expression of the INHBE gene, or to prepare drugs for the prevention or treatment of diseases, symptoms or disorders associated with abnormal expression of the INHBE gene.

[0413] (3) To treat subjects with diseases that would benefit from reduced INHBE gene expression, or to prepare a medicine for treating subjects with diseases, conditions or disorders that would benefit from reduced INHBE gene expression.

[0414] This disclosure further provides the use of siRNA molecules (including siRNA modifiers, siRNA conjugates) or pharmaceutical compositions thereof in at least one of (1)-(3) above.

[0415] In this disclosure, "subject" includes humans or non-human animals, preferably vertebrates, and more preferably mammals. Subjects may include transgenic organisms. Most preferably, the subject is a human. Further, the subject has at least one of the following characteristics:

[0416] (1) Abnormal expression of the INHBE gene in vivo, more specifically, abnormally high expression of the INHBE gene;

[0417] (2) Suffering from a disease, condition or disorder associated with abnormal expression of the INHBE gene;

[0418] (3) People who have a disease that would benefit from reduced INHBE gene expression. Such as people who have or are predisposed to diseases, conditions or disorders associated with abnormal INHBE gene expression.

[0419] In this article, “diseases, conditions or disorders associated with abnormal expression of the INHBE gene” include, without limitation, obesity (such as abdominal obesity), non-alcoholic fatty liver disease, alcoholic fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, liver fibrosis, elevated liver enzyme levels (ALT, AST, ALP), hepatocellular carcinoma, hypercholesterolemia and related cardiovascular diseases (such as coronary heart disease), insulin resistance, impaired glucose tolerance, hyperglycemia, type II diabetes, and metabolic syndrome.

[0420] Double-stranded RNA and its modifications for inhibiting C5 gene expression

[0421] A further aspect of this disclosure provides a double-stranded RNA (dsRNA) for repressing the expression of the C5 gene. One strand of this dsRNA is an antisense strand, which is complementary to the mRNA sequence formed during the expression of the target gene (i.e., the C5 gene) to guide the cleavage of the target mRNA (i.e., the transcript of the C5 gene). The sense strand of the dsRNA includes a sequence that is at least partially complementary (including completely complementary) to the antisense strand to form a double-stranded region of the dsRNA.

[0422] In some embodiments, the double-stranded RNA serves as a substrate for the endonuclease Dicer, and is cleaved into small fragments of dsRNA, i.e., siRNA. In some embodiments, the double-stranded RNA is siRNA. The siRNA assembles to form an RNA-induced silencing complex (RISC), which cleaves the target mRNA and inhibits the expression of the C5 gene.

[0423] Based on the target sequence derived from human C5 mRNA (NM_001735.3), design double-stranded ribonucleic acid (e.g., siRNA) that binds to the target mRNA.

[0424] In some embodiments, the double-stranded ribonucleic acid for inhibiting C5 comprises a sense strand and an antisense strand, wherein the sense strand comprises sequence A, which differs from the nucleotide sequence shown in SEQ ID NO: 33 by no more than 3 (e.g., no more than 2 or 1) nucleotides, and the antisense strand comprises sequence B, which differs from the nucleotide sequence shown in SEQ ID NO: 34 by no more than 3 (e.g., no more than 2 or 1) nucleotides; the sequences A and B are at least partially reverse complementary to form the double-stranded region of the modified double-stranded ribonucleic acid;

[0425] 5'-AGUCAAAGAACUGUCAUACUA-3' (SEQ ID NO: 33),

[0426] 5'-UAGUAUGACAGUUCUUUGACUGC-3' (SEQ ID NO: 34).

[0427] In a specific implementation, the sense strand contains a nucleotide sequence as shown in SEQ ID NO: 33 as sequence A, and the antisense strand contains a nucleotide sequence as shown in SEQ ID NO: 34 as sequence B.

[0428] In some embodiments, at least one nucleoside residue at positions 2-8 of sequence B in the antisense strand is replaced by a nucleoside monomer of formula (II). In some embodiments, one, two, or three nucleoside residues at positions 4, 5, 6, or 7 of sequence B in the antisense strand are replaced by a nucleoside monomer of formula (II). In some embodiments, a nucleoside residue at position 6 or 7 of sequence B in the antisense strand is replaced by a nucleoside monomer of formula (II).

[0429] In some embodiments, the nucleoside residue at position 6 or 7 of sequence B in the antisense strand is replaced by a nucleoside monomer as shown in formula (II).

[0430] In a preferred embodiment, the nucleotides of sequence A and sequence B in the antisense and sense strands of the double-stranded ribonucleic acid used to inhibit C5 are both modified nucleotides.

[0431] In some embodiments, at least three sites (e.g., 3-7, such as 3, 4, 5, 6, or 7) at positions 2, 6, 8, 9, 14, and 16 of sequence B of the antisense strand are 2'-fluorinated nucleotides, in the direction from the 5' end to the 3' end, and each nucleotide at the other positions of sequence B of the antisense strand is independently one of non-2'-fluorinated nucleotides.

[0432] In some embodiments, at least three sites (e.g., 3-6, such as 3, 4, 5, or 6) at positions 7, 9, 10, and 11 of sequence A of the positive strand are 2'-fluorinated nucleotides, with each nucleotide at the other positions of sequence A of the positive strand being independently one of non-2'-fluorinated nucleotides.

[0433] In another specific embodiment, the antisense strand may optionally contain the nucleotide sequence E.

[0434] Preferably, the antisense chain includes EVP modification, etc.

[0435] In a specific embodiment, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy-modified nucleotide.

[0436] In a specific embodiment, following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 8, 9, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy-modified nucleotide; or,

[0437] In the direction from the 5' end to the 3' end, the nucleotides at positions 2, 8, 9, 14 and 16 of sequence B of the antisense strand are 2'-fluorinated nucleotides, the nucleotide at position 6 of sequence B of the antisense strand is GNA modified or the nucleoside residue at position 6 of sequence B of the antisense strand is replaced by the nucleoside monomer shown in formula (II), and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy modified nucleotide. Optionally, the nucleosides at positions 5 and 6 of sequence B of the antisense strand are linked by an amide bond.

[0438] In a specific implementation, the antisense strand in the modified double-stranded ribonucleic acid used to suppress C5 may have a structure selected from any of the following sequences:

[0439] 1) 5'-EVPusAfsguaUfgAfCfaguuCfuUfugacusgsc-3' (SEQ ID NO: 35);

[0440] or

[0441] 2) 5'- EVPusAfsgu(Aam)U NP1 gAfCfaguuCfuUfugacusgsc-3' (SEQ ID NO: 36);

[0442] In this context, lowercase letters a, u, c, and g represent nucleotides modified with a 2'-methoxy group; Af, Uf, Cf, and Gf represent nucleotides modified with a 2'-fluoride group; EVP indicates that the nucleotide adjacent to the right of this letter combination is a 5'-trans-vinylphosphonate nucleotide; s indicates that two adjacent nucleotides are linked by a phosphothioester bond; (Aam)U NP1 express: .

[0443] In a specific embodiment, the nucleotides at positions 7, 9, 10, and 11 of sequence A of the positive strand are 2'-fluoro-modified nucleotides, following the direction from the 5' end to the 3' end, and each nucleotide at other positions of sequence A of the positive strand is a 2'-methoxy-modified nucleotide.

[0444] In some embodiments, the justice chain includes one or more (invAbs), preferably one or two. In some embodiments, (invAbs) are located at the 5' end and / or the 3' end of the justice chain. For example, (invAbs) are located at the 5' end of the justice chain; or, (invAbs) are located at the 3' end of the justice chain; or, (invAbs) are located at both the 5' end and the 3' end of the justice chain, wherein each of the (invAbs) is independently one or two (invAbs), preferably one (invAb).

[0445] In a preferred embodiment, one or more (invAb) are added at the 5' end and / or the 3' end of the justice chain, for example, one or two. In some embodiments, one (invAb) is added at the 5' end of the justice chain. In some embodiments, one (invAb) is added at the 3' end of the justice chain. In some embodiments, one (invAb) is added at both the 5' end and the 3' end of the justice chain.

[0446] In some embodiments, the positive chain contains phosphothioester bonds located at the positions described below, along the direction from the 5' end to the 3' end:

[0447] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0448] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0449] Between the first and second nucleotides starting at the 3' end of the positive strand;

[0450] Between the second and third nucleotides starting at the 3' end of the positive strand;

[0451] Between the reverse debased deoxyribose residue starting at the 5' end of the positive strand and the first nucleotide at the 5' end of the positive strand; and / or

[0452] The reverse debased deoxyribose residue starting at the 3' end of the positive strand is between the first nucleotide at the 3' end of the positive strand.

[0453] Preferably, the positive chain comprises a phosphothioester bond located at the following positions: between the reverse debased deoxyribose residue starting at the 5' end of the positive chain and the first nucleotide, between the first nucleotide and the second nucleotide, and between the reverse debased deoxyribose residue starting at the 3' end and the first nucleotide.

[0454] Preferably, the positive chain comprises a phosphothioester bond located at the following positions: between the first and second nucleotides starting at the 5' end of the positive chain, between the second and third nucleotides, and between the reverse debased deoxyribose residue starting at the 3' end and the first nucleotide.

[0455] Preferably, the positive chain comprises a phosphothioester bond located at the following positions: between the reverse debased deoxyribose residue starting at the 5' end of the positive chain and the first nucleotide, and between the first nucleotide and the second nucleotide.

[0456] In a specific embodiment, the sense strand in the modified double-stranded ribonucleic acid may have the following sequence:

[0457] 1)5'- asgsucaaAfgAfAfCfugucauacua-3' (SEQ ID NO: 37);

[0458] In this context, lowercase letters a, u, c, and g represent 2'-methoxy modified nucleotides; Af, Uf, Cf, and Gf represent 2'-fluorinated modified nucleotides; and s indicates that two adjacent nucleotides are linked by a phosphothioester bond. Optionally, if the 5' end of the positive strand does not have a reverse debased deoxyribose residue, the ribosome of the nucleotide has a 5' hydroxyl group, preferably having a 5' hydroxyl group.

[0459] In some embodiments, this disclosure provides a kit comprising: a) the double-stranded ribonucleic acid, its modifier, its conjugate, or a pharmaceutical composition thereof as described in this disclosure; and b) the following instructions for use:

[0460] (1) Inhibit C5 gene expression;

[0461] (2) Used for the prevention or treatment of diseases, conditions or disorders associated with abnormal expression of the C5 gene;

[0462] (3) For the treatment of subjects with diseases that would benefit from reduced C5 gene expression.

[0463] This disclosure provides for at least one use of the modified double-stranded ribonucleic acid, or its conjugates, or pharmaceutical compositions thereof described herein:

[0464] (1) Inhibit C5 gene expression, or prepare drugs for inhibiting C5 gene expression;

[0465] (2) To prevent or treat diseases, symptoms or disorders associated with abnormal expression of the C5 gene, or to prepare drugs for the prevention or treatment of diseases, symptoms or disorders associated with abnormal expression of the C5 gene.

[0466] (3) To treat subjects with diseases that would benefit from reduced C5 gene expression, or to prepare a medicine for treating subjects with diseases, conditions or disorders that would benefit from reduced C5 gene expression.

[0467] This disclosure further provides the use of siRNA molecules (including siRNA modifiers, siRNA conjugates) or pharmaceutical compositions thereof in at least one of (1)-(3) above.

[0468] In this disclosure, "subject" includes humans or non-human animals, preferably vertebrates, and more preferably mammals. Subjects may include transgenic organisms. Most preferably, the subject is a human. Further, the subject has at least one of the following characteristics:

[0469] (1) Abnormal expression of C5 gene in vivo, more specifically, abnormally high expression of C5 gene;

[0470] (2) Having a disease, condition or disorder associated with abnormal expression of the C5 gene;

[0471] (3) People who have a disease that would benefit from reduced C5 gene expression. Such as people who have or are predisposed to diseases, conditions or disorders associated with abnormal C5 gene expression.

[0472] In this article, "diseases, conditions, or disorders associated with abnormal C5 gene expression" include, without limitation, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, generalized myasthenia gravis, thromboembolism, neuromyelitis optica, antibody-mediated kidney transplant rejection, Guillain-Barré syndrome, antineutrophil cytoplasmic antibody-associated vasculitis, amyotrophic lateral sclerosis, Parkinson's disease, autoimmune encephalitis, IgG4-related disorders, asthma, antiphospholipid antibody syndrome, and ischemia-reperfusion injury. Typical hemolytic uremic syndrome, multifocal motor neuropathy, multiple sclerosis, thrombotic thrombocytopenic purpura, traumatic brain injury, cold agglutinin disease, dermatomyositis, hemolytic uremic syndrome associated with Shigella-producing Escherichia coli, graft dysfunction, myocardial infarction, sepsis, atherosclerosis, septic shock, spinal cord injury, psoriasis, autoimmune hemolytic anemia, antiphospholipid syndrome, myocarditis, immune complex vasculitis, Goyan's disease, Kawasaki disease, and rheumatoid arthritis.

[0473] Double-stranded ribonucleic acid and its modifications for inhibiting PNPLA3 gene expression

[0474] A further aspect of this disclosure provides a double-stranded RNA (dsRNA) for repressing the expression of the PNPLA3 gene. One strand of this dsRNA is an antisense strand, which is complementary to the mRNA sequence formed during the expression of the target gene (i.e., the PNPLA3 gene) to guide the cleavage of the target mRNA (i.e., the transcript of the PNPLA3 gene). The sense strand of the dsRNA includes a sequence that is at least partially complementary (including completely complementary) to the antisense strand to form a double-stranded region of the dsRNA.

[0475] In some embodiments, the double-stranded RNA serves as a substrate for the endonuclease Dicer, and is cleaved into small fragments of dsRNA, i.e., siRNA. In some embodiments, the double-stranded RNA is siRNA. The siRNA assembles to form an RNA-induced silencing complex (RISC), which cleaves the target mRNA and inhibits the expression of the PNPLA3 gene.

[0476] Based on the target sequence derived from human PNPLA3 mRNA (NM_025225.3), a double-stranded ribonucleic acid (e.g., siRNA) is designed to bind to the target mRNA.

[0477] In some embodiments, the double-stranded ribonucleic acid for inhibiting PNPLA3 comprises a sense strand and an antisense strand, wherein the sense strand comprises sequence A, which differs from the nucleotide sequence shown in SEQ ID NO: 39 by no more than 3 (e.g., no more than 2 or 1) nucleotides, and the antisense strand comprises sequence B, which differs from the nucleotide sequence shown in SEQ ID NO: 40 by no more than 3 (e.g., no more than 2 or 1) nucleotides; sequence A and sequence B are at least partially reverse complementary to form the double-stranded region of the modified double-stranded ribonucleic acid;

[0478] 5'-CGAGGAUAUUUGGAUGCAU-3' (SEQ ID NO: 39),

[0479] 5'-AUGCAUCCAAAUAUCCUCGAA-3' (SEQ ID NO: 40).

[0480] In a specific implementation, the sense strand contains a nucleotide sequence as shown in SEQ ID NO: 39 as sequence A, and the antisense strand contains a nucleotide sequence as shown in SEQ ID NO: 40 as sequence B.

[0481] In some embodiments, at least one nucleoside residue at positions 2-8 of sequence B in the antisense strand is replaced by a nucleoside monomer of formula (II). In some embodiments, one, two, or three nucleoside residues at positions 4, 5, 6, or 7 of sequence B in the antisense strand are replaced by a nucleoside monomer of formula (II). In some embodiments, a nucleoside residue at position 6 or 7 of sequence B in the antisense strand is replaced by a nucleoside monomer of formula (II).

[0482] In some embodiments, the nucleoside residue at position 6 or 7 of sequence B in the antisense strand is replaced by a nucleoside monomer as shown in formula (II).

[0483] In a preferred embodiment, the nucleotides of sequence A and sequence B in the antisense and sense strands of the double-stranded ribonucleic acid used to inhibit PNPLA3 are both modified nucleotides.

[0484] In some embodiments, at least three sites (e.g., 3-7, such as 3, 4, 5, 6, or 7) of the nucleotides at positions 2, 6, 7, 10, 14, and 16 of sequence B of the antisense strand are 2'-fluorinated nucleotides, in the direction from the 5' end to the 3' end, and each nucleotide at the other positions of sequence B of the antisense strand is independently one of non-2'-fluorinated nucleotides.

[0485] In some embodiments, at least three sites (e.g., 3-6, such as 3, 4, 5, or 6) of the 3rd, 5th, 7th, 8th, 9th, 10th, or 11th positions of the sequence A of the positive strand are 2'-fluorinated nucleotides, in the direction from the 5' end to the 3' end, and each nucleotide at the other positions of the sequence A of the positive strand is independently one of non-2'-fluorinated nucleotides.

[0486] In another specific embodiment, the antisense strand may optionally contain the nucleotide sequence E.

[0487] Preferably, the antisense chain includes EVP modification, etc.

[0488] In a specific embodiment, the nucleotides at positions 2, 6, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy-modified nucleotide.

[0489] In a specific embodiment, following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified nucleotides, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy-modified nucleotide; or,

[0490] In the direction from the 5' end to the 3' end, the nucleotides at positions 2, 14, and 16 of sequence B of the antisense strand are 2'-fluorinated nucleotides, the nucleotide at position 6 of sequence B of the antisense strand is GNA modified, and each nucleotide at other positions of sequence B of the antisense strand is a 2'-methoxy modified nucleotide or the nucleoside residue at position 6 of sequence B of the antisense strand is replaced by a nucleoside monomer as shown in formula (II); optionally, the nucleosides at positions 5 and 6 of sequence B of the antisense strand are linked by an amide bond.

[0491] In a specific embodiment, the antisense strand in the modified double-stranded ribonucleic acid used to suppress PNPLA3 may have a structure selected from any of the following sequences:

[0492] 1) 5'-EVPasUfsgcaUfccaaauaUfcCfucgsasa-3' (SEQ ID NO: 41); or

[0493] 2) 5'-EVPasUfsgc(Aam)U NP1 ccaaauaUfcCfucgsasa-3' (SEQ ID NO: 42);

[0494] In this context, lowercase letters a, u, c, and g represent nucleotides modified with a 2'-methoxy group; Af, Uf, Cf, and Gf represent nucleotides modified with a 2'-fluoride group; EVP indicates that the nucleotide adjacent to the right of this letter combination is a 5'-trans-vinylphosphonate nucleotide; s indicates that two adjacent nucleotides are linked by a phosphothioester bond; (Aam)U NP1 express: .

[0495] In a specific embodiment, the nucleotides at positions 7, 9, 10, and 11 of sequence A of the positive strand are 2'-fluoro-modified nucleotides, following the direction from the 5' end to the 3' end, and each nucleotide at other positions of sequence A of the positive strand is a 2'-methoxy-modified nucleotide.

[0496] In some embodiments, the justice chain includes one or more (invAbs), preferably one or two. In some embodiments, (invAbs) are located at the 5' end and / or the 3' end of the justice chain. For example, (invAbs) are located at the 5' end of the justice chain; or, (invAbs) are located at the 3' end of the justice chain; or, (invAbs) are located at both the 5' end and the 3' end of the justice chain, wherein each of the (invAbs) is independently one or two (invAbs), preferably one (invAb).

[0497] In a preferred embodiment, one or more (invAb) are added at the 5' end and / or the 3' end of the justice chain, for example, one or two. In some embodiments, one (invAb) is added at the 5' end of the justice chain. In some embodiments, one (invAb) is added at the 3' end of the justice chain. In some embodiments, one (invAb) is added at both the 5' end and the 3' end of the justice chain.

[0498] In some embodiments, the positive chain contains phosphothioester bonds located at the positions described below, along the direction from the 5' end to the 3' end:

[0499] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0500] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0501] Between the first and second nucleotides starting at the 3' end of the positive strand;

[0502] Between the second and third nucleotides starting at the 3' end of the positive strand;

[0503] Between the reverse debased deoxyribose residue starting at the 5' end of the positive strand and the first nucleotide at the 5' end of the positive strand; and / or

[0504] The reverse debased deoxyribose residue starting at the 3' end of the positive strand is between the first nucleotide at the 3' end of the positive strand.

[0505] Preferably, the positive chain comprises a phosphothioester bond located at the following positions: between the reverse debased deoxyribose residue starting at the 5' end of the positive chain and the first nucleotide, between the first nucleotide and the second nucleotide, and between the reverse debased deoxyribose residue starting at the 3' end and the first nucleotide.

[0506] Preferably, the positive chain comprises a phosphothioester bond located at the following positions: between the first and second nucleotides starting at the 5' end of the positive chain, between the second and third nucleotides, and between the reverse debased deoxyribose residue starting at the 3' end and the first nucleotide.

[0507] Preferably, the positive chain comprises a phosphothioester bond located at the following positions: between the reverse debased deoxyribose residue starting at the 5' end of the positive chain and the first nucleotide, and between the first nucleotide and the second nucleotide.

[0508] In a specific embodiment, the sense strand in the modified double-stranded ribonucleic acid may have a structure selected from any of the following sequences:

[0509] 1)5'- csgsaggaUfaUfUfUfggaugcau-3' (SEQ ID NO: 43);

[0510] In this context, lowercase letters a, u, c, and g represent 2'-methoxy modified nucleotides; Af, Uf, Cf, and Gf represent 2'-fluorinated modified nucleotides; and s indicates that two adjacent nucleotides are linked by a phosphothioester bond. Optionally, if the 5' end of the positive strand does not have a reverse debased deoxyribose residue, the ribosome of the nucleotide has a 5' hydroxyl group, preferably having a 5' hydroxyl group.

[0511] In some embodiments, this disclosure provides a kit comprising: a) the double-stranded ribonucleic acid, its modifier, its conjugate, or a pharmaceutical composition thereof as described in this disclosure; and b) the following instructions for use:

[0512] (1) Inhibit PNPLA3 gene expression;

[0513] (2) Used for the prevention or treatment of diseases, conditions or disorders associated with abnormal expression of the PNPLA3 gene;

[0514] (3) For the treatment of subjects with diseases that would benefit from reduced PNPLA3 gene expression.

[0515] This disclosure provides for at least one use of the modified double-stranded ribonucleic acid, or its conjugates, or pharmaceutical compositions thereof described herein:

[0516] (1) Inhibit PNPLA3 gene expression, or prepare drugs for inhibiting PNPLA3 gene expression;

[0517] (2) To prevent or treat diseases, symptoms or disorders associated with abnormal expression of the PNPLA3 gene, or to prepare drugs for the prevention or treatment of diseases, symptoms or disorders associated with abnormal expression of the PNPLA3 gene.

[0518] (3) To treat subjects with diseases that would benefit from reduced PNPLA3 gene expression, or to prepare a medicine for treating subjects with diseases, conditions or disorders that would benefit from reduced PNPLA3 gene expression.

[0519] This disclosure further provides the use of siRNA molecules (including siRNA modifiers, siRNA conjugates) or pharmaceutical compositions thereof in at least one of (1)-(3) above.

[0520] In this disclosure, "subject" includes humans or non-human animals, preferably vertebrates, and more preferably mammals. Subjects may include transgenic organisms. Most preferably, the subject is a human. Further, the subject has at least one of the following characteristics:

[0521] (1) Abnormal expression of PNPLA3 gene in vivo, more specifically, abnormally high expression of PNPLA3 gene;

[0522] (2) Having a disease, condition or disorder associated with abnormal expression of the PNPLA3 gene;

[0523] (3) People who have a disease that would benefit from reduced PNPLA3 gene expression. Such as people who have or are predisposed to diseases, conditions or disorders associated with abnormal PNPLA3 gene expression.

[0524] In this article, "diseases, conditions or disorders associated with abnormal expression of the PNPLA3 gene" include, without limitation, NAFLD, alcoholic fatty liver disease, alcoholic steatohepatitis, hepatic steatosis, cirrhosis, liver cancer, liver fibrosis, hepatitis, liver injury, and hepatocellular necrosis; NAFLD is selected from any one or a combination of the following: non-alcoholic fatty liver disease and non-alcoholic steatohepatitis; hepatitis is selected from any one or a combination of the following: chronic hepatitis and HCV hepatitis.

[0525] Double-stranded ribonucleic acid or its modifications or conjugates

[0526] This disclosure also provides that the double-stranded ribonucleic acid or its modifications or conjugates (hereinafter referred to as double-stranded ribonucleic acid conjugates or conjugates) described in this application are obtained by conjugating and linking the double-stranded ribonucleic acid or its modifications provided in this disclosure with a conjugating group.

[0527] In this disclosure, the sense strand and antisense strand of the conjugate form a double-stranded region of the double-stranded ribonucleic acid conjugate, and the sense strand of the double-stranded ribonucleic acid conjugate has a blunt end at the 3' end.

[0528] In some preferred embodiments, the double-stranded ribonucleic acid conjugate is obtained by conjugating a double-stranded ribonucleic acid or a modification thereof with a conjugating group. Specifically, the sense and antisense strands of the double-stranded ribonucleic acid or its modification are at least partially complementary to form the double-stranded region of the double-stranded ribonucleic acid or the modified double-stranded ribonucleic acid, and the conjugating group is conjugated to the 3' end of the sense strand to form the double-stranded ribonucleic acid conjugate.

[0529] Optionally, if the 5' end of the positive strand does not have a reverse debased deoxyribose residue, the 5' nucleotide ribosome of the positive strand has a 5' hydroxyl group.

[0530] The conjugation site of siRNA and the conjugating group can be at the 3' or 5' end of the siRNA's sense strand, at the 5' end of the antisense strand, or within the siRNA's internal sequence. In some embodiments, the conjugation site of siRNA and the conjugating group is at the 3' end of the siRNA's sense strand.

[0531] In some embodiments, the conjugate group can be attached to the phosphate group, 2'-hydroxyl group, or base of the nucleotide. In some embodiments, the conjugate group can also be attached to the 3'-hydroxyl group, in which case the nucleotides are linked by a 2',5'-phosphodiester bond. When the conjugate group is attached to the end of the siRNA chain, it is usually attached to the phosphate group of the nucleotide; when the conjugate group is attached to the internal sequence of the siRNA, it is usually attached to the ribose ring or base, for example, by a phosphate ester bond or a thiophosphate bond. Various connection methods can be found in the reference: Muthiah Manoharan et al. siRNA conjugates carrying sequentiallyassembled trivalent N-acetylgalactosamine linked through nucleosides elicitrobust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015,10(5): 1181-7.

[0532] In this disclosure, the conjugation group can be a ligand conventionally used in the field of siRNA drug delivery. In some embodiments, the conjugation group can be selected from one or more ligands formed from the following target molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g., vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as transmembrane peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid; receptor ligands expressed by hepatocytes, such as desialyl glycoprotein, desialyl sugar residues, lipoproteins (e.g., high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, and transferrin, etc.

[0533] In some embodiments, the conjugating group has a structure as shown in any of the formulas L1, L-III, L-IV, L-LV, L-VI, L-VII, L-VIII, and L-IX (wherein, phosphate ester bonds or thiophosphate ester bonds are exemplary shown in some conjugating groups):

[0534] (Formula LI);

[0535] (Formula L-III);

[0536] (Formula L-IV);

[0537] (LV style);

[0538] (Form L-VI);

[0539] (Form L-VII);

[0540] (Formula L-VIII);

[0541] (Formula L-IX).

[0542] Among them, the conjugation groups shown in formulas LI, L-III, L-IV, LV, L-VI, L-VII, L-VIII, and L-IX are GalNAc. GalNAc has liver targeting capability and can deliver siRNA molecules to liver tissue with high specificity, specifically inhibiting the high expression of target genes in the liver.

[0543] In some specific implementations, GalNAc is conjugated to the 3' end of the positive sense strand via a phosphodiester bond, resulting in an siRNA conjugate with the structure shown in Formula III:

[0544] (Equation III)

[0545] The double helix structure is the siRNA or siRNA modified as described in this article.

[0546] Pharmaceutical Composition

[0547] This disclosure also provides a pharmaceutical composition comprising one or more of the oligonucleotides (e.g., double-stranded ribonucleic acid) described herein, and their modifications and conjugates.

[0548] In some embodiments, the pharmaceutical composition contains one or more of the oligonucleotides (e.g., double-stranded ribonucleic acid) described herein as above, and their modifications and conjugates (e.g., modified siRNA) as the active ingredient and a pharmaceutically acceptable carrier. In this disclosure, the pharmaceutical composition is used to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and exerting its biological activity. The pharmaceutical compositions of this disclosure can be administered in any form, including by injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosal, oral (oral solid dosage form, oral liquid dosage form), rectal, inhalation, implantation, and topical (e.g., ocular) administration. Non-limiting examples of oral solid dosage forms include, but are not limited to, powders, capsules, lozenges, granules, tablets, etc. Non-limiting examples of oral or mucosal liquid dosage forms include, but are not limited to, suspensions, tinctures, elixirs, solutions, etc. Non-limiting examples of topical dosage forms include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Non-limiting examples of parenteral administration formulations include, but are not limited to, solutions for injection, dry powders for injection, suspensions for injection, and emulsions for injection. The pharmaceutical compositions disclosed herein can also be formulated into controlled-release or delayed-release dosage forms (e.g., liposomes or microspheres).

[0549] In this disclosure, the route of administration can be varied or modified in any applicable manner to meet the needs of the properties of the drug, the convenience of patients and healthcare professionals, and other relevant factors.

[0550] In some embodiments, this disclosure provides a kit comprising: a) the oligonucleotides (e.g., double-stranded ribonucleic acid, particularly siRNA), their modifications or conjugates, or pharmaceutical compositions thereof as described in this disclosure; and b) instructions for use as follows:

[0551] (1) Inhibit the expression of target genes;

[0552] (2) Used for the prevention or treatment of diseases, symptoms or disorders related to abnormal expression of target genes;

[0553] (3) Used to treat subjects with diseases that would benefit from reduced expression of the target gene.

[0554] Medical Use

[0555] This disclosure provides for at least one use of the oligonucleotides (e.g., double-stranded ribonucleic acid, particularly siRNA), their modifications or conjugates, or pharmaceutical compositions thereof described herein:

[0556] (1) Inhibit the expression of target genes, or prepare drugs for inhibiting the expression of target genes;

[0557] (2) To prevent or treat diseases, symptoms or disorders related to abnormal expression of target genes, or to prepare drugs for the prevention or treatment of diseases, symptoms or disorders related to abnormal expression of target genes.

[0558] (3) To treat subjects with diseases that would benefit from reduced expression of the target gene, or to prepare a medicine for treating subjects with diseases, conditions or disorders that would benefit from reduced expression of the target gene.

[0559] This disclosure further provides the use of siRNA molecules (including siRNA modifiers, siRNA conjugates) or pharmaceutical compositions thereof in at least one of (1)-(3) above.

[0560] Modifiers of siRNA or conjugates thereof or pharmaceutical compositions thereof disclosed herein cause inhibition of target gene expression by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, thereby achieving treatment for diseases, conditions, or disorders related to abnormal expression of the target gene.

[0561] In some embodiments, this disclosure provides a method for inhibiting the expression of intracellular target genes, comprising contacting the oligonucleotides (e.g., double-stranded ribonucleic acid) described in this disclosure or their modifications or conjugates or pharmaceutical compositions thereof with cells.

[0562] Furthermore, a method for inhibiting the expression of target genes in cells is to introduce the siRNA molecule (including siRNA modifiers or conjugates thereof) or a pharmaceutical composition thereof described in this disclosure into cells.

[0563] In some embodiments, the cells are in vivo or in vitro cells. In some specific embodiments, the cells are in the body of a subject.

[0564] In some embodiments, this disclosure provides methods for preventing or treating diseases, including administering to a subject the oligonucleotides (e.g., double-stranded ribonucleic acid) described in this disclosure, or their modifications or conjugates, or pharmaceutical compositions thereof.

[0565] In some embodiments, this disclosure provides methods for preventing or treating a disease, including administering to a subject an effective amount of the oligonucleotide (e.g., double-stranded ribonucleic acid) described in this disclosure, or a modification or conjugate thereof, or a pharmaceutical composition thereof. In some embodiments, the effective amount is a therapeutically effective amount.

[0566] Administration to subjects can be performed via any suitable route known in the art. In some embodiments of this disclosure, the routes include, but are not limited to, oral or parenteral routes, including at least one of intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and local administration (including oral and sublingual administration). In some embodiments of this disclosure, the frequency of administration may be once or more daily, weekly, monthly, or annually. The dosage of the modified siRNA or its conjugates described herein can be determined based on various parameters, particularly the subject's age, weight, and sex.

[0567] Furthermore, a method for preventing or treating disease is to administer to a subject the siRNA molecule (including siRNA modifiers, siRNA conjugates) or a pharmaceutical composition thereof as described in this disclosure.

[0568] In this disclosure, "subject" includes humans or non-human animals, preferably vertebrates, and more preferably mammals. Subjects may include transgenic organisms. Most preferably, the subject is a human. Further, the subject has at least one of the following characteristics:

[0569] (1) Abnormal expression of target genes in vivo, more specifically, abnormally high expression of target genes;

[0570] (2) Suffering from a disease related to abnormal expression of the target gene;

[0571] (3) People who have a disease that would benefit from reduced expression of the target gene. For example, people who have or are prone to have a disease that is associated with abnormal expression of the target gene.

[0572] In this article, target genes include, but are not limited to, one or more of the following genes: SOD1, CFB, SCAP, INHBE, C5, PNPLA3, TTR, PCSK9, APOC3, or C3. Detailed Implementation

[0573] definition

[0574] For convenience, several terms used herein, in the specification, embodiments, and appended claims are collected herein. Unless otherwise stated or implied by the context, the following terms and phrases include the meanings provided below. Unless otherwise expressly stated or obvious from the context, the following terms and phrases do not exclude the meanings that the term or phrase already has in the art to which it pertains. The definitions provided are intended to aid in the description of particular embodiments and are not intended to limit the claimed invention, as the scope of this application is defined only by the claims. Moreover, unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms.

[0575] In the claims and / or specification of this disclosure, the words “a” or “an” or “the” may mean “one”, but may also mean “one or more”, “at least one”, and “one or more”.

[0576] As used in the claims and specification, the words “comprising,” “having,” “including,” or “containing” mean included or open-ended and do not exclude additional, uncited elements or method steps.

[0577] Throughout this application, the term "about" means that a value includes the standard deviation of the error of the apparatus or method used to determine that value. The numerical ranges and parameters used to define this disclosure are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains a standard deviation due to the aforementioned test methods or apparatus. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified by the term "about." Here, "about" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0578] The gene sequence and mRNA sequence of the term "target gene" as used in the context of this disclosure may be obtained, for example, from the following sources: GenBank, UniProt, Human Mendelian Inheritance Online (OMIM), etc.

[0579] In this article, the term "target gene" can refer to a wild-type gene or a gene mutant with sequence variations. Many sequence variations in target genes have been identified and can be found, for example, in NCBIdbSNP and UniProt (see, for example, ncbi.nlm.nih.gov / snp).

[0580] In this document, the terms "peptide" and "protein" are used interchangeably to refer to a string of at least two amino acid residues linked together by covalent bonds (e.g., peptide bonds), and can be recombinant, natural, or synthetic peptides. Peptides can be linear or branched, can contain modified amino acids, and can be separated by non-amino acid segments. The term also includes amino acid polymers that have been modified (e.g., through disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with labeled components).

[0581] As used in the context of this disclosure, the term "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene, including mRNA as a product of RNA processing of the primary transcription product.

[0582] In some embodiments, the target gene is the CFB gene, the SCAP gene, or the INHBE gene. In some embodiments, the target portion of the gene sequence (i.e., the portion corresponding to the target sequence in the mRNA sequence) will be at least long enough to act as a substrate for iRNA-guided cleavage at or near the nucleotide sequence portion of the mRNA molecule formed during transcription of the target gene.

[0583] In this technical field, "G", "C", "A", "T" and "U" typically represent the nucleobases of guanine, cytosine, adenine, thymine, and uracil, respectively. However, it is also generally known in the art that each of "G", "C", "A", "T" and "U" typically also represents a nucleotide containing guanine, cytosine, adenine, thymine, and uracil as nucleobases, respectively. This is a common practice in representing deoxyribonucleic acid (DNA) sequences and / or ribonucleic acid (RNA) sequences. Therefore, in the context of this disclosure, the meanings of "G", "C", "A", "T", and "U" include all the aforementioned possible scenarios. However, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide (as further detailed below) or a substituted portion. In this application, "nucleotide", "ribonucleotide", and "ribonucleic acid" are used interchangeably, and "deoxyribonucleic acid" and "2'-deoxyribonucleic acid" are used interchangeably. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be substituted with other moieties without substantially altering the base-pairing properties of an oligonucleotide (including a nucleotide having such a substitution moiety). For example, without limitation, a nucleotide including inosine as its base can base-pair with a nucleotide including adenine, cytosine, or uracil. Therefore, a nucleotide containing uracil, guanine, or adenine can be substituted in the nucleotide sequence of the dsRNA characterized in this disclosure with a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in an oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU swing base pairing with the target mRNA. Sequences containing such substitution moieties are suitable for the compositions and methods characterized in this disclosure.

[0584] In this application, "5'-nucleotide" refers to a nucleotide in which the phosphate group is attached to the 5' carbon of a pentose sugar, and it is the main type of nucleotide that exists freely in organisms. "3'-nucleotide" refers to a nucleotide in which the phosphate group is attached to the 3' carbon of a pentose sugar, and may include, for example, adenosine-3'-phosphate, guanosine-3'-phosphate, cytidine-3'-phosphate, uridine-3'-phosphate, 2'-deoxythymidine-3'-phosphate, 2'-O-methyladenosine-3'-phosphate, 2'-O-methyladenosine-3'-thiophosphate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroadenosine-3'-thiophosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methylguanosine-3'-phosphate. The terms glycoside-3'-thiophosphate, 2'-fluoroguanosine-3'-phosphate, 2'-fluoroguanosine-3'-thiophosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylcytidine-3'-thiophosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'-thiophosphate, 2'-O-methyluridine-3'-phosphate, 2'-O-methyluridine-3'-thiophosphate, 2'-fluorouridine-3'-phosphate, 2'-fluorouridine-3'-thiophosphate, and 2'-deoxythymidine-3'-thiophosphate are used. This definition can be applied to modified or unmodified nucleoside phosphoramidamide monomers.

[0585] The terms “iRNA,” “RNAi reagent,” “iRNA agent,” and “RNA interference agent” as used in the context of this disclosure are used interchangeably and refer to terms defined herein that contain siRNA and mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates, for example, the expression of target genes in cells (such as the cells of a subject, such as a mammalian subject).

[0586] The terms “double-stranded ribonucleic acid,” “double-stranded RNA (dsRNA) molecule,” and “dsRNA” as used in the context of this disclosure are used interchangeably. The term “dsRNA” refers to a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, described as having “sense” and “antisense” orientation relative to a target gene, such as a target gene. In some embodiments, double-stranded ribonucleic acid (dsRNA) triggers the degradation of target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).

[0587] Typically, the majority of nucleotides on each strand of a dsRNA molecule are double-stranded ribonucleic acid (BRNA), but as detailed herein, each or both strands may also include one or more non-ribonucleic acids, such as deoxyribonucleotides and / or modified nucleotides. Additionally, as used herein, “double-stranded ribonucleic acid” can include chemically modified ribonucleotides, phosphate backbones, etc. These modifications can include all types of modifications disclosed herein or known in the art.

[0588] As used in the context of this disclosure, the term "isonucleotide" refers to a compound formed by altering the position of a base on the ribose ring, for example, a compound formed by attaching a base to the 2' or 3' position of the ribose ring instead of the 1' position.

[0589] In some embodiments, the double-stranded ribonucleic acid disclosed herein is siRNA, which interacts with the mRNA sequence transcribed from the target gene (e.g., the mRNA sequence transcribed from the target gene) to guide the cleavage of the target RNA. Not wishing to be bound by theory, long double-stranded RNA introduced into cells is broken down into siRNA by a type III endonuclease called Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer (a ribonuclease III-like enzyme) processes dsRNA into short 19-23 base pairs of interfering RNA with a characteristic dibase 3' overhang (Bernstein et al., (2001) Nature 409:363). These siRNAs are then incorporated into the RNA-inducible silencing complex (RISC), where one or more helicases unwind the siRNA duplex, enabling complementary antisense strands to guide target recognition (Nykanen et al., (2001) Cell 107:309). Once bound to a suitable target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15:188).

[0590] In the context of this disclosure, a "nucleotide difference" between two nucleotide sequences refers to a change in the type of bases at the same position of the nucleotides compared to the latter. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, then a nucleotide difference at that position is considered to exist between the two nucleotide sequences. In some embodiments, replacing the nucleotide at the original position with a nucleotide analog can also be considered as a nucleotide difference at that position.

[0591] As used in the context of this disclosure, the terms "protruding nucleotide" and "protruding end" refer to one or more unpaired nucleotides that protrude from the double-stranded structure of a dsRNA when one 3' end of one strand of the double-stranded RNA extends beyond the 5' end of the other strand, or vice versa. "Blunt end" or "flat-ended" means that there are no unpaired nucleotides at that end of the double-stranded RNA, i.e., no nucleotide protrusion. A "flat-ended" double-stranded RNA is a dsRNA that is double-stranded throughout its entire length, i.e., without nucleotide protrusions at either end of the molecule.

[0592] In this article, the term "antisense strand" refers to a double-stranded RNA strand containing a region that is substantially complementary to a target sequence (e.g., mRNA derived from a human target gene). Mismatches at the terminal regions are generally tolerable, provided that the complementary region is not perfectly complementary to the target sequence, and if mismatches do occur, they are typically within one or more terminal regions, such as 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends.

[0593] In this article, the term "sense chain" refers to a double-stranded RNA containing a nucleic acid chain that is substantially complementary to the antisense chain region.

[0594] In this article, unless otherwise specified, the term "difference" or "differentiation" refers to changes in the nucleotide sequence between two strands being compared, resulting from the insertion, deletion, or substitution of nucleotides.

[0595] In this document, the terms “complementary” or “reverse complementary” are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired in a complementary manner with the bases of the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair consists of one purine and one pyrimidine. 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, and the sequence of the complementary strand can be inferred from its sequence. Correspondingly, “mismatch” in the art means, in a double-stranded nucleic acid, that the bases at corresponding positions are not paired in a complementary manner.

[0596] In this article, the term "at least partially complementary" means that at least 20% but less than 100% of the residues in one of the two nucleotide sequences are complementary to the residues in the other nucleotide sequence. In some cases, this complementarity may reach a higher percentage, such as at least 50%, 70%, 80%, 90%, 95%, or 98% or higher. When the complementarity of two sequences reaches a certain level (e.g., more than 50%), they are able to hybridize with each other under standard or stringent hybridization conditions.

[0597] In this article, the terms "substantially complementary" or "substantially anticomplementary" refer to the presence of no more than three base mismatches between the two nucleotide sequences involved, i.e., one, two, or three base mismatches between the two nucleotide sequences involved; "completely complementary" or "completely anticomplementary" refers to the presence of no base mismatches between the two nucleotide sequences.

[0598] In this article, the terms “complementary / anticomplementary,” “fully complementary / fully anticomplementary,” and “substantially complementary / substantially anticomplementary” can be used relative to base pairings between the sense and antisense strands of dsRNA, or between the antisense strand of dsRNA and the target sequence, as will be understood from the context in which they are used.

[0599] In this article, the term "chemical modification" or "modification" refers to all structural changes that occur to nucleic acids, including but not limited to the addition, removal, or substitution of chemical parts, as well as any structural changes introduced by chemical, biochemical, enzymatic, or other means.

[0600] In this document, when referring to the modification of nucleotides, unless otherwise stated, "2'-... modification" refers to a modification present at the 2' position of the ribosome in the nucleotide. Therefore, in this document, when referring to the modification of nucleotides, unless otherwise stated, "2'-... modified nucleotide" refers to a nucleotide formed by a modification present at the 2' position of the ribosome in the nucleotide.

[0601] In this article, "non-2'-fluorinated nucleotides" refers to nucleotides or nucleotide analogs that are modified by a nucleotide other than having the hydroxyl group at the 2' position of the ribose group of the ribonucleotide replaced by a fluorine group.

[0602] In the foregoing and hereinafter, particularly in the description of methods for preparing double-stranded ribonucleic acid, modified double-stranded ribonucleic acid, or their conjugates or pharmaceutical compositions, unless otherwise specified, a nucleoside monomer refers to a modified or unmodified nucleoside phosphorus amide monomer used in phosphorus amide solid-phase synthesis, depending on the type and sequence of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphorus amide solid-phase synthesis is a method known to those skilled in the art for RNA synthesis. All nucleoside monomers used in this disclosure are commercially available.

[0603] In this article, "stereoisomers" refers to isomers with the same molecular formula and functional groups, but different spatial arrangements of atoms or functional groups in the molecule. Stereoisomers include cis-trans isomers and chiral isomers. Chiral isomers are further divided into two main categories: enantiomers and diastereomers.

[0604] In this article, "enantiomers" refers to compounds with the same molecular formula and functional groups that are isomers due to differences in the spatial arrangement of atoms or functional groups. The compounds form stereoisomers that are mirror images of each other and cannot be superimposed.

[0605] In this article, "diastereomers" refers to compounds with the same molecular formula and functional groups that are isomers due to different spatial arrangements of atoms, and these compounds are stereoisomers that are not mirror images of each other.

[0606] In this application, the structural formulas of "compound", "chemical group", "nucleoside", "nucleotide", and "nucleotide derivative" contain linear covalent bonds. "Can represent being coplanar with the paper. In this application, when the atoms connected by a straight covalent bond have stereoisomers, the arrangement of the atoms connected by the straight covalent bond can include being coplanar with the paper, facing outwards from the paper, facing inwards from the paper, or a mixture of various arrangements."

[0607] Unless otherwise specified, use wedge-shaped solid line keys ( ) and wedge-shaped dashed key ( The absolute configuration of a solid center is represented by a straight solid line key ( ). ) and straight dashed key ( The relative configuration of the center of a solid is represented by a wavy line ( ). ) indicates a wedge-shaped solid line key ( ) or wedge-shaped dashed key ( ), or use wavy lines ( ) indicates a straight solid line key ( ) and straight dashed key ( In this article, the term “inhibition” may be used interchangeably with “reduction,” “silence,” “downregulation,” “suppression,” and other similar terms, and includes any level of inhibition.

[0608] In this document, the term "suppressing the expression of a target gene" includes suppressing the expression of any target gene (such as, for example, a mouse target gene, a rat target gene, a monkey target gene, or a human target gene) as well as variants (e.g., naturally occurring variants) or mutants of the target gene. Therefore, the target gene can be a wild-type target gene, a mutant target gene, or, in the case of genetically manipulated cells, cell groups, or organisms, a transgenic target gene.

[0609] In this document, the expression "inhibition of target gene expression" includes inhibition of any level of target gene expression, such as at least partial inhibition of target gene expression, including inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0610] In this text, the term "independently" means that at least two groups (or ring systems) in a structure with the same or similar value ranges can have the same or different meanings under specific circumstances. For example, if substituent X and substituent Y are each independently hydrogen, hydroxyl, alkyl, or aryl, then when substituent X is hydrogen, substituent Y can be hydrogen, hydroxyl, alkyl, or aryl; similarly, when substituent Y is hydrogen, substituent X can be hydrogen, hydroxyl, alkyl, or aryl. The terms "each...independently selected" are interchangeable with "...independently selected" and "...independently selected" and should be interpreted broadly. They can mean that in different groups, the specific options expressed by the same symbol do not affect each other, or that in the same group, the specific options expressed by the same symbol do not affect each other.

[0611] In this article, the term "phosphate derivatives" refers to a class of compounds derived from phosphoric acid (H3PO4) or its anions (such as PO4). 3- Compounds derived from phosphates include phosphates, phosphate esters, and phosphates. For example, these compounds form compounds with different chemical properties by removing or replacing one or more atoms (such as hydrogen atoms) in a phosphate molecule.

[0612] In this text, the term "key" refers to a single bond unless otherwise specified.

[0613] In this disclosure, the term "reactive phosphorus group" generally refers to a functional group containing a phosphorus atom and possessing high chemical reactivity. In some embodiments, the reactive phosphorus group is a phosphorus-containing group that can be removed by reacting with other compounds. For example, in this disclosure, the reactive phosphorus group may be selected from... and .

[0614] In this document, the terms “optional” or “optionally” mean that the event or condition described below may or may not occur, including both the occurrence and non-occurrence of the event or condition. For example, the ethyl group “optionally” being halogenated means that the ethyl group may be unsubstituted (-CH2CH3), monosubstituted (e.g., -CH2CH2F), polysubstituted (e.g., -CHFCH2F, -CH2CHF2, etc.), or fully substituted (-CF2CF3). Those skilled in the art will understand that for any group containing one or more substituents, no substitution or substitution pattern that is spatially impossible and / or cannot be synthesized is introduced.

[0615] The terms "combination" and "linkage" are used interchangeably to refer to the connection of two or more chemical parts, each with a specific function, to each other in a non-covalent or covalent manner; correspondingly, a "combination" refers to a compound formed by the non-covalent or covalent connection of the respective chemical parts. In this application, conjugates connected to each other in a covalent manner are preferred. Hereinafter, the term "alkyl" includes straight-chain, branched, or cyclic saturated alkyl groups. For example, alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclohexyl, and similar groups. Exemplarily, "C 1-6 The "C" in "alkyl" 1-6 "" refers to a group consisting of 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight chain, branched chain or cyclic form.

[0616] In this document, the term "alkoxy" refers to an alkyl group that is attached to the remainder of a molecule by an oxygen atom (-O-alkyl), wherein the alkyl group is as defined herein. Non-limiting examples of alkoxy groups and substituted alkoxy groups include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, etc.

[0617] In this document, the term "cycloalkyl" refers to a fully saturated carbon ring that may exist in the form of a monocyclic, bridged, or spirocyclic ring. Unless otherwise indicated, the carbon ring is typically a 3- to 6-membered ring. Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0618] In this article, the terms “halogen,” “halogenated,” or “halogen” refer to fluorine, chlorine, bromine, or iodine.

[0619] In this article, the term "hydroxyl group" refers to the -OH group.

[0620] In this disclosure, the interchangeable use of "substituted" and "substituted" means that any one or more hydrogen atoms on a specific atom in the given structure are substituted by a substituent, provided that the valence state of the specific atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted. Unless otherwise stated, a substituted group may have one substituent at each substituted position of the group. When more than one position in the given structural formula can be substituted by one or more substituents selected from a specific group, then the substituents may be substituted at each substituted position in the same or different ways.

[0621] The term "substituent" as used herein includes all substituents mentioned in the context, including but not limited to the terms halogen, "alkyl," "alkoxy," "cycloalkyl," "heteroalkyl," "alkenyl," "alkynyl," etc., and their corresponding non-limiting or exemplary groups. Some non-limiting examples of said "substituent" include C 1-6 Alkyl, C 1-6 Alkoxy groups, C atoms containing oxygen atoms 1-6 Heteroalkyl, C 2-6 alkenyl, C 2-6 Alkyne groups, etc.

[0622] C in this article m-n This means that the part has an integer number of carbon atoms within a given range. For example, "C 1-6 "" means that the group can have 1, 2, 3, 4, 5, or 6 carbon atoms. For example, C 1-3 This means that the group can have 1 carbon atom, 2 carbon atoms, or 3 carbon atoms.

[0623] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted by two Rs, each R has an independent option.

[0624] Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of that group can be connected to other groups by chemical bonds. When the chemical bond connection is non-directional and the connectable site contains H atoms, the number of H atoms at that site will decrease accordingly with the number of chemical bonds connected, resulting in a group with a corresponding valence. The chemical bonds connecting the site to other groups can be straight solid line bonds (…). Straight dashed key ( ) or wavy line ( )express.

[0625] In this article, when a letter refers to an object or quantity, it is case-sensitive.

[0626] In this article, the term "room temperature" refers to a temperature range of 15-30°C, such as 20-25°C (or approximately 20°C, 25°C).

[0627] In this document, the term "treatment" means, after a person has contracted a disease, exposing (e.g., administering medication) a subject to the modified double-stranded RNA, its conjugates, or pharmaceutical compositions of this disclosure, thereby alleviating the symptoms of the disease compared to when the subject is not exposed, without implying the necessity to completely suppress the symptoms of the disease, such as improving or eliminating the disease or one or more symptoms associated with the disease. Contracting a disease means the appearance of disease symptoms in the body.

[0628] In this document, the term "prevention" means: to reduce the symptoms of a disease before the onset of the disease by exposing (e.g., administering medication) a subject to the modified double-stranded RNA, its conjugates, or pharmaceutical compositions thereof disclosed herein, compared to the absence of exposure, without implying the necessity of completely suppressing the disease.

[0629] In this document, the terms "effective amount" or "effective dose" refer to an amount or dose of the modified double-stranded ribonucleic acid, its conjugates, or pharmaceutical compositions of this disclosure that, when administered to a patient in a single or multiple dose, produces the intended effect in a patient requiring treatment or prevention. The effective amount can be readily determined by a physician skilled in the art by considering a variety of factors, such as: the species of the mammal; its size, age, and general health; the specific disease involved; the degree or severity of the disease; the individual patient's response; the specific antibody administered; the administration method; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies. The term "therapeutic effective amount" refers to an amount or dose of the modified double-stranded ribonucleic acid, its conjugates, or pharmaceutical compositions of this disclosure that, when administered to a patient in a single or multiple dose, produces the intended therapeutic effect in a patient requiring treatment or prevention. The dosage of the modified double-stranded ribonucleic acid, its conjugates, or pharmaceutical compositions of this disclosure may depend on variables such as the type and severity of the disease or condition, the general health status of the specific subject or patient, the relative biological potency of the selected compound, the excipient formulation used, and the route of administration. The effective dose can be obtained by extrapolation from the dose-response curve derived from an in vitro or animal model testing system.

[0630] In this document, the terms "pharmaceuticalally acceptable excipient" or "pharmaceuticalally acceptable carrier" refer to excipient materials widely used in the pharmaceutical manufacturing industry. The primary purpose of using excipients is to provide a pharmaceutical composition that is safe to use, stable in nature, and / or has specific functionalities, and also to provide a method for dissolving the active ingredient at a desired rate or promoting the effective absorption of the active ingredient in the body of the administered subject after administration of the drug to a subject. Pharmaceutically acceptable excipients can be inert fillers or functional ingredients that provide a function to the pharmaceutical composition (e.g., stabilizing the overall pH of the composition or preventing degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, sweeteners, etc.

[0631] In this document, the term "pharmaceutical composition" refers to a mixture of one or more of the modified double-stranded ribonucleic acids of this disclosure, or conjugates thereof, with pharmaceutically acceptable excipients / carriers. The purpose of a pharmaceutical composition is to facilitate the administration of the oligonucleotides (e.g., double-stranded ribonucleic acids, particularly siRNA), or modifications thereof, or conjugates thereof, of this disclosure to an organism; it may refer to a composition containing an active ingredient for the treatment of a disease or for use in in vitro cell culture experiments.

[0632] The pharmaceutical compositions disclosed herein, when used for the prevention and / or treatment of diseases, generally refer to unit-dose form and can be formulated into solid, semi-solid, liquid, or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. The pharmaceutical compositions can be prepared using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, and / or lyophilization processes.

[0633] In this disclosure, the route of administration of the oligonucleotides (e.g., double-stranded ribonucleic acid, particularly siRNA), their modifications or conjugates, or pharmaceutical compositions thereof described herein can be varied or modified in any applicable manner to meet the needs of the drug's properties, patient and healthcare personnel convenience, and other relevant factors. Typical routes of administration include, but are not limited to, oral, rectal, local, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0634] As used in the context of this disclosure, the terms “individual,” “patient,” or “subject” include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0635] For purposes of description and disclosure, all patents, patent applications and other identified publications are expressly incorporated herein by reference. Any reference to these publications herein does not constitute an endorsement that such publication is part of the general knowledge in the art.

[0636] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0637] The present application is described in detail below through examples, but these examples do not constitute any adverse limitation on the present application. The compounds of this application can be prepared by various synthetic methods well known to those skilled in the art, including but not limited to the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of this application. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of this application without departing from the spirit and scope of this application, and all such changes and modifications should fall within the protection scope of this application.

[0638] Unless otherwise specified in the embodiments, conditions should be performed according to conventional conditions in the art or conditions recommended by the manufacturer. Unless otherwise stated, all reagents and reagent consumables used in this application whose manufacturers are not specified are products that can be obtained through commercial channels.

[0639] Example

[0640] This application uses the following abbreviations:

[0641] Ac represents acetyl; Boc represents tert-butoxycarbonyl; TsCl represents p-toluenesulfonyl chloride; TMSCl represents trimethylchlorosilane; (i-BuCO)2O represents isobutyric anhydride; DCM represents dichloromethane; THF represents tetrahydrofuran; DMF represents N,N-dimethylformamide; MeOH represents methanol; TBAF represents tetrabutylammonium fluoride; DIPEA represents N,N-diisopropylethylamine; PBS represents phosphate buffer; HATU represents 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; DMP represents Dys-Martin oxidant; Pd / C represents palladium on carbon; DMTrCl represents 4,4'-bismethoxytriphenylmethyl chloride; TBD represents 1,5,7-triazabicyclo[4.4.0]decene-5-ene.

[0642] Commercially available compounds are listed under supplier catalog names.

[0643] Example 1: Compound DT-U NP1 Preparation

[0644]

[0645] Synthesis of compound 1a: Uracil (6.35 g, 56.62 mmol, 1.2 eq), N,O-bis(trimethylsilylacetamide) (24.0 g, 117.98 mmol, 2.5 eq), and acetonitrile (60 mL) were added sequentially to a reaction flask. After purging with N2, the mixture was stirred at 80 °C for 2 h. Then, 1,3-diacetoxy-2-(diacetoxymethoxy)propane (11.72 g, 47.19 mmol, 1 eq) was added. The mixture was cooled to 0 °C in an ice bath, and then trimethylsilyl trifluoromethanesulfonate (15.73 g, 70.79 mmol, 1.5 eq) was added. The mixture was reacted overnight at room temperature. The reaction was quenched by adding saturated sodium bicarbonate aqueous solution / DCM (100 mL / 100 mL). The filter cake was washed with DCM, and the filtrate was separated to obtain the DCM phase. The DCM phase was washed twice with saturated sodium chloride (100 mL), dried, filtered, and evaporated to dryness. The solution was purified by silica gel column chromatography (DCM / methanol: 20 / 1 (v / v)) to give 1a (13 g, yield: 91.83%). MS: m / z 300.8 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ11.39 (s, 1H), 7.72 (d, J = 7.9 Hz, 1H), 5.66 (d, J = 7.9 Hz, 1H), 5.20 (s,2H), 4.15 (m, J = 10.6, 2.7 Hz, 2H), 4.07 – 4.00 (m, 3H), 2.01 (s, 6H).

[0646] Synthesis of compound 1b: 1a (7.00 g, 23.32 mmol, 1.0 eq), methanol (70 mL), and sodium methoxide (3.00 g, 55.98 mmol, 2.4 eq) were added sequentially to a reaction flask, and the reaction was allowed to proceed for 1.5 h at room temperature. The pH was adjusted to 7 with 4N dioxane hydrochloride, and the mixture was concentrated under vacuum to obtain 1b (8 g). MS: m / z 216.8 [M+H] + .

[0647] Synthesis of compound 1c: 1b (8.00 g, 37.03 mmol, 1.0 eq) and pyridine (100 mL) were added to a reaction flask. The mixture was cooled to 0 °C, and TsCl (7.41 g, 38.88 mmol, 1.05 eq) was slowly added. The mixture was then allowed to stand at room temperature overnight. Water (100 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (100 mL), washed once with saturated sodium chloride (200 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The purified ethyl acetate was then purified by silica gel column chromatography to give 1c (2.9 g, yield: 21.17%). MS: m / z 371.0 [M+H] + .

[0648] 1 H NMR (400 MHz, DMSO-d6) δ 11.32 (d, J = 2.2 Hz, 1H), 7.77 (d, J =8.0 Hz, 2H), 7.62 (d, J = 7.9 Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 5.61 (m, J =7.9, 2.1 Hz, 1H), 5.11 (m, J = 10.6 Hz, 2H), 4.89 (m, J = 5.4 Hz, 1H), 4.13(m, J = 10.6, 2.9 Hz, 1H), 3.95 (m, J = 10.6, 6.6 Hz, 1H), 3.81 (m, J = 5.8,2.8 Hz, 1H), 3.39 (d, J = 2.8 Hz, 2H), 2.44 (s, 3H).

[0649] Synthesis of compound 1d: 1c (5.00 g, 13.51 mmol, 1.0 eq) and 25 wt% ammonia (50 mL) were added to a sealed tube. The mixture was sealed and reacted at 80 °C for 5 h. The mixture was then concentrated under vacuum and lyophilized to obtain crude product 1d (4.4 g). Proceed directly to the next step. MS: m / z 216.0 [M+H] + .

[0650] Synthesis of compound 1e: 1d (4.00 g, 18.60 mmol, 1.0 eq), triethylamine (4.71 g, 46.51 mmol, 2.5 eq), and ethanol (80 mL) were added sequentially to a reaction flask, followed by dropwise addition of Boc anhydride (4.47 g, 20.48 mmol, 1.1 eq). The reaction was allowed to proceed to the next step at room temperature for 2 h after the addition was complete. The product was then purified by silica gel column chromatography (dichloromethane / methanol: 25 / 1 (v / v)) after vacuum concentration to give 1e (4.2 g, yield: 71.67%). MS: m / z 313.8 [MH] - .

[0651] 1 H NMR (400 MHz, DMSO-d6) δ 11.38 – 11.18 (m, 1H), 7.53 (d, J = 7.9Hz, 1H), 6.70 (m, J = 5.9 Hz, 1H), 5.60 (m, J = 7.8, 2.0 Hz, 1H), 5.24 – 5.03(m, 2H), 4.72 (m, J = 5.5 Hz, 1H), 3.53 (m, J = 5.5 Hz, 1H), 3.35 (m, J =13.8, 11.3, 8.2 Hz, 3H), 2.91 (m, J = 13.9, 6.3 Hz, 1H), 1.38 (s, 9H).

[0652] Compound DT-U NP1 Synthesis: Ie (240 mg, 0.76 mmol, 1 eq), anhydrous dichloromethane (10 mL), and triethylamine (230.7 mg, 2.28 mmol, 3 eq) were added sequentially to a reaction flask. After the system was fully replaced with N2, N,N-diisopropylphosphonamide (2-cyanoethyl) ester (270.37 mg, 1.14 mmol, 1.5 eq) was added. The mixture was stirred at room temperature for 2 h, and monitored by TLC. After the reaction was complete, the reaction system was evaporated to dryness, and anhydrous dichloromethane was added for wet loading. The mixture was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1 (v / v)) to obtain compound DT-U. NP1 (390 mg, yield: 99.4%). MS: m / z 450.0 [MN(C3H7)2+OH+NH4] + . 1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 7.68 (d, J = 1.3 Hz, 1H), 6.80 (m, J = 5.5 Hz, 1H), 5.61 (m, J = 7.9, 1.1 Hz, 1H), 5.25 – 5.09 (m, 2H),3.80 – 3.69 (m, 3H), 3.62 – 3.49 (m, 4H), 3.11 (s, 1H), 3.02 – 2.94 (m, 1H),2.78 (m, J = 5.9 Hz, 2H), 1.38 (s, 11H), 1.15 (m, J = 6.4 Hz, 12H).

[0653] Example 2: Compound DT-A NP1 Preparation

[0654]

[0655] In Example 1, uracil was replaced with N6-benzoyladenine, and the operation and steps were the same as in Example 1, to obtain compound DT-A. NP1 . MS: m / z 577.4[MN(C3H7)2+OH+NH4] + .

[0656] Example 3: Compound DT-G NP1 Preparation

[0657]

[0658] Synthesis of compounds 3a-3b: Uracil in Example 1 was replaced with ganciclovir, and the procedures and steps were the same as those for the synthesis of 1a and 1b in Example 1, yielding compound 3b. MS: m / z 255.2 [M+H] + .

[0659] Synthesis of compound 3c: Compound 3b (5.29 g, 20.8 mmol, 1 eq) was dissolved in pyridine (212 mL), and TMSCl (32.5 mL, 255.8 mmol, 12.3 eq) was added dropwise. The mixture was stirred at 25 °C for 2 hours. Then (i-BuCO)2O (34.5 mL, 208.1 mmol, 10 eq) was added, and the mixture was stirred at 25 °C for 2 hours. The reaction system was cooled to 0 °C in an ice bath, 30 mL of water was added, and the mixture was stirred for 5 minutes. Then 25 wt% ammonia (30 mL) was added, and the mixture was stirred for 5 minutes. After the reaction was complete, pyridine was removed by rotary evaporation. A saturated aqueous solution of ammonium chloride (100 mL) was added, and the mixture was extracted three times with ethyl acetate (100 mL). The organic phases were combined and washed twice with a saturated aqueous solution of sodium chloride (50 mL). The mixture was then dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: water / acetonitrile = 78 / 22 (v / v)) to give a white solid compound 3c (4.8 g, yield 70.9%). MS: m / z 325.2 [M+H] + .

[0660] Synthesis of compound 3d-3: Replace 1d with 3c in Example 1, and follow the same procedures as in Example 1 to obtain compound DT-G. NP1 . MS: m / z 559.4.[MN(C3H7)2+OH+NH4] + .

[0661] Example 4: Compound DT-C NP1 Preparation

[0662]

[0663] In Example 1, uracil was replaced with N4-acetylcytosine, and the operation method and steps were the same as in Example 1, to obtain compound DT-C. NP1 . MS: m / z 491.4[MN(C3H7)2+OH+NH4] + .

[0664] Example 5: Compound DT-T NP1 Preparation

[0665]

[0666] Replacing uracil with thymine in Example 1, and following the same procedures as in Example 1, compound DT-T was obtained. NP1 . MS: m / z 464.4[MN(C3H7)2+OH+NH4] + .

[0667] Example 6: Compound (Aam-U) np1 Preparation of di

[0668] (1) (Aam-U np1 Synthesis of di-P1

[0669]

[0670] At room temperature, (Aam-Unp1)di-P1-A1 (42.0 g, 61.11 mmol, 1 eq.; N6-benzoyl-5'-O-DMT-2'-methoxyadenosine, CAS No. 110764-72-2) was added to a reaction flask, followed by ethyl acetate (300 mL) as solvent, and then DMP (31.10 g, 73.33 mmol, 1.2 eq.). The reaction was carried out at 35 °C for 12 h, and mass spectrometry confirmed the complete reaction. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under vacuum to obtain compound (Aam-Unp1)di-P1-A2 (62.8 g). MS: m / z 686.2 [M+H] + .

[0671] At room temperature, (Aam-Unp1)di-P1-A2 (62.8 g, 91.65 mmol, 1 eq.) was added to a reaction flask, followed by THF (500 mL) as a solvent, and then ethoxyformylmethylenetriphenylphosphine (47.89 g, 137.47 mmol, 1.5 eq.). The reaction was allowed to proceed overnight at room temperature, and mass spectrometry confirmed the completeness of the reaction. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under vacuum to obtain compound (Aam-Unp1)di-P1-A3 (85.6 g). MS: m / z 756.4 [M+H] + .

[0672] At room temperature, compound (Aam-Unp1)di-P1-A3 (85.6 g, 113.18 mmol, 1 eq.) was added to a reaction flask, followed by dichloromethane (600 mL) as a solvent, and then trifluoroacetic acid (64.53 g, 565.90 mmol, 5 eq.). The reaction was allowed to proceed overnight at room temperature, and mass spectrometry confirmed the completeness of the reaction. The reaction mixture was then poured into a saturated sodium bicarbonate aqueous solution (500 mL), adjusted to pH 7 with sodium bicarbonate, extracted three times with dichloromethane (500 mL), dried over anhydrous sodium sulfate with stirring, filtered, and evaporated to dryness. The purified compound (Aam-Unp1)di-P1-A4 (7.2 g) was obtained by silica gel column chromatography (dichloromethane / methanol = 10 / 1, v / v). MS: m / z 454.2 [M+H] + .

[0673] Compound (Aam-Unp1)di-P1-A4 (3.0 g, 6.61 mmol, 1 eq.) was added to a reaction flask at room temperature. THF (45 mL) was added as a solvent, followed by Pd / C (6.0 g, 10% purity). The mixture was purged three times with hydrogen and reacted at room temperature for 48 h. Mass spectrometry analysis confirmed the reaction was complete. The reaction mixture was filtered through a diatomaceous earth liner, and the filtrate was concentrated under vacuum to obtain compound (Aam-Unp1)di-P1-A5 (2.8 g). MS: m / z 456.2 [M+H] + .

[0674] Compound (Aam-Unp1)di-P1-A5 (2.8 g, 6.14 mmol, 1 eq.) was added to the reaction flask under ice bath conditions. Pyridine (40 mL) was added as a solvent, followed by the slow addition of DMTrCl (3.12 g, 9.21 mmol, 1.5 eq.). After the addition was complete, the mixture was allowed to react overnight at room temperature, and mass spectrometry confirmed the completeness of the reaction. The reaction mixture was then poured into water (50 mL), extracted three times with ethyl acetate (50 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The purified compound was then purified by column chromatography (dichloromethane / methanol = 10 / 1, v / v) to give compound (Aam-Unp1)di-P1-A6 (3.2 g). MS: m / z 758.4 [M+H] + .

[0675] At room temperature, compound (Aam-Unp1)di-P1-A6 (2.0 g, 2.65 mmol, 1 eq.) was added to a reaction flask, followed by methanol / water (4 / 1, 20 mL) as solvent, and then TBD (1.1 g, 7.96 mmol, 3 eq.). The reaction was allowed to proceed for 3 h at room temperature, and mass spectrometry confirmed the complete reaction. The reaction mixture was then poured into water (30 mL), extracted three times with dichloromethane (30 mL), dried over anhydrous sodium sulfate with stirring, filtered, and evaporated to dryness to obtain compound (Aam-Unp1)di-P1 (2.5 g). MS: m / z 730.2 [M+H] + .

[0676] (2) Synthesis of (Aam-Unp1)di-P2

[0677]

[0678] Compound 1e (13.0 g, 41.3 mmol, 1 eq.) from Example 1, imidazole (4.2 g, 61.9 mmol, 1.5 eq.), 130 mL of dichloromethane, and tert-butyldiphenylchlorosilane (17 g, 61.9 mmol, 1.5 eq.) were added sequentially to a reaction flask in an ice bath, and the reaction was carried out at 25 °C for 7 h. The reaction mixture was filtered and concentrated, then extracted three times with water (200 mL) and ethyl acetate (300 mL). The combined ethyl acetate phases were washed twice with saturated sodium chloride water. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: DCM:MeOH = 20:1, v:v) to give compound (Aam-Unp1)di-P2-A1 (9.0 g, yield: 40.4%). MS: m / z 554.1 [M+H] + .

[0679] Compound (Aam-Unp1)di-P2-A1 (5.0 g, 9.02 mmol, 1 eq.), 15 mL of trifluoroacetic acid, and 15 mL of dichloromethane were added sequentially to a reaction flask, and the reaction was carried out at 25 °C for 4 h. The reaction mixture was concentrated and purified by column chromatography (eluent: DCM:MeOH = 00:1) to give (Aam-Unp1)di-P2 (3.2 g, yield: 80.0%). MS: m / z 454.1 [M+H] + .

[0680] (3) Synthesis of (Aam-Unp1)di

[0681]

[0682] At room temperature, (Aam-Unp1)di-P1 (679.0 mg, 0.93 mmol, 1 eq.), (Aam-Unp1)di-P2 (422.0 mg, 0.93 mmol, 1 eq.), HATU (531.1 mg, 1.4 mmol, 1.5 eq.), and DIPEA (361.0 mg, 2.79 mmol, 3 eq.) were weighed sequentially, and DMF (5 mL) was added as a solvent. The reaction was carried out at room temperature for 3 h. The reaction was monitored by TLC until it was complete. The reaction system was poured into water (10 mL), extracted three times with ethyl acetate (15 mL), washed twice with saturated sodium chloride aqueous solution (20 mL), and dried with anhydrous sodium sulfate. The ethyl acetate phase was filtered and evaporated to dryness. The solution was purified by silica gel column chromatography (dichloromethane / methanol = 15 / 1, v / v) to obtain compound (Aam-Unp1)di-A1 (421 mg, yield: 38.8%).

[0683] At room temperature, compound (Aam-Unp1)di-A1 (500 mg, 0.43 mmol, 1 eq.), THF (6 mL), and TBAF (449 mg, 1.72 mmol, 4 eq.) were added to a reaction flask. The reaction was allowed to proceed overnight at room temperature. Mass spectrometry analysis confirmed the reaction was complete. The reaction mixture was then concentrated and purified by silica gel column chromatography (DCM / methanol = 10 / 1, v / v) to give compound (Aam-Unp1)di-A2 (244 mg). MS: m / z 927.6 [M+H] + .

[0684] At room temperature, compound DT(aU) was added to the reaction flask. NP1 (Aam-Unp1)di (364 mg, 0.393 mmol, 1 eq.) was prepared using dichloromethane (2 mL) as solvent. After complete N2 purging, triethylamine (119.3 mg, 1.179 mmol, 3 eq.) was added under ice bath conditions. After 10 min, N,N-diisopropylphosphoramide (2-cyanoethyl) ester (CAS No. 89992-70-1) and chlorphosphine reagent (186 mg, 0.786 mmol, 2 eq.) were added, and the reaction was maintained under ice bath conditions for 6 h. Mass spectrometry confirmed the reaction was complete. The reaction mixture was then evaporated to dryness and purified by silica gel column chromatography (dichloromethane / methanol = 60 / 1, v / v) to give compound (Aam-Unp1)di (400 mg). MS: m / z 1044.0 [MN(C3H7)2+OH+H] + .

[0685] The preparation of the siRNA conjugates described in this application will be further illustrated below with more examples, but these examples do not constitute any limitation on this application. The sequences and structures related to the preparation of the siRNA conjugates are listed in Tables 2 to 4.

[0686] Table 2: Exemplary nucleic acid sequences, their target genes, and off-target genes

[0687]

[0688] Table 3: Exemplary modified double-stranded ribonucleic acid conjugates

[0689]

[0690] In Table 3, lowercase letters a, u, c, g represent nucleotides modified with 2'-methoxy groups; Af, Uf, Cf, Gf represent nucleotides modified with 2'-fluoride groups; lowercase letter s indicates two adjacent nucleotides, and nucleotides are linked to (invAb) by a phosphothioester bond; EVP: trans-vinylphosphonic acid; invAb: reverse debasing deoxyribose residue;C , U This indicates GNA-modified cytosine and uracil nucleotides; T indicates: thymine deoxyribonucleotide; (Aam)U NP1 U NP1 and C NP1 The structure is shown in Table 4;

[0691] L96 has the structure shown in the figure below:

[0692]

[0693] Table 4 Nucleotide residues containing the nucleoside monomers of this application

[0694]

[0695] Preparation Example 1: Synthesis of siRNA conjugates

[0696] Nucleoside monomers are linked sequentially from 3' to 5' along the nucleotide arrangement using the conventional solid-phase phosphoramide method. Each linkage of a nucleoside monomer involves four steps: deprotection, coupling, oxidation or sulfidation, and capping. When two nucleotides are linked using a phosphate ester, the linkage of the next nucleoside monomer involves these four steps. When two nucleotides are linked using a thiophosphate ester, the linkage of the next nucleoside monomer involves these four steps. This application selects nucleotide monomers based on the target sequence. The selected nucleotide monomers are those commonly used by those skilled in the art; for example, the nucleotide monomer for synthesizing oligonucleotides can be, but is not limited to, adenosine-3′-O-phosphoramide. It should be understood that these monomers, when present in oligonucleotides, are linked together via 5'-3' phosphodiester bonds or 5'-3' thiophosphate groups. When, for example, the 3' position of the last nucleotide in the 5' to 3' direction is a hydroxyl group, this is achieved using conventional methods in the art.

[0697] The synthesis conditions for the siRNA in this application are as follows:

[0698] (1) When synthesizing sequences containing only conventional monomers

[0699] The deprotection conditions include: a reaction temperature of 25°C, a reaction time of 70 seconds, a deprotection reagent selected from a dichloromethane solution of dichloroacetic acid (3% v / v), and a molar ratio of the deprotection reagent to the protecting group on the solid support of 5:1.

[0700] The coupling reaction conditions included: a reaction temperature of 25°C, a reaction time of 600 seconds, a coupling reagent selected from a 0.25 M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT), and a molar ratio of nucleic acid sequence to nucleoside monomer linked on the solid-phase support of 1:10.

[0701] The oxidation reaction conditions included: a reaction temperature of 25°C, a reaction time of 15 seconds, an oxidizing agent selected from 0.05 M tetrahydrofuran solution, and a molar ratio of the oxidizing agent to the nucleic acid sequence linked on the solid-phase support in the coupling step of 30:1.

[0702] The sulfidation reaction conditions include: a reaction temperature of 25°C, a reaction time of 300 seconds, a sulfidation reagent selected from hydrogenated xanthanin, and a molar ratio of the sulfidation reagent to the nucleic acid sequence linked on the solid-phase support in the coupling step of 120:1.

[0703] The capping reaction conditions included: a reaction temperature of 25°C, a reaction time of 15 seconds, and the capping reagent being a mixed solution of CapA (10% acetic anhydride acetonitrile solution) and CapB (10% N-methylimidazolium pyridine / acetonitrile solution) in a molar ratio of 1:1. The molar ratio of the capping reagent to the nucleic acid sequence linked on the solid-phase support was acetic anhydride:N-methylimidazolium:the nucleic acid sequence linked on the solid-phase support was 1:1:1.

[0704] (2) Synthesizing a special monomer containing the present application (using DT-U) NP1 DT-A NP1 DT-G NP1 DT-C NP1 Or DT-T NP1 (Single sequence) sequence

[0705] The above method is still used for regular monomers in the sequence, but when special monomers (such as DT-U) are involved... NP1 After coupling and capping, the deprotection process requires removing the solid-phase support intermediate from the synthesis column, adding a 10% (v / v) trifluoroacetic acid dichloromethane solution, reacting at 25°C for 30 min, centrifuging, discarding the supernatant, and repeating this operation four times. Then, this solid-phase support intermediate is repacked into the synthesis column, and the remaining base sequence is synthesized using conventional methods.

[0706] (3) Synthesize a sequence containing the special monomer of this application (using the (Aam-Unp1)di sequence).

[0707] The sequence using (Aam-Unp1)di is still processed using the conventional method with regular monomers.

[0708] After linking all nucleoside monomers, the nucleic acid sequences linked on the solid-phase support were sequentially subjected to ammonolysis, purification, and desalting to obtain the siRNA sense and antisense strands. Finally, the two strands were heated and annealed to obtain the product.

[0709] Preparation Example 2: Synthesis of siRNA conjugate (GalNAc-siRNA)

[0710] 2.1 The siRNA conjugate has the structure shown in Formula III:

[0711]

[0712] Formula III

[0713] 2.2 Synthesis process of siRNA conjugates

[0714] The first step involves reacting DMTr-L96 with succinic anhydride to obtain compound L96-A:

[0715]

[0716] Preparation process: DMTr-L96, succinic anhydride, 4-dimethylaminopyridine, and diisopropylethylamine were added to dichloromethane and stirred at 25°C for 24 hours. The reaction solution was then washed with 0.5 M triethylamine phosphate, and the aqueous phase was washed three times with dichloromethane. The combined organic phases were evaporated to dryness under reduced pressure to obtain the crude product. Then, column chromatography was used to purify the product to obtain pure L96-A.

[0717] The second step involves reacting L96-A with NH2-SPS to obtain L96-B:

[0718]

[0719] Preparation process: L96-A, O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), and diisopropylethylamine (DIPEA) were mixed and dissolved in acetonitrile. The mixture was stirred at room temperature for 5 minutes to obtain a homogeneous solution. Aminomethyl resin (NH2-SPS, 100-200 mesh) was added to the reaction solution, and the reaction was initiated at 25°C on a shaker. After 18 hours of reaction, the mixture was filtered. The filter cake was washed successively with dichloromethane and acetonitrile to obtain the filter cake. The obtained filter cake was subjected to a capping reaction with a CapA / CapB mixed solution to obtain L96-B, which is the solid-phase support containing the conjugated molecules.

[0720] The third step is the synthesis of siRNA conjugates:

[0721] Using L96-B as a solid-phase support, the sense strand of the siRNA conjugate was synthesized according to the conventional monomer siRNA synthesis method described above. The antisense strand of the siRNA conjugate was synthesized using the siRNA synthesis method described above. Annealing was then performed to generate the siRNA conjugate of this application.

[0722] Ammonolysis, purification, desalting, and annealing methods are well known in the art. For example, ammonolysis is performed by contacting the nucleotide sequence linked to a solid-phase support with concentrated ammonia (e.g., a 25wt%-28wt% ammonia solution); purification is performed by chromatography; desalting is performed by reversed-phase chromatography; and the corresponding siRNA conjugate in Table 3 of this application is obtained by gradually cooling after mixing the sense and antisense strands in equimolar ratios under different stringent conditions.

[0723] Experimental Example 1

[0724] In vitro cell experiments on the anti-off-target activity of siRNA modifiers and conjugates targeting CFB.

[0725] Hep3B cells, purchased from ATCC, catalog number HB-8064;

[0726] Lipofectamine RNAiMax transfection reagent, purchased from Invitrogen, catalog number 13778-150;

[0727] Opti-medium: serum-reduced culture medium, purchased from Gibco, catalog number 31985-070;

[0728] EMEM culture medium: ATCC, catalog number 30-2003;

[0729] TaqMan™ gene expression premix, purchased from Applied Biosystems, catalog number 4369016;

[0730] VAMNE Magnetic Cell / Tissue Total RNA Kit, purchased from Novizan, catalog number RMA101-C2;

[0731] Phosphate Buffer Saline (PBS), purchased from Gibco, catalog number 10010-023;

[0732] GAPDH probe, purchased from Thermo, part number 4326317E (VIC);

[0733] CFB TaqMan probe, purchased from Thermo, part number 4351370; ID: Hs00156060_m1 (FAM);

[0734] ZCCHC17 primers were purchased from Yaoyuan Biotechnology (Shanghai) Co., Ltd.

[0735] Experimental steps:

[0736] 1. Human hepatocellular carcinoma cells (Hep3B) were seeded in fresh Opti-medium medium in 96-well plates and cultured for 24 hours. The cultured cells were then resuspended in EMEM medium without PS (penicillin-streptomycin mixture) to a density of 5.55 × 10⁻⁶ cells / well. 4 A 90 μL / well cell suspension was spread into each well of a 96-well plate, resulting in 5000 cells per well.

[0737] 2. The dry powder of the siRNA to be tested was centrifuged at low temperature and high speed, and then dissolved in ultrapure distilled water to prepare a 100 μM siRNA stock solution.

[0738] 3. Prepare a siRNA solution with a final concentration of 10 nM.

[0739] (1) Preparation of 10 μM siRNA stock solution: Take 2 μL of the 100 μM siRNA stock solution prepared above and add 18 μL of ultrapure distilled water to obtain a 10 μM siRNA stock solution.

[0740] (2) Preparation of 0.1 nM siRNA transfection dilution: Take 2 μL of 10 μM siRNA stock solution and add 98 μL of ultrapure distilled water to obtain a 200 nM siRNA dilution; then add an equal volume of Lipofectamine RNAiMax transfection reagent dilution (add 3 μL of Lipofectamine RNAiMax transfection reagent to 97 μL of Opti-medium to obtain Lipofectamine RNAiMax transfection reagent dilution) to obtain a 100 nM siRNA transfection dilution;

[0741] (3) Take 10 μL of the above 100 nM siRNA transfection dilution solution and add it to a 96-well plate containing Hep3B cells to obtain a final siRNA concentration of 10 nM.

[0742] Other final concentrations of siRNA solutions (2.5 nM, 0.625 nM, 0.156 nM, 0.039 nM, 0.001 nM, 0.0025 nM, and 0.0006 nM) should be prepared following the steps described above.

[0743] 4. After transfection, cells were cultured for 48 hours, with two replicates for each concentration (10 nM, 2.5 nM, 0.625 nM, 0.156 nM, 0.039 nM, 0.001 nM, 0.0025 nM and 0.0006 nM).

[0744] 5. RNA extraction using the VAMNE Magnetic Cell / Tissue Total RNA Kit:

[0745] 6. Reverse transcribe the RNA into cDNA, following these steps:

[0746] a) Prepare the reverse transcription reaction mixture as shown in the table below and mix thoroughly. Keep all reagents on ice throughout the procedure:

[0747]

[0748] b) 42℃ for 2 minutes;

[0749] c) Prepare the second-step reverse transcription reaction mixture as shown in the table below and mix thoroughly;

[0750]

[0751] d) Perform the reverse transcription procedure as described below.

[0752] 50℃, 15 min; 85℃, 5 s; stand at 4℃.

[0753] 7. Perform real-time PCR analysis

[0754] a) Prepare the qPCR reaction mixture as shown in the table below. Throughout the procedure, keep all reagents on ice:

[0755]

[0756] b) Perform the qPCR procedure as described below.

[0757] 50℃, 2 min, 95℃, 10 min; 95℃, 15 s, 60℃, 1 min, 40 cycles.

[0758] 8. Results Analysis

[0759] a) Use Quant Studio 7 software with default settings to automatically calculate the Ct value;

[0760] b) Calculate the relative expression level of the gene using the following formula:

[0761] ΔCt = Ct (target gene) –Ct (ACTB)

[0762] ΔCt = ΔCt (test sample group) - ΔCt (Mock group), where the Mock group represents the group without siRNA compared to the test sample group;

[0763] mRNA expression relative to the Mock group = 2 -ΔΔCt

[0764] Inhibition rate (%) = (Relative expression level of mRNA in the Mock group – Relative expression level of mRNA in the test sample group) / Relative expression level of mRNA in the Mock group × 100%.

[0765] Using the log value of siRNA concentration as the X-axis and the percentage inhibition rate as the Y-axis, the dose-response curve was fitted using the "log (inhibitor) vs. response variable slope" function module of GraphPadPrism 8 software to obtain the IC50 value of each siRNA. 50 value.

[0766] The fitting formula is: Y = Bottom + (Top – Bottom) / (1 + 10^(logIC)) 50 –X)*HillSlope))

[0767] Where: Top represents the percentage inhibition rate at the top plateau, and the standard for the Top of the curve is generally between 80% and 120%; Bottom represents the percentage inhibition rate at the bottom plateau, and the Bottom of the curve is generally between -20% and 20%; HillSlope represents the slope of the percentage inhibition rate curve.

[0768] The experimental results are shown in Table 5 below.

[0769] Table 5

[0770]

[0771] The results showed that the sequence N-ER-FY010124M51L96 had good inhibitory activity against the target gene CFB, but its activity against gene ZCCHC17 was comparable to its on-target activity, and the off-target activity / on-target activity IC50 ratio was [not specified]. 50 The ratio is 1.06, indicating a high risk of off-target effects and potential safety concerns. In N-ER-FY010124M51L96, the uracil nucleotide U at position 6 of the antisense strand should be replaced with U... NP1 After (N-ER-FY010124M61L96), the target activity was still maintained, but the activity against the off-target gene ZCCHC17 was significantly reduced, compared with GNA monomer modification (N-ER-FY010124M60L96). NP1 The activity was not reduced after the introduction of monomers, but the activity was reduced by 5 times after the introduction of GNA.

[0772] Experimental Example 2: In vitro cell experiments demonstrating the anti-off-target activity of siRNA modifiers and conjugates targeting SCAP.

[0773] Experimental materials:

[0774] Huh7 cells were purchased from JCRB cell bank, catalog number JCRB0403;

[0775] PBS, purchased from MACGENE, catalog number CC006;

[0776] Trypsin, purchased from Gibco, catalog number 15400054;

[0777] Lipo® RNAiMAX transfection reagent, purchased from Invitrogen, catalog number 13778-150;

[0778] RNA extraction kit (RNeasy® 96 Kit), purchased from QIAGEN, catalog number 74106;

[0779] Fastking RT Kit (with gDNase), purchased from TIANGEN, item number KR116-02;

[0780] Reverse transcription kit, purchased from Vazyme, catalog number R312-02;

[0781] Opti-medium: serum-reduced culture medium, purchased from Gibco, catalog number 31985-070;

[0782] SCAP probe, purchased from Thermo, part number Hs00378725_m1 (FAM);

[0783] GAPDH probe, purchased from Thermo, part number 4326317E;

[0784] TaqMan™ gene expression premix, purchased from Applied Biosystems, catalog number 4369016;

[0785] TRIQK and SCOC primers were purchased from Yaoyuan Biotechnology (Shanghai) Co., Ltd.

[0786] Experimental methods:

[0787] 1. Take Huh7 cells, wash them twice with PBS, add trypsin to digest them, adjust the cell density, and then seed them into 96-well plates at a density of 20,000 cells per well, with 100 µL of culture medium per well. Incubate the Huh7 cells overnight in a 5% CO2, 37°C incubator.

[0788] 2. The dry powder of the siRNA to be tested was centrifuged at low temperature and high speed, and then dissolved in ultrapure distilled water to prepare a 100 μM siRNA stock solution.

[0789] 3. Prepare a siRNA solution with a final concentration of 1 nM.

[0790] (1) Preparation of 10 μM siRNA stock solution: Take 2 μL of the above 100 μM siRNA stock solution and add 18 μL of ultrapure distilled water to obtain a 10 μM siRNA stock solution;

[0791] (2) Preparation of 1 nM siRNA transfection dilution: Take 2 μL of 10 μM siRNA stock solution and add 18 μL of ultrapure distilled water to obtain a 1 μM siRNA dilution; take 2 μL of 1 μM siRNA and add 98 μL of ultrapure distilled water to obtain a 20 nM siRNA dilution. Add an equal volume of Lipofectamine RNAiMax transfection reagent dilution (add 3 μL of Lipofectamine RNAiMax transfection reagent to 97 μL of Opti-medium to obtain Lipofectamine RNAiMax transfection reagent dilution) to obtain a 10 nM siRNA transfection dilution.

[0792] (3) Take 10 μL of the above 10 nM siRNA dilution solution and add it to a 96-well plate containing Huh7 cells to obtain a final siRNA concentration of 1 nM.

[0793] Other final concentrations of siRNA solution (0.1 nM) should be prepared following the steps described above.

[0794] 4. After transfection, cells were cultured for 48 hours, with two replicates for each concentration (1 nM and 0.1 nM).

[0795] 5. Extract total RNA from the transfected Huh7 cells obtained in step 4 according to the RNeasy® 96 Kit product instructions.

[0796] 6. Reverse transcribe the extracted total RNA into cDNA using a Fastking RT Kit (containing gDNase), following these steps:

[0797] a) Remove gDNA with gDNase according to the table below.

[0798]

[0799] 42℃, 2 min; 4℃, stand.

[0800] b) Add the reagents described below to the system obtained in step a) and perform reverse transcription:

[0801]

[0802] 50℃, 15 min; 85℃, 5 s.

[0803] c) Store the reverse transcription product obtained in step b) at -20°C for real-time PCR analysis.

[0804] 7. Perform real-time PCR analysis

[0805] a) Prepare the real-time PCR reaction mixture as shown in the table below. All reagents should be kept on ice throughout the entire procedure:

[0806]

[0807] b) Perform the qPCR procedure as follows:

[0808] 50℃, 2 minutes; 95℃, 10 minutes;

[0809] 95℃, 15 seconds, 60℃, 1 minute (this operation is repeated 40 times).

[0810] 8. Results Analysis

[0811] a) Use Quant Studio 7 software with default settings to automatically calculate the Ct value;

[0812] b) Calculate the relative expression level of the gene using the following formula:

[0813] ΔCt = Ct (target gene) – Ct (GAPDH)

[0814] ΔCt = ΔCt (test sample group) - ΔCt (Mock group), where the Mock group represents the group without siRNA compared to the test sample group;

[0815] mRNA expression relative to the Mock group = 2 -ΔΔCt .

[0816] Inhibition rate = (mRNA expression level in the Mock group – mRNA expression level in the test sample group) / mRNA expression level in the Mock group × 100%

[0817] The results are shown in Table 6 below.

[0818] Table 6

[0819]

[0820] The results showed that the sequence N-ER-FY022041M44L96 exhibited inhibitory activity against the target gene SCAP and off-target genes TRIQK and SCOC at both 1 nM and 0.1 nM doses. Furthermore, it showed considerable inhibitory activity against both SCAP and TRIQK at these doses, indicating a high risk of off-target effects and potential safety concerns. Introducing a GNA monomer to form the sequence N-ER-FY022041M5L96 retained the activity against the target gene SCAP, but completely lost its inhibitory activity against the off-target genes TRIQK and SCOC. Replacing the GNA monomer in the N-ER-FY022041M5L96 sequence with the monomer U from this application further demonstrated its effectiveness. NP1 The N-ER-FY022041M70L96 sequence was subsequently formed. This siRNA conjugate showed an increase of approximately 10% in on-target activity at both concentration points, while exhibiting no inhibitory activity against TRIQK and SCOC, and a low off-target risk.

[0821] Experimental Example 3: In vitro cell experiments demonstrating the anti-off-target activity of siRNA modifiers and conjugates targeting INHBE.

[0822] Experimental materials:

[0823] Huh7 cells were purchased from JCRB cell bank, catalog number JCRB0403;

[0824] PBS, purchased from MACGENE, catalog number CC006;

[0825] Trypsin, purchased from Gibco, catalog number 15400054;

[0826] Lipo® RNAiMAX transfection reagent, purchased from Invitrogen, catalog number 13778-150;

[0827] RNA extraction kit (RNeasy® 96 Kit), purchased from QIAGEN, catalog number 74106;

[0828] VAMNE Magnetic Cell / Tissue Total RNA Kit, purchased from Novizan, catalog number RMA101-C2;

[0829] Reverse transcription kit, purchased from Vazyme, catalog number R312-02;

[0830] Opti-medium: serum-reduced culture medium, purchased from Gibco, catalog number 31985-070;

[0831] INHBE probe, purchased from Thermo, part number Hs00368884_g1 (FAM);

[0832] GAPDH probe, purchased from Thermo, part number 4326317E;

[0833] LMAN1 primers, FKBP1A and GAPDH primers were synthesized by Yaoyuan Biotechnology (Shanghai) Co., Ltd.

[0834] TaqMan™ gene expression premix, purchased from Applied Biosystems, catalog number 4369016.

[0835] Experimental methods:

[0836] 1. Take Huh7 cells, wash them twice with PBS, add trypsin to digest them, adjust the cell density, and then seed them into 96-well plates at a density of 20,000 cells per well, with 100 µL of culture medium per well. Incubate the Huh7 cells overnight in a 5% CO2, 37°C incubator.

[0837] 2. The dry powder of the siRNA to be tested was centrifuged at low temperature and high speed, and then dissolved in ultrapure distilled water to prepare a 100 μM siRNA stock solution.

[0838] 3. Prepare a siRNA solution with a final concentration of 1 nM.

[0839] (1) Preparation of 10 μM siRNA stock solution: Take 2 μL of the above 100 μM siRNA stock solution and add 18 μL of ultrapure distilled water to obtain a 10 μM siRNA stock solution;

[0840] (2) Preparation of 1 nM siRNA transfection dilution: Take 2 μL of 10 μM siRNA stock solution and add 18 μL of ultrapure distilled water to obtain a 1 μM siRNA dilution; take 2 μL of 1 μM siRNA and add 98 μL of ultrapure distilled water to obtain a 20 nM siRNA dilution. Add an equal volume of Lipofectamine RNAiMax transfection reagent dilution (add 3 μL of Lipofectamine RNAiMax transfection reagent to 97 μL of Opti-medium to obtain Lipofectamine RNAiMax transfection reagent dilution) to obtain a 10 nM siRNA transfection dilution.

[0841] (3) Take 10 μL of the above 10 nM siRNA dilution solution and add it to a 96-well plate containing Huh7 cells to obtain a final siRNA concentration of 1 nM.

[0842] Other final concentrations of siRNA solution (0.1 nM) should be prepared following the steps described above.

[0843] 4. After transfection, cells were cultured for 48 hours, with two replicates for each concentration (1 nM and 0.1 nM).

[0844] 5. Extract total RNA from the transfected Huh7 cells obtained in step 4 according to the RNeasy® 96 Kit product instructions.

[0845] 6. Reverse transcribe the extracted total RNA into cDNA using a Fastking RT Kit (containing gDNase), following these steps:

[0846] a) Remove gDNA with gDNase according to the table below.

[0847]

[0848] 42℃, 2 min; 4℃, stand.

[0849] b) Add the reagents described below to the system obtained in step a) and perform reverse transcription:

[0850]

[0851] 50℃, 15 min; 85℃, 5 s.

[0852] c) Store the reverse transcription product obtained in step b) at -20°C for real-time PCR analysis.

[0853] 7. Perform real-time PCR analysis

[0854] a) Prepare the real-time PCR reaction mixture as shown in the table below. All reagents should be kept on ice throughout the entire procedure:

[0855]

[0856] b) Perform the qPCR procedure as follows:

[0857] 50℃, 2 minutes; 95℃, 10 minutes;

[0858] 95℃, 15 seconds, 60℃, 1 minute (this operation is repeated 40 times).

[0859] 8. Results Analysis

[0860] a) Use Quant Studio 7 software with default settings to automatically calculate the Ct value;

[0861] b) Calculate the relative expression level of the gene using the following formula:

[0862] ΔCt = Ct (target gene) – Ct (GAPDH)

[0863] ΔCt = ΔCt (test sample group) - ΔCt (Mock group), where the Mock group represents the group without siRNA compared to the test sample group;

[0864] mRNA expression relative to the Mock group = 2 -ΔΔCt .

[0865] Inhibition rate = (mRNA expression level in the Mock group – mRNA expression level in the test sample group) / mRNA expression level in the Mock group × 100%

[0866] The results showed that sequences N-ER-FY046134M11L96 and N-ER-FY046134M47L96 exhibited good inhibitory activity against the target gene INHBE and off-target genes LMAN1 and FKBP1A at 1 nM and 0.1 nM, respectively, but also had a high off-target risk, posing safety concerns. These two modified sequences were introduced into the monomer C of this application. NP1 The resulting N-ER-FY046134M70L96 and N-ER-FY046134M71L96 siRNA conjugates enhanced on-target activity at both concentration points while exhibiting no inhibitory activity against LMAN1 and FKBP1A, with a low off-target risk.

[0867] Experiment Example 4: In vitro anti-off-target activity test of siRNA modifiers and conjugates targeting C5

[0868] 1. Experimental materials:

[0869] HepG2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number SCSP-510.

[0870] RNA extraction kit, purchased from QIAGEN, catalog number 74106;

[0871] Lipo®RNAiMAX 2000 transfection reagent, purchased from Invitrogen, catalog number 11668019;

[0872] MEM medium, purchased from Gibco, catalog number 11090-081;

[0873] The reverse transcription kit (HiScript® Ⅲ 1st Strand cDNA Synthesis Kit (+gDNA wiper)) was purchased from Vazyme, catalog number R312-02;

[0874] TaqMan™ gene expression premix, purchased from Applied Biosystems, catalog number 4369016;

[0875] Opti-MEM: serum-reduced culture medium, purchased from Gibco, catalog number 31985-070;

[0876] C5 probe, purchased from Thermo, part number Hs00156197_m1 (FAM);

[0877] GAPDH probe, purchased from Thermo, part number 4326317E;

[0878] The DPY19L1 primer and FANCG primer were synthesized by Yaoyuan Biotechnology (Shanghai) Co., Ltd.

[0879] 2. Experimental Methods:

[0880] 2.1 HepG2 cells were seeded in 96-well plates in Opti-MEM medium and cultured for 24 hours. The cultured cells were then resuspended in PS-free (penicillin-streptomycin mixture) MEM medium to a density of 1.11 × 10⁻⁶ cells / well. 5 A cell suspension of 10,000 cells / mL was spread into a 96-well plate, with 90 μL of cell suspension added to each well, resulting in 10,000 cells / well.

[0881] 2.2 The dry powder of the siRNA to be tested was centrifuged at low temperature and high speed, and then dissolved in ultrapure distilled water to prepare a 10 μM siRNA stock solution.

[0882] 2.3 Take the 10 μM siRNA stock solution prepared above and dilute it with enzyme-free water to the required concentration of siRNA dilution (e.g., 200 nM siRNA dilution W).

[0883] 2.4 Transfection of HepG2 cells

[0884] (1) Take 3 μL of Lipo® RNAiMAX transfection reagent and add 97 μL of Opti-MEM to obtain Lipo® RNAiMAX transfection reagent dilution; mix the Lipo® RNAiMAX transfection reagent dilution with the 200 nM siRNA dilution W prepared in step 2.3 at a volume ratio of 1:1, let stand for 15 minutes, and add 10 μL of the transfection mixture to a 96-well plate to transfect the HepG2 cells cultured in step 2.1 (final volume 100 μL, the concentration of siRNA in this system is 10 nM).

[0885] The preparation of siRNA solutions of other concentrations (2.5 nM, 0.625 nM, 0.156 nM, 0.039 nM, 0.001 nM, 0.0024 nM, and 0.0006 nM) is the same as the experimental method described above.

[0886] The cells were cultured for 48 hours after transfection; two replicates were set up for each concentration (10 nM, 2.5 nM, 0.625 nM, 0.156 nM, 0.039 nM, 0.001 nM, 0.0024 nM and 0.0006 nM).

[0887] 2.5 Extract total RNA from HepG2 cells obtained in step 2.4 according to the RNA extraction kit instructions.

[0888] 2.6 The total RNA obtained in step 2.5 was reverse transcribed into cDNA using a reverse transcription kit, following these steps:

[0889] a) Remove gDNA using gDNAase according to the table below;

[0890]

[0891] 42°C, 2 minutes; 4°C, stand.

[0892] b) Add the reagents described below to the system obtained in step a) and perform reverse transcription:

[0893]

[0894] 50°C, 15 minutes; 85°C, 5 seconds.

[0895] c) Store the reverse transcription product obtained in step b) at 4°C for real-time PCR analysis.

[0896] 2.7 Perform real-time PCR analysis

[0897] a) Prepare the qPCR reaction mixture as shown in the table below. Keep all reagents on ice throughout the entire process.

[0898]

[0899] b) Perform the qPCR procedure as described below.

[0900] 50°C, 2 minutes; 95°C, 10 minutes;

[0901] 95°C, 15 seconds, 60°C, 1 minute (this operation is repeated 40 times).

[0902] 2.8 Results Analysis

[0903] a) Use Quant Studio 6 Flex software with default settings to automatically calculate the Ct value;

[0904] b) Calculate the relative expression level of the gene using the following formula:

[0905] ΔCt = Ct (target gene) –Ct (GAPDH)

[0906] ΔCt = ΔCt (sample group) - ΔCt (mock group)

[0907] mRNA expression relative to the Mock group = 2 -ΔΔCt .

[0908] Mock group: Compared to the test sample group, the group without siRNA was not added.

[0909] Inhibition rate (%) = (Relative expression level of mRNA in the Mock group – Relative expression level of mRNA in the test sample group) / Relative expression level of mRNA in the Mock group × 100%

[0910] IC 50 Calculation process: Using the log value of siRNA concentration as the X-axis and the percentage inhibition rate as the Y-axis, the dose-response curve was fitted using the "log (inhibitor) vs. response variable slope" function module of GraphPad Prism 8 software to obtain the IC50 of each siRNA conjugate. 50 value.

[0911] The fitting formula is: Y = Bottom + (Top - Bottom) / (1 + 10^(logIC)) 50 -X)*HillSlope))

[0912] Where: Top represents the percentage inhibition rate at the top plateau, and the standard for the Top of the curve is generally between 80% and 120%; Bottom represents the percentage inhibition rate at the bottom plateau, and the Bottom of the curve is generally between -20% and 20%; HillSlope represents the slope of the percentage inhibition rate curve.

[0913] 3. The experimental results are shown in Table 7 below.

[0914] Table 7

[0915]

[0916] The results showed that N-ER-FY008085M9L96 exhibited considerable inhibitory activity against the target gene C5 and off-target genes DPY19L1 and FANCG, but also had a high off-target risk, posing safety concerns. The U introduced in this application... NP1 The on-target activity of N-ER-FY008085M88L96 is comparable to that of N-ER-FY008085M9L96 without its introduction, and its inhibitory activity against off-target genes DPY19L1 and FANCG is very weak, meaning that the off-target risk of DPY19L1 and FANCG is low.

[0917] Experimental Example 5: In vitro anti-off-target activity assay of siRNA modifiers and conjugates targeting PNPLA3.

[0918] 1. Experimental materials:

[0919] HepG2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number SCSP-510.

[0920] RNA extraction kit, purchased from QIAGEN, catalog number 74106;

[0921] Lipo®RNAiMAX 2000 transfection reagent, purchased from Invitrogen, catalog number 11668019;

[0922] MEM medium, purchased from Gibco, catalog number 11090-081;

[0923] The reverse transcription kit (HiScript® Ⅲ 1st Strand cDNA Synthesis Kit (+gDNA wiper)) was purchased from Vazyme, catalog number R312-02;

[0924] TaqMan™ gene expression premix, purchased from Applied Biosystems, catalog number 4369016;

[0925] Opti-MEM: serum-reduced culture medium, purchased from Gibco, catalog number 31985-070;

[0926] PNPLA3 primers, PHYH primers, and GAPDH primers were synthesized by Yaoyuan Biotechnology (Shanghai) Co., Ltd.

[0927] 2. Experimental Methods:

[0928] 2.1 HepG2 cells were seeded in 96-well plates in Opti-MEM medium and cultured for 24 hours. The cultured cells were then resuspended in PS-free (penicillin-streptomycin mixture) MEM medium to a density of 1.11 × 10⁻⁶ cells / well. 5A cell suspension of 10,000 cells / mL was spread into a 96-well plate, with 90 μL of cell suspension added to each well, resulting in 10,000 cells / well.

[0929] 2.2 The dry powder of the siRNA to be tested was centrifuged at low temperature and high speed, and then dissolved in ultrapure distilled water to prepare a 10 μM siRNA stock solution.

[0930] 2.3 Take the 10 μM siRNA stock solution prepared above and dilute it with enzyme-free water to the required concentration of siRNA dilution (e.g., 200 nM siRNA dilution W).

[0931] 2.4 Transfection of HepG2 cells

[0932] (1) Take 3 μL of Lipo® RNAiMAX transfection reagent and add 97 μL of Opti-MEM to obtain Lipo® RNAiMAX transfection reagent dilution; mix the Lipo® RNAiMAX transfection reagent dilution with the 200 nM siRNA dilution W prepared in step 2.3 at a volume ratio of 1:1, let stand for 15 minutes, and add 10 μL of the transfection mixture to a 96-well plate to transfect the HepG2 cells cultured in step 2.1 (final volume 100 μL, the concentration of siRNA in this system is 10 nM).

[0933] The preparation of siRNA solutions of other concentrations (2.5 nM, 0.625 nM, 0.156 nM, 0.039 nM, 0.001 nM, 0.0024 nM, and 0.0006 nM) is the same as the experimental method described above.

[0934] The cells were cultured for 48 hours after transfection; two replicates were set up for each concentration (10 nM, 2.5 nM, 0.625 nM, 0.156 nM, 0.039 nM, 0.001 nM, 0.0024 nM and 0.0006 nM).

[0935] 2.5 Extract total RNA from HepG2 cells obtained in step 2.4 according to the RNA extraction kit instructions.

[0936] 2.6 The total RNA obtained in step 2.5 was reverse transcribed into cDNA using a reverse transcription kit, following these steps:

[0937] a) Remove gDNA using gDNAase according to the table below;

[0938]

[0939] 42°C, 2 minutes; 4°C, stand.

[0940] b) Add the reagents described below to the system obtained in step a) and perform reverse transcription:

[0941]

[0942] 50°C, 15 minutes; 85°C, 5 seconds.

[0943] c) Store the reverse transcription product obtained in step b) at 4°C for real-time PCR analysis.

[0944] 2.7 Perform real-time PCR analysis

[0945] a) Prepare the qPCR reaction mixture as shown in the table below. Keep all reagents on ice throughout the entire process.

[0946]

[0947] b) Perform the qPCR procedure as described below.

[0948] 50°C, 2 minutes; 95°C, 10 minutes;

[0949] 95°C, 15 seconds, 60°C, 1 minute (this operation is repeated 40 times).

[0950] 2.8 Results Analysis

[0951] a) Use Quant Studio 6 Flex software with default settings to automatically calculate the Ct value;

[0952] b) Calculate the relative expression level of the gene using the following formula:

[0953] ΔCt = Ct (target gene) –Ct (GAPDH)

[0954] ΔCt = ΔCt (sample group) - ΔCt (mock group)

[0955] mRNA expression relative to the Mock group = 2 -ΔΔCt .

[0956] Mock group: Compared to the test sample group, the group without siRNA was not added.

[0957] Inhibition rate (%) = (Relative expression level of mRNA in the Mock group – Relative expression level of mRNA in the test sample group) / Relative expression level of mRNA in the Mock group × 100%

[0958] IC 50Calculation process: Using the log value of siRNA concentration as the X-axis and the percentage inhibition rate as the Y-axis, the dose-response curve was fitted using the "log (inhibitor) vs. response variable slope" function module of GraphPad Prism 8 software to obtain the IC50 of each siRNA conjugate. 50 value.

[0959] The fitting formula is: Y = Bottom + (Top - Bottom) / (1 + 10^(logIC)) 50 -X)*HillSlope))

[0960] Where: Top represents the percentage inhibition rate at the top plateau, and the standard for the Top of the curve is generally between 80% and 120%; Bottom represents the percentage inhibition rate at the bottom plateau, and the Bottom of the curve is generally between -20% and 20%; HillSlope represents the slope of the percentage inhibition rate curve.

[0961] 3. The experimental results are shown in Table 8 below.

[0962] Table 8

[0963]

[0964] The results showed that N-ER-FY006052M8L96 exhibited considerable inhibitory activity against both the target gene PNPLA3 and the off-target gene PHYH, but also had a high off-target risk, posing safety concerns. (This application incorporates U...) NP1 The on-target activity of N-ER-FY006052M112L96 is comparable to that of N-ER-FY006052M8L966 without its introduction, but its inhibitory activity against the off-target gene PHYH is very weak, meaning that the risk of PHYH off-target is low.

[0965] The above specific embodiments are merely illustrative of the content of this application and do not constitute a limitation thereof. For those skilled in the art, any modifications, alterations, or equivalent substitutions made without departing from the spirit and substance of this application should be considered to fall within the protection scope of this application.

Claims

1. A modification of a double-stranded ribonucleic acid (BRNA) or a conjugate thereof, said BRNA comprising a sense strand and an antisense strand, each strand having 15-40 nucleotides; wherein at least one nucleotide at position 2-8 of the antisense strand is replaced by a nucleotide monomer of formula (II) in the 5' to 3' direction: Equation (II), in, X is selected from O or S; R2 is selected from hydrogen, deuterium, or C. 1-3 alkyl; Wherein, Base represents an unmodified or modified nucleobase, the unmodified or modified nucleobase having a structure selected from any of the following: , , ,or , Among them, R4, R5, R6, R7, R8, R9, R 10 Each is independently selected from hydrogen, deuterium, and C. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, 3-6 membered cycloalkyl, or -(C=O)C 1-6 alkyl.

2. The modified double-stranded ribonucleic acid or its conjugate as described in claim 1, wherein, X is O; Alternatively, R2 is selected from hydrogen, deuterium, or methyl, preferably, R2 is hydrogen; Or, R 10 Selected from hydrogen, deuterium, and C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, 3-6 membered cycloalkyl, or -(C=O)C 1-6 alkyl; Or, R4, R5, R6, R7, R8, R9, R 10 Each is independently selected from hydrogen, deuterium, and C. 1-3 Alkyl, C 1-3 Alkoxy, C 1-3 Halogenated alkyl, 3-6 membered cycloalkyl, or -(C=O)C 1-3 alkyl, Or, R4, R5, R6, R7, R8, R9, R 10 Each is independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, methoxyethyl, cyclopropyl, fluoromethyl, difluoromethyl, or acetyl; Alternatively, R4 or R5 can be independently selected from hydrogen or C. 1-3 Alkyl groups, preferably hydrogen or methyl groups; Alternatively, both R4 and R5 may be hydrogen; or R4 may be hydrogen and R5 may be methyl. Alternatively, R6 and R7 are hydrogen; Alternatively, R8 and R9 are hydrogen; Or, R 10 It can be hydrogen or methyl.

3. The modified double-stranded ribonucleic acid or its conjugate as described in claim 1 or 2, wherein, The Base is selected from any of the following: , , or , R5, R7, or R9 are each independently selected from hydrogen or C. 1-3 Alkyl, R 10 Selected from hydrogen or C 1-6 Alkyl; preferably, R 10 Selected from hydrogen or C 1-3 alkyl.

4. The modified double-stranded ribonucleic acid or its conjugate as described in any one of claims 1-3, wherein, The nucleoside monomer represented by formula (II) is selected from one or more of the following: Preferably, the nucleoside monomer represented by formula (II) is selected from one or more of the following: 。 5. A modification of the double-stranded ribonucleic acid or a conjugate thereof as described in any one of claims 1-4, wherein, In the direction from the 5' end to the 3' end, one, two, three, four, or five nucleoside residues at positions 2, 3, 4, 5, 6, 7, or 8 of the antisense strand are replaced by nucleoside monomers represented by formula (II).

6. A modification of the double-stranded ribonucleic acid or a conjugate thereof as described in any one of claims 1-5, wherein, The nucleotide sequence of the sense strand of the double-stranded ribonucleic acid contains sequence A, and / or the nucleotide sequence of the antisense strand of the double-stranded ribonucleic acid contains sequence B; Preferably, the modified double-stranded ribonucleic acid further comprises at least one of the following chemical modifications: (1) Modification of at least one nucleotide in the positive strand, (2) Modification of the phosphodiester bond at at least one position in the positive chain. (3) Modification of at least one nucleotide in the antisense strand, (4) Modification of the phosphodiester bond at at least one position in the antisense chain; Optionally, along the 5' end to the 3' end direction, the positive strand of the modified double-stranded ribonucleic acid includes the following modification: at least three of the ribonucleotides at positions 3-11 of sequence A of the positive strand are 2'-fluoro modified ribonucleotides; Preferably, along the 5' end to the 3' end, the positive strand of the double-stranded ribonucleic acid modification includes the following modification: at least three of the ribonucleotides at positions 3, 5, 7, 8, 9, 10, and 11 of sequence A of the positive strand are 2'-fluorinated ribonucleotides; preferably, at least three of the ribonucleotides at positions 3, 5, 7, 8, 9, and 11 of sequence A of the positive strand are 2'-fluorinated ribonucleotides. and / or Along the 5' end to the 3' end, the antisense strand of the double-stranded ribonucleic acid modification includes the following modification: at least three of the ribonucleotides at positions 2-16 of sequence B of the antisense strand are 2'-fluoro modified ribonucleotides; Preferably, along the 5' end to the 3' end, the antisense strand of the double-stranded ribonucleic acid modification includes the following modification: at least three of the ribonucleotides at positions 2, 6, 7, 8, 9, 10, 14 and 16 of sequence B of the antisense strand are 2'-fluoro modified ribonucleotides. More preferably, along the 5' end to the 3' end direction, the antisense strand of the double-stranded ribonucleic acid modification includes the following modification: at least three of the ribonucleotides at positions 2, 6, 7, 10, 14 and 16 of sequence B of the antisense strand are 2'-fluoro modified ribonucleotides.

7. The modified double-stranded ribonucleic acid or its conjugate as described in claim 6, wherein, The ribonucleotides at other positions in sequence A of the positive strand are 2'-methoxy modified ribonucleotides; or, the positive strand is further modified with one or more nucleotide derivatives. and / or The ribonucleotides at other positions in sequence B of the antisense strand are 2'-methoxy modified ribonucleotides; Optionally, the 6th or 7th position of sequence B of the antisense strand is GNA or a nucleoside monomer represented by formula (II); Optionally, the nucleoside modified with GNA or the nucleoside monomer of formula (II) has a phosphodiester bond, thiophosphate diester bond or amide bond with one or both of its adjacent nucleosides.

8. The modified double-stranded ribonucleic acid or its conjugate as described in claim 6 or 7, wherein, Along the direction from the 5' end to the 3' end, the positive strand of the double-stranded ribonucleic acid modification includes the following modification: the ribonucleotides at positions 5, 7, 8 and 9 of sequence A of the positive strand are 2'-fluoro-modified ribonucleotides; optionally, the ribonucleotides at other positions of sequence A of the positive strand are 2'-methoxy-modified ribonucleotides. Alternatively, along the 5' end to 3' end direction, the positive strand of the double-stranded ribonucleic acid modification includes the following modification: the ribonucleotides at positions 3, 7, 8 and 9 of sequence A of the positive strand are 2'-fluoro-modified ribonucleotides; optionally, the ribonucleotides at other positions of sequence A of the positive strand are 2'-methoxy-modified ribonucleotides. Alternatively, along the 5' end to 3' end direction, the positive strand of the double-stranded ribonucleic acid modification includes the following modification: the ribonucleotides at positions 3, 7, 9 and 11 of sequence A of the positive strand are 2'-fluoro-modified ribonucleotides; optionally, the ribonucleotides at other positions of sequence A of the positive strand are 2'-methoxy-modified ribonucleotides. Alternatively, along the 5' end to the 3' end direction, the positive strand of the double-stranded ribonucleic acid modification includes the following modification: the ribonucleotides at positions 7, 9, 10, and 11 of sequence A of the positive strand are 2'-fluoro-modified ribonucleotides; optionally, the ribonucleotides at other positions of sequence A of the positive strand are 2'-methoxy-modified ribonucleotides. and / or Along the 5' end to the 3' end, the antisense strand of the double-stranded ribonucleic acid modification includes the following modifications: the ribonucleotides at positions 2, 6, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; optionally, sequence B of the antisense strand may have one or more nucleotide derivative modifications, and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides; Along the 5' end to the 3' end, the antisense strand of the double-stranded ribonucleic acid modification includes the following modification: the ribonucleotides at positions 2, 7, 10, and 14 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; optionally, sequence B of the antisense strand may have one or more nucleotide derivative modifications, and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides; Along the 5' end to the 3' end, the antisense strand of the double-stranded ribonucleic acid modification includes the following modification: the ribonucleotides at positions 2, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; optionally, sequence B of the antisense strand may have one or more nucleotide derivative modifications, and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides; Along the 5' end to 3' end direction, the antisense strand of the double-stranded ribonucleic acid modification includes the following modification: the ribonucleotides at positions 2, 10, and 14 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; optionally, sequence B of the antisense strand has one or more nucleotide derivative modifications, and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides; Along the 5' to 3' direction, the antisense strand of the double-stranded ribonucleic acid modification includes the following modification: the ribonucleotides at positions 2, 6, 8, 9, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; optionally, sequence B of the antisense strand has one or more nucleotide derivative modifications, and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides; Along the 5' to 3' direction, the antisense strand of the double-stranded ribonucleic acid modification includes the following modifications: the ribonucleotides at positions 2, 8, 9, 14, and 16 of sequence B of the antisense strand are 2'-fluoro-modified ribonucleotides; optionally, sequence B of the antisense strand has one or more nucleotide derivative modifications, and the ribonucleotides at the remaining positions of sequence B of the antisense strand are 2'-methoxy-modified ribonucleotides.

9. A modification of the double-stranded ribonucleic acid or a conjugate thereof as described in any one of claims 1-8, wherein, The conjugate contains a conjugating group; Optionally, the conjugated group has a structure as shown in any of the formulas L1, L-III, L-IV, L-LV, L-VI, L-VII, L-VIII, and L-IX: (Formula LI); (Formula L-III); (Formula L-IV); (LV style); (Form L-VI); (Form L-VII); (Formula L-VIII); (Formula L-IX).

10. The use of the modified double-stranded ribonucleic acid according to any one of claims 1-9 in at least one of the following applications: (1) Inhibit the expression of target genes, or prepare drugs for inhibiting the expression of target genes; (2) To prevent or treat diseases, symptoms or disorders related to abnormal expression of target genes, or to prepare drugs for the prevention or treatment of diseases, symptoms or disorders related to abnormal expression of target genes. (3) To treat subjects with diseases that would benefit from reduced expression of the target gene, or to prepare a medicine for treating subjects with diseases, conditions or disorders that would benefit from reduced expression of the target gene.

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