Modified nucleoside monomers and double-stranded ribonucleic acids

Abstract

The present invention relates to the field of biopharmaceuticals, and more particularly to a modified nucleoside monomer compound and a double-stranded ribonucleic acid containing the same, which has reduced off-target effects or improved activity of inhibiting gene expression, as well as its use in the production of a drug for inhibiting the expression of a target gene in mammalian cells and in a composition containing the drug.

Description

Detailed Description of the Invention

[0001] This application claims priority to two prior patent applications, filed with the State Intellectual Property Office of China on January 20, 2023, bearing patent application number 202310199895.5, and filed on November 14, 2023, bearing patent application number 202311510663.3, both entitled "Modified nucleoside monomers and double-stranded ribonucleic acids." Both prior applications are incorporated herein by reference in their entireties.

[0002] [Technical Field] The present invention relates to the field of biopharmaceuticals, and more particularly to a modified nucleoside monomer compound and a double-stranded ribonucleic acid containing the same, which has reduced off-target effects or improved activity of inhibiting gene expression, as well as its use in the production of a drug for inhibiting the expression of a target gene in mammalian cells and in a composition containing the drug.

[0003] [Background technology] RNA interference or "RNAi" was a term first proposed by the Fire team and was used to describe the observation that double-stranded RNA (dsRNA) can block gene expression, that short dsRNAs direct gene-specific post-transcriptional silencing in many organisms (including vertebrates), and provided a new tool for studying gene function (Fire et al., Nature., (1998), 391(6669):806-811; Elbashir et al., Genes Dev. [Genes and Development], (2001), 15, 188-200). The endonuclease Dicer in the cytoplasm of host cells cleaves dsRNA into multiple small RNA fragments (approximately 21-23 bp) with specific lengths and structures, i.e., siRNAs. The siRNAs are directly involved in the RNAi process. Specifically, RNAi is mediated by the RNA-induced silencing complex (RISC). RISC specifically binds to the corresponding mRNA and, with its nuclease function, cleaves the mRNA at the binding site, resulting in immediate degradation of the cleaved mRNA.

[0004] RNAi drugs degrade the target mRNA gene, thereby reducing the target protein expressed by the target gene, thereby achieving the purpose of treating protein-related diseases.In contrast, antibody drugs treat diseases at the protein level through affinity with the target protein, while RNAi is controlled at the level of mRNA, the precursor of protein expression, and belongs to a more upstream therapeutic technology.

[0005] According to Nature Reviews Drug Discovery, currently approved drugs interact with approximately 700 proteins (encoded by approximately 0.05% of the genome). However, over 80% of pathogenic proteins associated with human diseases cannot be targeted by conventional small molecule drugs or biopolymer drugs, making them undruggable protein targets. RNA-targeted drugs, such as RNAi, offer advantages such as a rich number of target candidates, short development times, long-lasting efficacy, and a high success rate in clinical development. They can treat diseases at the post-transcriptional level and achieve breakthroughs against difficult-to-drug special protein targets. They are expected to overcome unmet medical needs, including genetic and other intractable diseases, and to develop therapeutics for "undruggable" and "untargable" diseases. Therefore, given the enormous therapeutic needs for RNA-targeted drugs, the industry believes that RNA-targeted drugs are expected to become the third wave of modern new drugs, following small molecule drugs and antibody drugs.

[0006] Currently, RNAi-targeted drugs are the mainstream direction of RNA-targeted drugs, with most projects in preclinical or early clinical research, offering enormous development potential. However, although siRNA has great prospects in drug development, multiple intracellular and extracellular obstacles limit its widespread clinical application. For example, unmodified siRNA has drawbacks such as less than ideal stability, relatively poor pharmacokinetic characteristics, relatively weak gene silencing effect, and the possibility of inducing off-target effects. Furthermore, the phosphodiester bond of siRNA is easily damaged by RNases and phosphatases. Once systemically administered and entering the circulation, systemic endonucleases or exonucleases rapidly degrade siRNA into fragments, preventing the accumulation of intact therapeutic siRNA in the desired tissues.

[0007] Off-target effects of siRNAs are a major challenge that must be overcome in the development of RNAi-targeted drugs, particularly those caused by a mechanism known as miRNA. Off-target effects occur when siRNA drugs (i.e., RNAi drugs) silence target genes, but the argonaute protein bound to the siRNA prevents the target gene from being silenced by the RNAi effect, instead processing the siRNA as a miRNA (microRNA) (Lam et al., Molecular Therapy Nucleic Acids, (2015), 4, e252). Off-target effects caused by siRNAs result from the complementarity of the seed region (positions 2–9 from the 5′ end) of the antisense strand loaded by the RNAi RISC with one or more bases in the mRNA of a non-target gene. Off-target effects in siRNAs have been reported in several studies and can affect the expression of multiple genes depending on the sequence of the seed region, severe enough to cause up to 30% of positive results in phenotypic screening based on siRNAs.

[0008] Therefore, applying chemical modifications to adjust the design of siRNA to enhance the gene silencing efficacy of siRNA gene therapy, or to maximize the elimination or reduction of siRNA off-target effects without impairing the gene silencing efficacy of siRNA gene therapy, has become an important direction of research and development worldwide.

[0009] Chinese invention patent application CN107075516A discloses a double-stranded RNA (dsRNA) reagent capable of inhibiting the expression of a target gene. The sense strand of the dsRNA reagent contains at least one thermolabile nucleotide, and at least one such thermolabile nucleotide appears opposite the seed region (positions 2-8) of the antisense strand. However, the disclosed monomer structure is artificially synthesized and is not a chemical structure naturally occurring in the human body, which may result in physiological toxicity. Furthermore, the overall synthesis involves a relatively large number of steps, resulting in high production costs, a complex process, and high demands on technical expertise.

[0010] Summary of the Invention Based on the various shortcomings of the prior art, the present invention employs simpler chemical synthesis steps and methods (fewer than the disclosed technical steps) to modify the structure of some of the natural ribonucleoside monomers, in particular, through intensive research into the structure of cytosine ribonucleoside, and then modifies its structure and incorporates it into dsRNA, thereby improving specific binding or maintaining specific binding while minimizing non-specific binding between siRNA and target sites, thereby reducing off-target effects.

[0011] In one aspect, the present invention provides a modified nucleoside monomer, having the structure of formula (I): [ka] Among them, the base is selected from the following structures: [ka] R3, R4, R5, and R6 each independently represent an amino protecting group, hydrogen, or C 1-3The alkyl group is selected from alkyl groups (methyl, ethyl, propyl, isopropyl), methoxyethyl, cyclopropyl, fluoromethyl, difluoromethyl, trifluoromethyl, and acetyl groups, with the condition that R3 and R4 are not simultaneously hydrogen atoms, and R5 and R6 are not simultaneously hydrogen atoms; X is selected from F or -OMe; R1 and R2 are each independently selected from H, a hydroxy protecting group, or a phosphoramidite group.

[0012] In some embodiments, R3, R4, R5, and R6 are each independently an amino protecting group, hydrogen, or C 1-3 It is selected from alkyl groups (methyl, ethyl, propyl, isopropyl) and cyclopropyl groups.

[0013] In some embodiments, R3, R4, R5, and R6 are each independently selected from an amino-protecting group, hydrogen, a methyl group, and a cyclopropyl group.

[0014] In some embodiments, R3 and R4 are each independently selected from a methyl group.

[0015] In some embodiments, one of R3 and R4 is selected from hydrogen and the other is selected from a cyclopropyl group.

[0016] In some embodiments, Base is [ka] Selected from.

[0017] The "amino-protecting group" is selected from benzoyl (Bz), acetyl (Ac), benzyloxycarbonyl (CBz), tert-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), p-methoxybenzyl (PMB), benzyl (Bn), trityl (Tr), p-toluenesulfonyl (Tos), phthaloyl (Pht), allyloxycarbonyl (Alloc), and trifluoroacetyl (Tfa). Preferred are benzoyl (Bz), acetyl (Ac), benzyloxycarbonyl (CBz), tert-butoxycarbonyl (Boc), and 9-fluorenylmethyloxycarbonyl (Fmoc).

[0018] In some embodiments, R1 and R2 are each independently selected from a hydroxy protecting group or a phosphoramidite group.

[0019] The "hydroxy-protecting group" is selected from methoxymethyl ether (MOM), 2-tetrahydropyran (THP), tert-butyldimethylchlorosilyl group (TBS), trimethylsilyl (TMS), triethylsilyl group (TES), trityl group (Tr), 4,4'-bismethoxytrityl group (DMTr), 4,4',4'-trimethoxytrityl group (TMTr), benzyl (Bn), benzoyl group (Bz), acetyl group (Ac), and p-methoxybenzyl group (PMB), and is preferably trityl (Tr), 4,4'-dimethoxytrityl group (DMTr), 4,4',4'-trimethoxytrityl group (TMTr), benzyl (Bn), benzoyl group (Bz), or acetyl group (Ac).

[0020] In some embodiments, R1 and R2 are each independently selected from DMTr or a phosphoramidite group.

[0021] In some embodiments, R1 is selected from DMTr and R2 is selected from a phosphoramidite group.

[0022] The "phosphoramidite group" has the following structure: [ka] wherein R7 and R8 are each independently selected from C 1-3 R9 is selected from straight or branched chain alkyl groups, 1-3 Straight or branched chain alkyl groups, C substituted with cyano (-CN) 1-3 It is selected from straight or branched chain alkyl groups.

[0023] In some embodiments, R1 is selected from DMTr and R2 is [ka] Selected from.

[0024] In some embodiments, R1 and R2 are both selected from H.

[0025] In some embodiments, R1 is selected from DMTr and R2 is selected from H.

[0026] In some embodiments, R1 is selected from H and R2 is [ka] Selected from.

[0027] In some embodiments, the modified nucleoside monomer is [ka] Selected from.

[0028] In another aspect, the present invention further relates to providing a solution for the treatment of diseases that can be regulated by downregulation of target genes by silencing the expression of the target genes using double-stranded ribonucleic acid (dsRNA).

[0029] The present invention provides a double-stranded ribonucleic acid capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the sense strand optionally comprising a ligand, and at least one of the 6th, 7th, or 8th positions from the 5' end of the antisense strand comprising a modified nucleoside monomer as shown below, i.e., [ka] Contains wherein R3, R4, R5, and R6 are each independently hydrogen, C 1-3 alkyl groups (methyl, ethyl, propyl, isopropyl), and cyclopropyl groups, with the proviso that R3 and R4 are not hydrogen atoms at the same time, and R5 and R6 are not hydrogen atoms at the same time; X is selected from F or -OMe; and the wavy line in the structure [ka] represents the site where the phosphate ester bond is formed.

[0030] In some embodiments, the antisense strand of the double-stranded ribonucleic acid of the present invention contains at least one of the following nucleoside monomers at positions 6, 7, or 8 from the 5' end: [ka] Contains wherein R3, R4, R5, and R6 are each independently selected from hydrogen, a methyl group, and a cyclopropyl group, provided that R3 and R4 are not both hydrogen, and R5 and R6 are not both hydrogen, and X is selected from F or -OMe.

[0031] In some embodiments, the antisense strand of the double-stranded ribonucleic acid of the present invention contains at least one of the following nucleoside monomers at positions 6, 7, or 8 from the 5' end: [ka] Contains X is selected from F or -OMe.

[0032] In some embodiments, the 6th position from the 5' end of the antisense strand of the double-stranded ribonucleic acid of the present invention is a nucleoside monomer. [ka] Contains:

[0033] In some embodiments, the 8th position from the 5' end of the antisense strand of the double-stranded ribonucleic acid of the present invention is a nucleoside monomer. [ka] Contains:

[0034] In some embodiments, the nucleoside monomer is located at position 7 from the 5' end of the antisense strand of the double-stranded ribonucleic acid of the present invention. [ka] Contains:

[0035] In some embodiments, the antisense strand of the double-stranded ribonucleic acid of the present invention has a nucleoside monomer at at least one of the 6th, 7th, or 8th positions from the 5' end. [ka] Contains:

[0036] In some embodiments, at least one of the 7th or 8th positions from the 5' end of the antisense strand of the double-stranded ribonucleic acid of the present invention is a nucleoside monomer. [ka] Contains:

[0037] In some embodiments, the double-stranded ribonucleic acid of the present invention has a nucleoside monomer at least at one of the 6th or 8th positions from the 5' end of the antisense strand. [ka] Contains:

[0038] In some embodiments, the double-stranded ribonucleic acid of the present invention has a nucleoside monomer at least at one of the 6th or 7th positions from the 5' end of the antisense strand. [ka] Contains:

[0039] In some embodiments, the antisense strand of the double-stranded ribonucleic acid of the present invention is at position 6 from the 5' end of the nucleoside monomer [ka] Contains:

[0040] In some embodiments, the sense strand of the double-stranded ribonucleic acid of the present invention contains a ligand.

[0041] In some embodiments, the ligand of the double-stranded ribonucleic acid of the present invention is linked to the 5'-end or 3'-end of the sense strand.

[0042] In some embodiments, the ligand of the double-stranded ribonucleic acid of the present invention is an asialoglycoprotein receptor (ASGPR) ligand.

[0043] In some embodiments, the ASGPR ligand of the double-stranded ribonucleic acid of the present invention is one or more GalNAc derivatives linked by a divalent or trivalent branched chain structure.

[0044] In some embodiments, illustrative examples of one or more GalNAc derivatives linked by a divalent or trivalent branched chain structure of the present invention include: [ka] This includes, but is not limited to, the following structures:

[0045] In some embodiments, the sense strand of the double-stranded ribonucleic acid of the present invention has 21 nucleotides and the antisense strand has 23 nucleotides.

[0046] In another aspect, the present invention provides a pharmaceutical composition comprising the double-stranded ribonucleic acid described above and a pharmaceutically acceptable carrier, excipient, or delivery vector.

[0047] In some embodiments, the pharmaceutical composition can be delivered by a liposome (e.g., liposome 2000), a viral vector, an extracellular vesicle (EV), a lipid nanoparticle (LNP), or by an ASGPR ligand (e.g., one or more GalNAc derivatives) at the 5'-end or 3'-end of the sense or antisense strand.

[0048] In some embodiments, the viral vector includes, but is not limited to, an adenovirus-associated virus (AAV), a lentivirus, an adenovirus, and a retrovirus.

[0049] In some embodiments, the pharmaceutical compositions can be administered by the following routes or forms: (1) oral (aqueous or non-aqueous solution or suspension), tablet (e.g., buccal, sublingual, and tablets targeted for systemic absorption), bolus, powder, granule, paste for administration to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, preferably by delivery using subcutaneous or intravenous methods.

[0050] In another aspect, the present invention further relates to the use of the above-described double-stranded ribonucleic acid or pharmaceutical composition in the manufacture of a medicament for inhibiting or silencing the expression of a target gene in a cell.

[0051] In some embodiments, the present invention relates to use of the double-stranded ribonucleic acid or pharmaceutical composition described above in the manufacture of a medicament for suppressing off-target effects caused by the antisense strand of the double-stranded ribonucleic acid.

[0052] In some embodiments, the present invention relates to the use of the double-stranded ribonucleic acid or pharmaceutical composition described above in the manufacture of a medicament for delivering the double-stranded ribonucleic acid to a specific target in a subject.

[0053] In another aspect, the present invention further relates to a method for inhibiting expression of a target gene in a cell, said method comprising: (a) introducing into a cell the double-stranded ribonucleic acid capable of inhibiting at least 40% of the expression of a target gene when contacted with the cell; (b) maintaining the cells obtained in step (a) for a sufficient time to inhibit expression of the target gene in the cells by degrading mRNA transcripts of the target gene.

[0054] In another aspect, the present invention further relates to a method for treating or preventing a disease associated with abnormal upregulation of target gene expression, said method comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of one or more of the above-described double-stranded ribonucleic acids.

[0055] In another embodiment, the present invention further relates to use of the above-described double-stranded ribonucleic acid for producing an inhibitor of the expression of a target gene.

[0056] In some embodiments, the target gene is selected from AGT, ANGPTL3, APOB, BRAF, CTNNB1, C5, CD274, FLT1, FLT2, KIF11, FXII, LPA, PCSK9 and RET, preferably the ANGPTL3 and PCSK9 genes, and most preferably the PCSK9 gene.

[0057] In another aspect, the present invention further relates to a method for inhibiting expression of a target gene in a cell, the method comprising: (a) introducing into a cell the double-stranded ribonucleic acid capable of inhibiting at least 40% of the expression of a target gene when contacted with the cell; (b) maintaining the cells obtained in step (a) for a sufficient time to inhibit expression of the target gene in the cells by degrading mRNA transcripts of the target gene.

[0058] In another aspect, the present invention further relates to a method for inhibiting expression of a target gene in a cell, said method comprising: (a) introducing into a cell the double-stranded ribonucleic acid capable of inhibiting at least 40% of target gene expression when contacted with a cell expressing PCSK9; (b) maintaining the cells obtained in step (a) for a sufficient time to inhibit expression of the target gene in the cells by degrading mRNA transcripts of the target gene.

[0059] In some embodiments, the present invention further relates to a vector for inhibiting expression of a target gene in a cell, comprising a regulatory sequence operably linked to a nucleotide sequence encoding at least one strand of the above-described double-stranded ribonucleic acid.

[0060] In some embodiments, the invention further relates to a cell comprising a vector that inhibits expression of a target gene in the cell, wherein the cell is typically a mammalian cell, such as a human cell.

[0061] In some embodiments, the present invention further relates to a pharmaceutical composition for inhibiting expression of a target gene, comprising one or more of the above double-stranded ribonucleic acids and a pharmaceutically acceptable carrier or delivery vector.

[0062] In some embodiments, the present invention further relates to methods for treating or preventing diseases associated with aberrant upregulation of target gene expression, comprising administering to a patient in need of such treatment, prevention, or management a therapeutically or prophylactically effective amount of one or more of the above-described double-stranded ribonucleic acids.

[0063] In some embodiments, the present invention provides a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of a target gene (e.g., PCSK9). The dsRNA comprises at least two sequences complementary to each other. The dsRNA comprises a sense strand having a first sequence and an antisense strand having a second sequence. The antisense strand comprises a nucleotide sequence substantially complementary to at least a portion of an mRNA encoding the target gene, and the length of the complementary region is less than 30 nucleotides, typically 19-24 nucleotides. When contacted with a cell expressing the target gene, the dsRNA can inhibit expression of at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the target gene.

[0064] In some embodiments, the target gene is selected from AGT, ANGPTL3, APOB, BRAF, CTNNB1, C5, CD274, FLT1, FLT2, KIF11, FXII, LPA, PCSK9, and RET, preferably the ANGPTL3, PCSK9, and LPA genes, more preferably the ANGPTL3 and PCSK9 genes, and most preferably the PCSK9 gene.

[0065] In some embodiments, the double-stranded ribonucleic acid comprises a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-AAUGACUUUUAUUGAGCUCUU-3' (SEQ ID NO: 1), and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-AAGAG / X / UCAAUAAAAGUCAUUCU-3' (SEQ ID NO: 2), where X is any of the modified nucleoside monomers described above. Optionally, conventional chemical modifications can be made to the sugar ring or phosphate linkage of the double-stranded ribonucleic acid to enhance stability.

[0066] In some other embodiments, the double-stranded ribonucleic acid comprises a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-AAUGACUUUUAUUGAGCUCUU-3' (SEQ ID NO: 1), and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-AAGAGCU / X / AAUAAAAGUCAUUCU-3' (SEQ ID NO: 3), where X is any of the modified nucleoside monomers described above. Optionally, conventional chemical modifications can be made to the sugar ring or phosphate linkage of the double-stranded ribonucleic acid to enhance stability.

[0067] In some embodiments, X is selected from M3C, 2M4C, M4C, and CP4C.

[0068] In some embodiments, the double-stranded ribonucleic acid comprises a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-CCACAACGCUUUUGGGGGUGA-3' (SEQ ID NO: 4), and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-UCACC / X / CCAAAAGCGUUGUGGGC-3' (SEQ ID NO: 5), where X is any of the modified nucleoside monomers described above. Optionally, conventional chemical modifications can be made to the sugar ring or phosphate linkage of the double-stranded ribonucleic acid to enhance stability.

[0069] In some embodiments, the double-stranded ribonucleic acid comprises a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-CCACAACGCUUUUGGGGGUGA-3' (SEQ ID NO: 4), and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-UCACCC / X / CAAAAGCGUUGUGGGC-3' (SEQ ID NO: 6), where X is any of the modified nucleoside monomers described above. Optionally, conventional chemical modifications can be made to the sugar ring or phosphate linkage of the double-stranded ribonucleic acid to enhance stability.

[0070] In some embodiments, the double-stranded ribonucleic acid comprises a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-CCACAACGCUUUUGGGGGUGA-3' (SEQ ID NO: 4), and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-UCACCCC / X / AAAAGCGUUGUGGGC-3' (SEQ ID NO: 7), where X is any of the modified nucleoside monomers described above. Optionally, conventional chemical modifications can be made to the sugar ring or phosphate linkage of the double-stranded ribonucleic acid to enhance stability.

[0071] In some embodiments, the double-stranded ribonucleic acid comprises a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-GAGAAUUUUGGUUGGGCCUAG-3' (SEQ ID NO: 8), and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-CUAGG / X / CCAACCAAAAUUCUCCA-3' (SEQ ID NO: 9), where X is any of the modified nucleoside monomers described above. Optionally, conventional chemical modifications can be made to the sugar ring or phosphate linkage of the double-stranded ribonucleic acid to enhance stability.

[0072] In some embodiments, the double-stranded ribonucleic acid comprises a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-GAGAAUUUUGGUUGGGCCUAG-3' (SEQ ID NO: 8), and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-CUAGGC / X / CAACCAAAAUUCUCCA-3' (SEQ ID NO: 10), where X is any of the modified nucleoside monomers described above. Optionally, conventional chemical modifications can be made to the sugar ring or phosphate linkage of the double-stranded ribonucleic acid to enhance stability.

[0073] In some embodiments, the double-stranded ribonucleic acid comprises a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-GAGAAUUUUGGUUGGGCCUAG-3' (SEQ ID NO: 8), and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-CUAGGCC / X / AACCAAAAUUCUCCA-3' (SEQ ID NO: 11), where X is any of the modified nucleoside monomers described above. Optionally, conventional chemical modifications can be made to the sugar ring or phosphate linkage of the double-stranded ribonucleic acid to enhance stability.

[0074] In some embodiments, the double-stranded ribonucleic acid comprises a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-CCCAGUGUCAGGUGGGAGUAC-3' (SEQ ID NO: 12), and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-GUACU / X / CCACCUGACACUGGGAU-3' (SEQ ID NO: 13), where X is any of the modified nucleoside monomers described above. Optionally, conventional chemical modifications can be made to the sugar ring or phosphate linkage of the double-stranded ribonucleic acid to enhance stability.

[0075] In some embodiments, the double-stranded ribonucleic acid comprises a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-CCCAGUGUCAGGUGGGAGUAC-3' (SEQ ID NO: 12), and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-GUACUC / X / CACCUGACACUGGGAU-3' (SEQ ID NO: 14), where X is any of the modified nucleoside monomers described above. Optionally, conventional chemical modifications can be made to the sugar ring or phosphate linkage of the double-stranded ribonucleic acid to enhance stability.

[0076] In some embodiments, the double-stranded ribonucleic acid comprises a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-CCCAGUGUCAGGUGGGAGUAC-3' (SEQ ID NO: 12), and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-GUACUCC / X / ACCUGACACUGGGAU-3' (SEQ ID NO: 15), where X is any of the modified nucleoside monomers described above. Optionally, conventional chemical modifications can be made to the sugar ring or phosphate linkage of the double-stranded ribonucleic acid to enhance stability.

[0077] In some embodiments, the double-stranded ribonucleic acid comprises a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-GUGAUUCAAGUUUAUGGAUAC-3' (SEQ ID NO: 16), and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-GUAUC / X / AUAAACUUGAAUCACAA-3' (SEQ ID NO: 17), where X is any of the modified nucleoside monomers described above. Optionally, conventional chemical modifications can be made to the sugar ring or phosphate linkage of the double-stranded ribonucleic acid to enhance stability.

[0078] In some embodiments, the double-stranded ribonucleic acid comprises a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-UGUGUAUUUGGAAGUUGUAUC-3' (SEQ ID NO: 18), and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-GAUACAA / X / UUCCAAAUACACAUA-3' (SEQ ID NO: 19), where X is any of the modified nucleoside monomers described above. Optionally, conventional chemical modifications can be made to the sugar ring or phosphate linkage of the double-stranded ribonucleic acid to enhance stability.

[0079] In some embodiments, the double-stranded ribonucleic acid comprises a sense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-GUGAAGAAAUGUUGUUACGAU-3' (SEQ ID NO: 20), and an antisense strand consisting of a nucleotide sequence having at least 90%, preferably 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence 5'-AUCGUAA / X / AACAUUUCUUCACUA-3' (SEQ ID NO: 21), where X is any of the modified nucleoside monomers described above. Optionally, conventional chemical modifications can be made to the sugar ring or phosphate linkage of the double-stranded ribonucleic acid to enhance stability.

[0080] In some embodiments, X is selected from M3C, 2M4C, M4C, and CP4C.

[0081] In some embodiments, the chemical modification of the sugar ring can be selected from a 2'-modification, and can further be selected from a 2'-F modification and a 2'-OMe modification.

[0082] In some embodiments, to enhance stability, the dsRNA can be conventionally chemically modified (mainly conventional chemical modifications of sugar rings and phosphate ester bonds).These chemical modifications include, but are not limited to, sugar ring 2'-modifications.Examples of sugar ring 2'-modifications include, but are not limited to, 2'-methoxyethyl, 2'-O-methyl (2'-OMe), 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-fluoro, or 2'-ara-F.Examples of conventional chemical modifications of phosphate ester bonds include, but are not limited to, replacing internucleotide phosphate ester bonds with other bonds, such as thiophosphate ester bonds.One or more of these modifications may be used.

[0083] In some embodiments, the double-stranded ribonucleic acid comprises:

[0084] Sense strand 5'-AfsAmsUfGmAfCmUfUmUfUfAfUmUfGmAfGmCfUmCfUmUf-3' (SEQ ID NO: 22) Antisense strand: 5'-AmsAfsGmAfGm / X / UmCfAmAfUmAmAmAfAmGfUmCfAmUfUmsCmsUm-3' (SEQ ID NO: 23) The X is any of the modified nucleoside monomers described above.

[0085] In some embodiments, the double-stranded ribonucleic acid comprises:

[0086] Sense strand: 5'-AfsAmsUfGmAfCmUfUmUfUfAfUmUfGmAfGmCfUmCfUmUf-3' (SEQ ID NO: 22) Antisense strand: 5'-AmsAfsGmAfGmCfUm / X / AmAfUmAmAmAfAmGfUmCfAmUfUmsCmsUm-3' (SEQ ID NO: 24) The X is any of the modified nucleoside monomers described above.

[0087] In some embodiments, the double-stranded ribonucleic acid comprises:

[0088] Sense strand: 5'-CmsCmsAmCmAmAmCfGmCfUfUfUmUmGmGmGmGmGmUmGmAm-3' (SEQ ID NO: 25) Antisense strand: 5'-UmsCfsAmCmCm / X / CmCmAmAmAmAmGmCfGmUfUmGmUmGmGmsGmsCm-3' (SEQ ID NO: 26) The X is any of the modified nucleoside monomers described above.

[0089] In some embodiments, the double-stranded ribonucleic acid comprises:

[0090] Sense strand: 5'-CmsCmsAmCmAmAmCfGmCfUfUfUmUmGmGmGmGmGmUmGmAm-3' (SEQ ID NO: 25) Antisense strand: 5'-UmsCfsAmCmCmCf / X / CmAmAmAmAmGmCfGmUfUmGmUmGmGmsGmsCm-3' (SEQ ID NO: 27) The X is any of the modified nucleoside monomers described above.

[0091] In some embodiments, the double-stranded ribonucleic acid comprises:

[0092] Sense strand: 5'-CmsCmsAmCmAmAmCfGmCfUfUfUmUmGmGmGmGmGmUmGmAm-3' (SEQ ID NO: 25) Antisense strand: 5'-UmsCfsAmCmCmCfCm / X / AmAmAmAmGmCfGmUfUmGmUmGmGmsGmsCm -3' (SEQ ID NO: 28) The X is any of the modified nucleoside monomers described above.

[0093] In some embodiments, the double-stranded ribonucleic acid comprises:

[0094] Sense strand: 5'-GmsAmsGmAmAmUmUfUmUfGfGfUmUmGmGmGmCmCmUmAmGm-3' (SEQ ID NO: 29) Antisense strand: 5'-CmsUfsAmGmGm / X / CmCmAmAmCmCmAmAfAmAfUmUmCmUmCmsCmsAm-3' (SEQ ID NO: 30) The X is any of the modified nucleoside monomers described above.

[0095] In some embodiments, the double-stranded ribonucleic acid comprises:

[0096] Sense strand: 5'-GmsAmsGmAmAmUmUfUmUfGfGfUmUmGmGmGmCmCmUmAmGm-3' (SEQ ID NO: 29) Antisense strand: 5'-CmsUfsAmGmGmCf / X / CmAmAmCmCmAmAfAmAfUmUmCmUmCmsCmsAm-3' (SEQ ID NO: 31) The X is any of the modified nucleoside monomers described above.

[0097] In some embodiments, the double-stranded ribonucleic acid comprises:

[0098] Sense strand: 5'-GmsAmsGmAmAmUmUfUmUfGfGfUmUmGmGmGmCmCmUmAmGm-3' (SEQ ID NO: 29) Antisense strand: 5'-CmsUfsAmGmGmCfCm / X / AmAmCmCmAmAfAmAfUmUmCmUmCmsCmsAm-3' (SEQ ID NO: 32) The X is any of the modified nucleoside monomers described above.

[0099] In some embodiments, the double-stranded ribonucleic acid comprises:

[0100] Sense strand: 5'-CmsCmsCmAmGmUmGfUmCfAfGfGmUmGmGmGmAmGmUmAmCm-3' (SEQ ID NO: 33) Antisense strand: 5'-GmsUfsAmCmUm / X / CmCmAmCmCmUmGmAfCmAfCmUmGmGmGmsAmsUm-3' (SEQ ID NO: 34) The X is any of the modified nucleoside monomers described above.

[0101] In some embodiments, the double-stranded ribonucleic acid comprises:

[0102] Sense strand: 5'-CmsCmsCmAmGmUmGfUmCfAfGfGmUmGmGmGmAmGmUmAmCm-3' (SEQ ID NO: 33) Antisense strand: 5'-GmsUfsAmCmUmCf / X / CmAmCmCmUmGmAfCmAfCmUmGmGmGmsAmsUm-3' (SEQ ID NO: 35) The X is any of the modified nucleoside monomers described above.

[0103] In some embodiments, the double-stranded ribonucleic acid comprises:

[0104] Sense strand: 5'-CmsCmsCmAmGmUmGfUmCfAfGfGmUmGmGmGmAmGmUmAmCm-3' (SEQ ID NO: 33) Antisense strand: 5'-GmsUfsAmCmUmCfCm / X / AmCmCmUmGmAfCmAfCmUmGmGmGmsAmsUm-3' (SEQ ID NO: 36) The X is any of the modified nucleoside monomers described above.

[0105] In some embodiments, the double-stranded ribonucleic acid comprises:

[0106] Sense strand: 5'-GmsUmsGmAmUmUmCfAmAfGfUfUmUmAmUmGmGmAmUmAmCm-3' (SEQ ID NO: 37) Antisense strand: 5'-GmsUfsAmUmCm / X / AmUmAmAmAmCmUmUfGmAfAmUmCmAmCmsAmsAm-3' (SEQ ID NO: 38) The X is any of the modified nucleoside monomers described above.

[0107] In some embodiments, the double-stranded ribonucleic acid comprises:

[0108] Sense strand: 5'-UmsGmsUmGmUmAmUfUmUfGfGfAmAmGmUmUmGmUmAmUmCm-3' (SEQ ID NO: 39) Antisense strand: 5'-GmsAfsUmAmCmAmAm / X / UmUmCmCmAmAfAmUfAmCmAmCmAmsUmsAm-3' (SEQ ID NO: 40) The X is any of the modified nucleoside monomers described above.

[0109] In some embodiments, the double-stranded ribonucleic acid comprises:

[0110] Sense strand: 5'-GmsUmsGmAmAmGmAfAmAfUfGfUmUmGmUmUmAmCmGmAmUm-3' (SEQ ID NO: 41) Antisense strand: 5'-AmsUfsCmGmUmAmAm / X / AmAmCmAmUmUfUmCfUmUmCmAmCmsUmsAm-3' (SEQ ID NO: 42) The X is any of the modified nucleoside monomers described above.

[0111] In some embodiments, X is selected from M3C, 2M4C, M4C, and CP4C.

[0112] In some embodiments, the sugar ring of X can be further modified, and the modifications include a 2'-F modification and a 2'-Ome modification.

[0113] The dsRNA of the present invention can also be expressed in vivo in cells using a recombinant viral vector. The recombinant viral vector of the present invention comprises a sequence encoding the dsRNA of the present invention and any promoter suitable for expressing the dsRNA sequence. Suitable promoters include, for example, U6 or H1 RNA pol III promoter sequences and cytomegalovirus promoters. The selection of other suitable promoters is within the skill of the art. The recombinant viral vector of the present invention can also comprise an inducible or regulatable promoter to express the dsRNA in a specific tissue or in a specific intracellular environment.

[0114] The dsRNA of the present invention can be expressed by recombinant viral vector as two separate complementary RNA molecules or a single RNA molecule with two complementary regions.Any viral vector that can accept the coding sequence that expresses dsRNA molecule can be used, for example, vectors derived from adenovirus (AV), adeno-associated virus (AAV), retrovirus (for example, lentivirus (LV), rhabdovirus, murine leukemia virus), herpesvirus and similar viruses.By pseudotyping viral vector with envelope protein or other surface antigen from other viruses, or by replacing different viral capsid protein, the tropism of the above-mentioned viral vector can be appropriately changed.

[0115] Definition: Unless otherwise specified, the term "alkyl group" refers to a monovalent saturated aliphatic hydrocarbon group that is a straight-chain or branched-chain group containing from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms (i.e., a C1-10 alkyl group), more preferably 1 to 8 carbon atoms (i.e., a C1-8 alkyl group), and even more preferably 1 to 6 carbon atoms (i.e., a C1-6 alkyl group); for example, a "C1-6 alkyl group" means that the group is an alkyl group and that the carbon chain has between 1 and 6 carbon atoms (specifically 1, 2, 3, 4, 5, or 6). Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, n-heptyl, n-octyl, etc. The term "alkyl chain" refers to a saturated aliphatic hydrocarbon straight or branched chain of carbon atoms, either end of which is connected to another atom.

[0116] Unless otherwise specified, the term "cycloalkyl group" refers to a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms, preferably 3 to 12 carbon atoms (i.e., a C3-12 cycloalkyl group), more preferably 3 to 10 carbon atoms (a C3-10 cycloalkyl group), and even more preferably 3 to 6 carbon atoms (a C3-6 cycloalkyl group), 4 to 6 carbon atoms (a C4-6 cycloalkyl group), or 5 to 6 carbon atoms (a C5-6 cycloalkyl group). Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclopropyl, 2-ethyl-cyclopentyl, dimethylcyclobutyl, and the like.

[0117] Unless otherwise specified, the term "alkoxy group" refers to an -O-alkyl group, wherein the definition of alkyl group is the same as above, i.e., containing 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms, and even more preferably 1 to 6 (specifically, 1, 2, 3, 4, 5, or 6) carbon atoms. Representative examples include, but are not limited to, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a 1-methylpropoxy group, a 2-methylpropoxy group, a tert-butoxy group, a pentyloxy group, a 1-methylbutoxy group, a 2-methylbutoxy group, a 3-methylbutoxy group, a 1,1-dimethylpropoxy group, a 1,2-dimethylpropoxy group, a 2,2-dimethylpropoxy group, a 1-ethylpropoxy group, and the like.

[0118] Unless otherwise specified, the term "halogen" or "halogenated" refers to F, Cl, Br, and I. The term "halogenated alkyl group" refers to an alkyl group, as defined above, in which one, two, or more hydrogen atoms or all hydrogen atoms have been replaced with halogen. Representative examples of halogenated alkyl groups include CCl, CF, CHCl, CHCl, CHBr, CHI, CHCF, CFCF, and the like.

[0119] Unless otherwise specified, the term "heterocyclyl group" refers to a saturated or partially unsaturated monocyclic, bicyclic, or polycyclic hydrocarbon substituent of non-aromatic structure containing 3 to 20 ring atoms, of which 1, 2, 3, or more are selected from N, O, or S, and the remaining ring atoms are C. Preferably, it contains 3 to 12 ring atoms, more preferably 3 to 10 ring atoms, or 3 to 8 ring atoms, or 3 to 6 ring atoms, or 4 to 6 ring atoms, or 5 to 6 ring atoms. The number of heteroatoms is preferably 1 to 4, more preferably 1 to 3 (i.e., 1, 2, or 3). Examples of monocyclic heterocyclyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyranyl, and the like. Polycyclic heterocyclyl groups include spirocyclic, fused-ring, and bridged-ring heterocyclyl groups. "Heterocycloalkyl group" refers to a saturated "heterocyclyl group" as defined above.

[0120] Unless otherwise specified, the term "aryl group" refers to monocyclic, bicyclic, and tricyclic aromatic carbocyclic ring systems containing 6 to 16 carbon atoms, or 6 to 14 carbon atoms, or 6 to 12 carbon atoms, or 6 to 10 carbon atoms, preferably 6 to 10 carbon atoms, and the term "aryl group" may be used interchangeably with the term "aromatic ring group." Examples of aryl groups may include, but are not limited to, phenyl, naphthyl, anthracyl, phenanthryl, or pyrenyl groups, and the like.

[0121] Unless otherwise specified, the terms "heteroaryl group," "heteroaromatic ring group," and "heteroaromatic ring group" refer to an aromatic monocyclic or polycyclic ring system containing a 5- to 12-membered structure, or preferably a 5- to 10-membered structure, a 5- to 8-membered structure, and more preferably a 5- to 6-membered structure, in which one, two, three, or more ring atoms are heteroatoms and the remaining atoms are carbon, the heteroatoms being independently selected from O, N, or S, with the number of heteroatoms preferably being one, two, or three. Illustrative examples of heteroaryl groups include furan, thienyl, oxazolyl, thiazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, thiadiazolyl, triazinyl, phthalazinyl, quinoline, isoquinoline, pteridine, purine, indolyl, isoindolyl, indazolyl, benzofuran, benzothiophenyl, benzophenone ... These include, but are not limited to, pyridyl groups, benzopyrimidine groups, benzopyrazinyl groups, benzimidazolyl groups, benzophthalazinyl groups, pyrrolo[2,3-b]pyridinyl, imidazo[1,2-a]pyridinyl, pyrazolo[1,5-a]pyridinyl, pyrazolo[1,5-a]pyrimidinyl, imidazo[1,2-b]pyridazinyl, [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[1,5-a]pyridinyl and the like.

[0122] Unless otherwise specified, the term "amino-protecting group" refers to a moiety that temporarily blocks an amine-reactive site in a compound; preferably, the amino-protecting group is selectively removable by chemical reaction and can be removed as needed using methods well known in the art. Common amino-protecting groups include, but are not limited to, benzoyl (Bz), acetyl (Ac), benzyloxycarbonyl (CBz), tert-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), p-methoxybenzyl (PMB), benzyl (Bn), trityl (Tr), p-toluenesulfonyl (Tos), phthaloyl (Pht), allyloxycarbonyl (Alloc), and trifluoroacetyl (Tfa). More preferably, the amino-protecting group is a benzoyl group (Bz), an acetyl group (Ac), a benzyloxycarbonyl group (CBz), a tert-butoxycarbonyl group (Boc), or a 9-fluorenylmethyloxycarbonyl group (Fmoc).

[0123] Unless otherwise specified, the term "hydroxy protecting group" refers to a moiety that temporarily blocks a hydroxy reactive site in a compound, and preferably, the hydroxy protecting group is selectively removable by chemical reaction and can be removed as needed using methods well known in the art. Common hydroxy protecting groups include, but are not limited to, methoxymethyl ether (MOM), 2-tetrahydropyran (THP), tert-butyldimethylchlorosilyl (TBS), trimethylsilyl (TMS), triethylsilyl (TES), trityl (Tr), 4,4'-bismethoxytrityl (DMTr), 4,4',4'-trimethoxytrityl (TMTr), benzyl (Bn), benzoyl (Bz), acetyl (Ac), and p-methoxybenzyl (PMB). More preferred hydroxyl protecting groups are trityl (Tr), 4,4'-bismethoxytrityl (DMTr), 4,4',4'-trimethoxytrityl (TMTr), benzyl (Bn), benzoyl (Bz), and acetyl (Ac).

[0124] "G," "C," "A," and "U" generally represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. However, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides or surrogate replacement moieties, as described in more detail below. It is well known to those skilled in the art that guanine, cytosine, adenine, and uracil can be substituted for other moieties without substantially altering the base-pairing properties of oligonucleotides containing nucleotides with such replacement moieties. Sequences containing such replacement moieties are embodiments of the present invention.

[0125] As used herein, "PCSK9" refers to the subtilisin precursor protein convertase 9 gene or protein (also known as FH3, HCHOLA3, NARC-1, or NARC1). The provided PCSK9 mRNA sequences are human: NM_174936, mouse: NM_153565, and rat: NM_199253.

[0126] The "target gene" as used herein is not limited to PCSK9, and currently, all genes that can be regulated by siRNA fall within the scope of the target gene defined in the present invention. For example, the target gene may be PCSK9, ANGPTL3, FXII, LPA, or APOB genes, preferably ANGPTL3 and PCSK9 genes, and most preferably PCSK9 gene.

[0127] As used herein, "LPA" refers to LP(A), also known as lipoprotein a, which is synthesized primarily in the liver. The protein encoded by this gene is a serine protease that can inhibit the activity of tissue plasminogen activator I.

[0128] As used herein, "FXII" refers to blood coagulation factor 12, a blood coagulation factor whose deficiency results in prolonged venous blood clotting time.

[0129] As used herein, "APOB" refers to apolipoprotein B, a protein in plasma lipoproteins whose primary function is to carry lipids. It can be classified into apoB48 and apoB100 subtypes. ApoB48 is one of the apolipoproteins in chylosporins (CM), and apoB100 is one of the apolipoproteins in very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL).

[0130] Unless otherwise specified, the term "complementary" as used herein to describe the relationship between a first nucleotide sequence and a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions to form a double helix structure, as understood by those skilled in the art. For example, such conditions may be stringent conditions, such as 400 mM NaCl, 40 mM PIPES pH 6.4, and 1 mM EDTA at 50°C or 70°C for 12-16 hours, followed by washing. Other conditions, such as physiologically relevant conditions that may be encountered in living organisms, may also be used. Those skilled in the art will be able to determine the optimal set of conditions for testing the complementarity of both sequences based on the ultimate application of the hybridized nucleotides.

[0131] This includes base pairing between an oligonucleotide or polynucleotide containing the first nucleotide sequence and an oligonucleotide or polynucleotide containing the second nucleotide sequence throughout the entire length of the first and second nucleotide sequences. Herein, these sequences are referred to as "fully complementary" to each other. However, when the first and second sequences are referred to as "substantially complementary" herein, the two sequences are either fully complementary or can form one or more, typically no more than four, three, or two mismatched base pairs upon hybridization while retaining their hybridization ability under conditions most relevant to their end application. However, if two oligonucleotides hybridize by design to form one or more single-stranded overhangs, such overhangs should not be considered mismatches in light of the definition of complementary. For example, in a dsRNA containing one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, and this case would still be considered "fully complementary" for purposes of the present invention.

[0132] As used herein, the terms "double-stranded ribonucleic acid," "double-stranded RNA," or "dsRNA" refer to a complex of ribonucleic acid molecules, which has a double-stranded structure and comprises two antiparallel and substantially complementary nucleic acid strands as described above. The two strands forming the double-stranded structure may be different portions of the same larger RNA molecule, or may be a single RNA molecule. When the two strands are a single RNA molecule, such a dsRNA is often referred to in the literature as siRNA ("small interfering RNA"). When the two strands are portions of a larger molecule and are linked by an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the other strand that form the double-stranded structure, the linked RNA strands are called "hairpin loops," "short hairpin RNAs," or "shRNAs." When the two strands are covalently linked in a form other than an uninterrupted chain between the 3'-end of one strand and the 5'-end of the other strand that form the double-stranded structure, the linked structure is called a "linker." The RNA strands may have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides of the shortest strand in dsRNA minus any overhangs present in the double strand. In addition to the double-stranded structure, dsRNA can contain one or more nucleotide overhangs. Furthermore, as used herein, "dsRNA" can include chemical modifications to ribonucleotides, modifications to multiple nucleotides, and all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, "dsRNA" encompasses all such modifications used in siRNA-type molecules.

[0133] The terms "dsRNA," "siRNA," and "iRNA reagent" are used interchangeably to refer to agents capable of mediating silencing of a target RNA (e.g., mRNA, e.g., a transcript of a gene encoding a protein). For convenience, such mRNA is also referred to herein as an mRNA awaiting silencing. Such a gene is also referred to as a target gene. Typically, the RNA awaiting silencing is an endogenous gene or a pathogen gene. RNA other than mRNA (e.g., TRNA) or viral RNA can also be targeted.

[0134] As used herein, "nucleotide overhang" refers to one or more unpaired nucleotides that protrude from the double-stranded structure of a dsRNA, such as when the 3'-end of one strand of the dsRNA exceeds the 5'-end of the other strand, or vice versa. "Flush end" or "blunt end" refers to the absence of unpaired nucleotides at the end of a dsRNA, i.e., the absence of nucleotide overhangs. "Blunt-ended" dsRNA refers to a double-stranded dsRNA throughout its entire length, i.e., the absence of nucleotide overhangs at either end of the molecule. For clarity, when determining whether an siRNA has an overhang or flush end, the chemical cap or non-nucleotide chemical moiety attached to the 3'-end or 5'-end of the siRNA is not taken into account.

[0135] The term "antisense strand" refers to one strand in dsRNA that comprises a region that is substantially complementary to a target sequence. As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (for example, a target sequence). If the complementary region is not completely complementary to the target sequence, the most permissible mismatch occurs in the terminal region, and when mismatch exists, it is usually located in the terminal region, or within 6, 5, 4, 3 or 2 nucleotides of, for example, the 5'-end and / or 3'-end.

[0136] As used herein, the term "sense strand" refers to the strand in a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.

[0137] The "conventional chemical modifications" of dsRNA described in the present invention mainly include conventional chemical modifications of sugar rings and phosphate bonds to enhance stability. Such chemical modifications include, but are not limited to, sugar ring 2'-modifications. Examples of sugar ring 2'-modifications include, but are not limited to, 2'-methoxyethyl, 2'-O-methyl (2'-OMe), 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-fluoro, or 2'-ara-F. Examples of conventional chemical modifications of phosphate bonds include, but are not limited to, replacing internucleotide phosphate bonds with other bonds, such as thiophosphate bonds. One or more of such modifications may be used.

[0138] As will be understood by those skilled in the art, the term "introducing into a cell" when used with dsRNA refers to a substance that is conveniently taken up or absorbed into a cell. Absorption or uptake of dsRNA can occur by unassisted diffusion or active cellular processes, or by the use of auxiliary reagents or equipment. This term is not limited to cells in vitro; dsRNA may also be "introduced into a cell," where the cell is part of a living organism. In this case, introduction into a cell includes delivery to the organism. For example, in in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection.

[0139] As used herein, a "pharmaceutical composition" comprises a pharmaceutically effective amount of dsRNA and a pharmaceutically acceptable carrier. As used herein, a "pharmacologically effective amount," "therapeutically effective amount," or "effective amount" refers to an amount of RNA that effectively produces the required pharmacological, therapeutic, or preventive result. For example, if a given clinical treatment that reduces a measurable parameter associated with a disease or disorder by at least 25% is considered to be an effective treatment, a therapeutically effective amount of a drug for treating the disease or disorder is the amount necessary to reduce the parameter by at least 25%. The term "pharmaceutically acceptable carrier" refers to a carrier used in administering a therapeutic agent. Such carriers include, but are not limited to, saline solution, buffered salt solution, glucose, water, glycerin, ethanol, and compositions thereof.

[0140] The term "silencing" refers to at least partially inhibiting the expression of a target gene.

[0141] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the changes in the number of down-regulated and up-regulated genes compared with modified dsRNA. FIG. 2 shows the effect of modified dsRNA on the % survival of target mRNA (PCSK9). FIG. 3 shows the effect of modified dsRNA on the % survival rate of target mRNA (ANGPTL3). FIG. 4 shows the effect of modified dsRNA on the % survival of target mRNA (LPA). FIG. 5 shows the contrasting changes in the number of down- and up-regulated genes with modified dsRNA (targeting the C5 gene). FIG. 6 shows the effect of modified dsRNA on the % survival of target mRNA (C5).

[0142] Working Example: [Table 1]

[0143] The present invention will be further described below with reference to specific examples. It should be understood that these examples are merely for the purpose of illustrating the present invention and do not limit the scope of the present invention. In the following examples, experimental methods for which specific conditions are not specified generally follow conventional conditions or conditions suggested by the manufacturer. Unless otherwise defined, all technical and scientific terms used in the specification have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described can all be applied to the method of the present invention. The preferred implementation methods and materials shown in the specification are provided for illustrative purposes only.

[0144] Example 1 Synthesis of Modified Nucleoside Monomer Compounds 1.1 Synthesis of 3-N-methyl-4-N-benzoyl-5'-(O-4,4'-bismethoxytrityl)-2'-fluoro-2'-deoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (5): [ka]

[0145] Synthesis of 3-N-methyl-2'-fluoro-2'-deoxycytidine (2) 2'-Fluoro-2'-deoxyuridine (1) (3.68 g, 15 mmol) was dissolved in dry DMF (40 mL), followed by the addition of iodomethane (1.9 mL, 30.3 mmol). The reaction mixture was stirred at room temperature for 24 h, after which the DMF was removed. The remaining crude product was azeotroped with toluene (2 × 80 mL) to remove residual DMF, and then redissolved in 15 mL of acetone. 40 mL of n-hexane was added, and the mixture was left at -20 °C for 1 h. The resulting precipitate was filtered, and the filter cake was washed with cold acetone / n-hexane and dried under vacuum to give compound 2 (2.57 g, 9.9 mmol, 66% yield) as a pale yellow solid. HRMS (ESI-TOF) [M+H] + = 260.0970.

[0146] 3-N-methyl-5'-(O-4,4'-bismethoxytrityl)-2'-fluoro-2'-deoxycytidine (3) Compound 2 (2.4 g, 9.3 mmol) was dissolved in 30 mL of dry pyridine, followed by the addition of 4,4'-bismethoxytrityl chloride (4.7 g, 14.0 mmol). The reaction was stirred overnight at room temperature, followed by the addition of 200 mL of dichloromethane. The organic phase was washed with 5% aqueous NaSO (2 x 80 mL), saturated sodium carbonate (80 mL), and saturated brine (80 mL), respectively, then dried over anhydrous sodium sulfate, filtered, and the solvent removed under reduced pressure. The remaining crude product was separated by column chromatography to give compound 3 (2.4 g, 4.3 mmol, 46% yield) as a white solid. HRMS (ESI-TOF) [M+H] + = 562.2281.

[0147] 3-N-methyl-4-N-benzoyl-5'-(O-4,4'-bismethoxytrityl)-2'-fluoro-2'-deoxycytidine (4) Compound 3 (2.3 g, 4.1 mmol) was dissolved in pyridine (2 × 50 mL) and excess water was removed by distillation under reduced pressure. It was then redissolved in 50 mL of dry pyridine. Trimethylsilyl chloride (TMSCl) (2.1 mL, 16.4 mmol) was added and stirred at room temperature for 1.5 hours. Benzoyl chloride (BzCl, 0.69 g, 4.92 mmol) was added and stirred at room temperature for 4 hours. After the reaction was complete, 10 mL of water was added and stirred at room temperature for 5 minutes. Aqueous ammonia (15 mL, 25-28% w / w) was then added. The reaction was stirred at room temperature for 15 minutes, after which the solvent was removed by distillation under reduced pressure. The remaining crude product was separated by column chromatography to give compound 4 (2.1 g, 3.20 mmol, 78% yield) as a white solid. HRMS (ESI-TOF) [M+H] + = 666.2546.

[0148] 3-N-methyl-4-N-benzoyl-5'-(O-4,4'-bismethoxytrityl)-2'-fluoro-2'-deoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (5) Compound 4 (533 mg, 0.8 mmol) was dissolved in 15 mL of dry dichloromethane, followed by the addition of N,N-diisopropylethylamine (0.40 mL, 3.2 mmol) and 2-cyanoethyl-N,N-diisopropylphosphoramidite chloride (0.24 mL, 1.60 mmol). The reaction mixture was stirred overnight at room temperature under nitrogen gas protection, quenched with water, and extracted with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography to give compound 5 (408 mg, 0.48 mmol, 60% yield) as a white solid. HRMS (ESI-TOF) [M+H] + = 866.3611.

[0149] 1.2 Synthesis of 4-N-methyl-5'-(O-4,4'-bismethoxytrityl)-2'-fluoro-2'-deoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (10) [ka]

[0150] 4-N-methyl-3',5'-ditert-butyldimethylsilyl-2'-fluoro-2'-deoxycytidine (7) Compound 6 (3.80 g, 8.0 mmol) was added to a sealed tube and dissolved in 100 mL of dry acetonitrile. After cooling the reaction to 0 °C, triethylamine (3.24 g, 32.0 mmol), p-toluenesulfonyl chloride (3.05 g, 16.0 mmol), and 4-dimethylaminopyridine (1.95 g, 16.0 mmol) were added. The reaction was stirred at 0 °C for 2-3 h, and then aqueous methylamine solution (320 mL, 25%-30% in water) was added. The tube was then closed, heated to 50 °C, and stirred for 48 h. After cooling to room temperature, the mixture was concentrated under reduced pressure. The crude product was purified by column chromatography to give compound 7 (1.80 g, 3.68 mmol, 46% yield) as a white solid. HRMS (ESI-TOF) [M+H] + = 487.2704.

[0151] 4-N-methyl-2'-fluoro-2'-deoxycytidine (8) Compound 7 (1.6 g, 3.28 mmol) was dissolved in 17 mL of tetrahydrofuran, followed by the addition of tetrabutylammonium fluoride (TBAF) (7.2 mL, 1 M in THF). The reaction was stirred at room temperature for 16 hours, then concentrated to give the crude product, which was separated by column chromatography to give compound 8 (0.55 g, 2.13 mmol, 65% yield) as a white solid. HRMS (ESI-TOF) [M+H] + = 260.0972.

[0152] 4-N-methyl-5'-(O-4,4'-bismethoxytrityl)-2'-fluoro-2'-deoxycytidine (9) Compound 8 (500 mg, 1.93 mmol) was dissolved in 6.5 mL of dry pyridine, followed by the addition of 4,4'-bismethoxytrityl chloride (0.97 g, 2.90 mmol). The reaction was stirred overnight at room temperature and then diluted with 40 mL of dichloromethane. The organic phase was washed with 5% aqueous NaSO (2 x 20 mL), saturated aqueous sodium carbonate (20 mL), and saturated brine (20 mL), respectively. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to give the crude product, which was then purified by column chromatography to give compound 9 (552.8 mg, 0.98 mmol, 51% yield) as a white solid. HRMS (ESI-TOF) [M+H] + = 562.2281.

[0153] 4-N-methyl-5'-(O-4,4'-bismethoxytrityl)-2'-fluoro-2'-deoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (10) Compound 9 (449.3 mg, 0.8 mmol) was dissolved in 15 mL of dry dichloromethane, followed by the addition of N,N-diisopropylethylamine (0.40 mL, 3.2 mmol) and 2-cyanoethyl-N,N-diisopropylphosphoramidite chloride (0.24 mL, 1.60 mmol). The reaction mixture was stirred overnight at room temperature under nitrogen gas protection, quenched with water, and extracted with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain compound 10 (370.0 mg, 0.50 mmol, 62% yield) as a white solid. HRMS (ESI-TOF) [M+H] + = 762.3351.

[0154] 1.3 Synthesis of 4-N,N-dimethyl-5'-(O-4,4'-bismethoxytrityl)-2'-fluoro-2'-deoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (14) [ka]

[0155] 4-N,N-Dimethyl-3',5'-ditert-butyldimethylsilyl-2'-fluoro-2'-deoxycytidine (11) Compound 6 (3.80 g, 8.0 mmol) was dissolved in 100 mL of dry acetonitrile in a sealed tube and cooled to 0 °C. Triethylamine (3.24 g, 32.0 mmol), p-toluenesulfonyl chloride (3.05 g, 16.0 mmol), and 4-dimethylaminopyridine (1.95 g, 16.0 mmol) were then added. The reaction mixture was stirred at 0 °C for 2-3 h, after which 200 mL of aqueous dimethylamine (40%) was added. The tube was then closed, heated to 50 °C, and stirred for 48 h. After cooling to room temperature and concentrating under reduced pressure, the crude product was separated by column chromatography to give compound 11 (2.33 g, 4.64 mmol, 58% yield) as a white solid. HRMS (ESI-TOF) [M+H] + = 502.2863.

[0156] 4-N,N-Dimethyl-2'-fluoro-2'-deoxycytidine (12) Compound 11 (2.0 g, 4.0 mmol) was dissolved in 20 mL of tetrahydrofuran, followed by the addition of tetrabutylammonium fluoride (TBAF) (8.8 mL, 1 M in THF) in THF (20 mL). The reaction was stirred at room temperature for 16 hours, then concentrated to give the crude product, which was separated by column chromatography to give compound 12 (961.9 mg, 3.5 mmol, 88% yield) as a white solid. HRMS (ESI-TOF) [M+H] + = 273.1117.

[0157] 4-N,N-dimethyl-5'-(O-4,4'-bismethoxytrityl)-2'-fluoro-2'-deoxycytidine (13) Compound 12 (527.4 mg, 1.93 mmol) was dissolved in 6.5 mL of dry pyridine, followed by the addition of 4,4'-bismethoxytrityl chloride (0.97 g, 2.90 mmol). The reaction mixture was stirred overnight at room temperature and then diluted with 40 mL of dichloromethane. The organic phase was washed with 5% aqueous NaSO (2 x 20 mL), saturated aqueous sodium carbonate (20 mL), and saturated brine (20 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to give the crude product, which was then purified by column chromatography to give compound 13 (722.1 mg, 1.25 mmol, 65% yield) as a white solid. Molecular formula: C 32 H 34 FN3O6. HRMS (ESI-TOF) [M+H] + = 576.2439.

[0158] 4-N,N-Dimethyl-5'-(O-4,4'-bismethoxytrityl)-2'-fluoro-2'-deoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (14) Compound 13 (460.5 mg, 0.8 mmol) was dissolved in 15 mL of dry dichloromethane, followed by the addition of N,N-diisopropylethylamine (0.40 mL, 3.2 mmol) and 2-cyanoethyl-N,N-diisopropylphosphoramidite chloride (0.24 mL, 1.60 mmol). The reaction mixture was stirred overnight at room temperature under nitrogen gas protection, quenched with water, and extracted with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was then purified by column chromatography to give compound 14 (413.3 mg, 0.54 mmol, 68% yield) as a white solid. HRMS (ESI-TOF) [M+H] + = 776.3521.

[0159] 1.4 Synthesis of 4-N,N-dimethyl-5'-(O-4,4'-bismethoxytrityl)-2'-methoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (19) [ka]

[0160] 4-N,N-Dimethyl-3',5'-ditert-butyldimethylsilyl-2'-methoxycytidine (16) Compound 15 (3.89 g, 8.0 mmol) was dissolved in 100 mL of dry acetonitrile in a sealed tube and cooled to 0 °C. Triethylamine (3.24 g, 32.0 mmol), p-toluenesulfonyl chloride (3.05 g, 16.0 mmol), and 4-dimethylaminopyridine (1.95 g, 16.0 mmol) were added. The reaction was stirred at 0 °C for 2-3 h, and then 200 mL of aqueous dimethylamine solution (40% in water) was added. The tube was closed, heated to 50 °C, and stirred for 48 h. After cooling to room temperature and concentrating under reduced pressure, the crude product was separated by column chromatography to give compound 16 (2.50 g, 4.88 mmol, 61% yield) as a white solid. HRMS (ESI-TOF) [M+H] + = 514.3081.

[0161] 4-N,N-dimethyl-2'-methoxycytidine (17) Compound 16 (2.05 g, 4.0 mmol) was dissolved in 20 mL of tetrahydrofuran, followed by the addition of tetrabutylammonium fluoride (TBAF) (8.8 mL, 1 M in THF) in THF (20 mL). The reaction was stirred at room temperature for 16 hours, then concentrated to give the crude product, which was separated by column chromatography to give compound 17 (912.4 mg, 3.2 mmol, 80% yield) as a white solid. HRMS (ESI-TOF) [M+H] + = 286.1316.

[0162] 4-N,N-Dimethyl-5'-(O-4,4'-bismethoxytrityl)-2'-methoxycytidine (18) Compound 17 (541.8 mg, 1.90 mmol) was dissolved in 6.5 mL of dry pyridine, followed by the addition of 4,4'-bismethoxytrityl chloride (0.97 g, 2.90 mmol). The reaction was stirred overnight at room temperature and then diluted with 40 mL of dichloromethane. The organic phase was washed with 5% aqueous NaSO (2 × 20 mL), saturated aqueous sodium carbonate (20 mL), and saturated brine (20 mL), respectively. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to give the crude product, which was then purified by column chromatography to give compound 18 (745.8 mg, 1.27 mmol, 67% yield) as a white solid. HRMS (ESI-TOF) [M+H] + = 588.2651.

[0163] 4-N,N-Dimethyl-5'-(O-4,4'-bismethoxytrityl)-2'-methoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (19) Compound 18 (469.8 mg, 0.8 mmol) was dissolved in 15 mL of dry dichloromethane, followed by the addition of N,N-diisopropylethylamine (0.40 mL, 3.2 mmol) and 2-cyanoethyl-N,N-diisopropylphosphoramidite chloride (0.24 mL, 1.60 mmol). The reaction mixture was stirred overnight at room temperature under nitrogen gas protection, quenched with water, and extracted with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product, which was then purified by column chromatography to give compound 19 (440.9 mg, 0.56 mmol, 70% yield) as a white solid. Molecular formula: C 42 H 54 N5O8P. HRMS (ESI-TOF) [M+H] + = 788.3689.

[0164] 1.5 Synthesis of 4-N-cyclopropyl-5'-(O-4,4'-bismethoxytrityl)-2'-fluoro-2'-deoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (23) [ka]

[0165] 5'-(O-4,4'-bismethoxytrityl)-2'-fluoro-2'-deoxycytidine (21) Compound 20 (5.0 g, 20.31 mmol) was dissolved in 75 mL of dry pyridine, followed by the addition of 4,4'-bismethoxytrityl chloride (10.32 g, 30.46 mmol). The reaction was stirred overnight at room temperature and then separated by column chromatography to give compound 21 (6.68 g, 1.25 mmol, 60% yield) as a white solid. HRMS (ESI-TOF) [M+H] + = 549.2234.

[0166] 4-N-cyclopropyl-5'-(O-4,4'-bismethoxytrityl)-5'-(O-4,4'-bismethoxytrityl)-2'-fluoro-2'-deoxycytidine (22) Compound 21 (4.0 g, 7.29 mmol) was dissolved in 40 mL of dry pyridine, followed by the addition of trimethylchlorosilane (TMSCl) (1.84 mL, 14.58 mmol). The reaction was stirred at room temperature for 2 hours and then designated as Solution A and prepared for use. Another reaction flask was taken, and 40 mL was added each time, followed by dilution with a total of 200 mL of dry dichloromethane. The organic phase was washed with 5% aqueous NaSO (2 × 40 mL), saturated aqueous sodium carbonate (40 mL), and saturated brine (40 mL), respectively. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. Pyridine, triethylamine (1.39 mL, 72.9 mmol), and 1,2,4-triazole (1.51 g, 21.87 mmol) were added, followed by phosphorus oxychloride (3.35 g, 21.87 mmol). The reaction was stirred at room temperature for 2 hours and then designated as Solution B. Solutions A and B were mixed homogeneously and stirred for 4 hours. Cyclopropylamine (2.52 mL, 36.45 mmol) was then added, and the mixture was stirred for 16 hours. The mixture was then diluted with 200 mL of dichloromethane. The organic phase was washed with saturated aqueous sodium carbonate (40 mL) and saturated brine (40 mL), respectively. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The resulting crude product was dissolved in 40 mL of tetrahydrofuran, and tetrabutylammonium fluoride (TBAF) (14.58 mL, 1M in THF) was added. The reaction mixture was stirred at room temperature for 16 hours, concentrated, and the crude product was separated by column chromatography to obtain compound 22 (3.0 g, 5.1 mmol, 70% yield) as a white solid. HRMS (ESI-TOF) [M+Na] + = 609.2417.

[0167] 4-N-Cyclopropyl-5'-(O-4,4'-bismethoxytrityl)-2'-fluoro-2'-deoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (23) Compound 22 (1.0 g, 1.7 mmol) was dissolved in 10 mL of dry dichloromethane, followed by the addition of N,N-diisopropylethylamine (0.89 mL, 5.1 mmol) and 2-cyanoethyl-N,N-diisopropylphosphoramidite chloride (0.57 mL, 2.55 mmol). The reaction mixture was stirred overnight at room temperature under nitrogen gas protection, quenched with water, and extracted with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was then purified by column chromatography to give compound 23 (937.6 mg, 1.19 mmol, 70% yield) as a white solid. HRMS (ESI-TOF) [M+H] + = 788.3521.

[0168] 1.6 Synthesis of 3-N-methyl-4-N-benzoyl-5'-(O-4,4'-bismethoxytrityl)-2'-methoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (28) [ka]

[0169] 3-N-methyl-2'-methoxycytidine (25) 2'-Methoxycytidine (24) (3.86 g, 15 mmol) was dissolved in dry DMF (40 mL), followed by the addition of iodomethane (1.9 mL, 30.3 mmol). The reaction mixture was stirred at room temperature for 24 h, after which the DMF was removed. The remaining crude product was azeotroped with toluene (2 x 80 mL) to remove residual DMF, and then redissolved in 15 mL of acetone. 40 mL of n-hexane was added, and the mixture was left at -20 °C for 1 h. The resulting precipitate was filtered, and the filter cake was washed with cold acetone / n-hexane and dried under vacuum to give compound 25 (2.81 g, 10.4 mmol, 69% yield) as a pale yellow solid. Chemical formula: C 11 H 17 N3O5. HRMS (ESI-TOF) [M+H] + = 272.1151.

[0170] 3-N-methyl-5'-(O-4,4'-bismethoxytrityl)-2'-methoxycytidine (26) Compound 25 (2.5 g, 9.2 mmol) was dissolved in 30 mL of dry pyridine, followed by the addition of 4,4'-bismethoxytrityl chloride (4.7 g, 14.0 mmol). The reaction was stirred overnight at room temperature, followed by the addition of 200 mL of dichloromethane. The organic phase was washed with 5% aqueous NaSO (2 x 80 mL), saturated sodium carbonate (80 mL), and saturated brine (80 mL), respectively, then dried over anhydrous sodium sulfate, filtered, and the solvent removed under reduced pressure. The remaining crude product was separated by column chromatography to give compound 26 (2.6 g, 4.6 mmol, 50% yield) as a white solid. Molecular formula: C 32 H 35 N3O7. HRMS (ESI-TOF) [M+H] + = 574.2511.

[0171] 3-N-methyl-4-N-benzoyl-5'-(O-4,4'-bismethoxytrityl)-2'-methoxycytidine (27) Compound 26 (2.29 g, 4.0 mmol) was dissolved in pyridine (2 × 50 mL) and excess water was removed by distillation under reduced pressure. The mixture was then redissolved in 50 mL of dry pyridine. Trimethylsilyl chloride (TMSCl) (2.1 mL, 16.4 mmol) was added and stirred at room temperature for 1.5 hours. Benzoyl chloride (BzCl, 0.69 g, 4.92 mmol) was added and stirred at room temperature for 4 hours. After the reaction was complete, 10 mL of water was added and stirred at room temperature for 5 minutes. Aqueous ammonia (15 mL, 25-28% w / w) was then added. The reaction was stirred at room temperature for 15 minutes, after which the solvent was removed by distillation under reduced pressure. The remaining crude product was separated by column chromatography to give compound 27 (2.0 g, 2.95 mmol, 74% yield) as a white solid. Molecular formula: C 39 H 39 NO8. HRMS (ESI-TOF) [M+H] + = 678.2729.

[0172] 3-N-methyl-4-N-benzoyl-5'-(O-4,4'-bismethoxytrityl)-2'-methoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (28) Compound 27 (541.8 mg, 0.8 mmol) was dissolved in 15 mL of dry dichloromethane, followed by the addition of N,N-diisopropylethylamine (0.40 mL, 3.2 mmol) and 2-cyanoethyl-N,N-diisopropylphosphoramidite chloride (0.24 mL, 1.60 mmol). The reaction mixture was stirred overnight at room temperature under nitrogen gas protection, quenched with water, and extracted with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to give compound 28 (422.3 mg, 0.48 mmol, 60% yield) as a white solid. Molecular formula: C 48 H 56 N5O9P. HRMS (ESI-TOF) [M+H] + = 878.3851.

[0173] 1.7 Synthesis of 4-N-dimethyl-5'-(O-4,4'-bismethoxytrityl)-2'-methoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (32) [ka]

[0174] 5'(O-4,4'-bismethoxytrityl)-2'-methoxycytidine (30) Compound 29 (5.2 g, 20.1 mmol) was dissolved in 75 mL of dry pyridine, followed by the addition of 4,4'-bismethoxytrityl chloride (10.32 g, 30.5 mmol). The reaction mixture was stirred overnight at room temperature and then separated by column chromatography to give compound 30 (7.01 g, 12.5 mmol, 62% yield) as a white solid. Molecular formula: C 31 H 32 NO8. HRMS (ESI-TOF) [M+H] + = 561.2165.

[0175] 4-N-methyl-5'-(O-4,4'-bismethoxytrityl)-2'-methoxycytidine (31) Compound 30 (4.0 g, 7.21 mmol) was dissolved in 40 mL of dry pyridine, followed by the addition of trimethylchlorosilane (TMSCl) (1.84 mL, 14.58 mmol). The reaction was stirred at room temperature for 2 hours and then designated Solution A and prepared for use. Another reaction flask was added, 40 mL each time, and diluted with a total of 200 mL of dry dichloromethane. The organic phase was washed with 5% aqueous NaSO (2 × 40 mL), saturated aqueous sodium carbonate (40 mL), and saturated brine (40 mL), respectively. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. Pyridine, triethylamine (1.39 mL, 72.9 mmol), and 1,2,4-triazole (1.51 g, 21.87 mmol) were added, followed by phosphorus oxychloride (3.35 g, 21.87 mmol). The reaction was stirred at room temperature for 2 hours and then designated Solution B. Solutions A and B were mixed homogeneously and stirred for 4 hours. After that, the methylamine solution was added, and stirring was continued for 16 hours. The mixture was then diluted with 200 mL of dichloromethane. The organic phase was washed with saturated aqueous sodium carbonate (40 mL) and saturated brine (40 mL), respectively. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The resulting crude product was dissolved in 40 mL of tetrahydrofuran, and tetrabutylammonium fluoride (TBAF) (14.58 mL, 1 M in THF) was added. The reaction mixture was stirred at room temperature for 16 hours, then concentrated to obtain the crude product. The crude product was separated by column chromatography to obtain compound 31 (3.0 g, 5.2 mmol, 73% yield) as a white solid. Molecular formula: C 32 H 35 N3O7. HRMS (ESI-TOF) [M+Na] + = 597.2384.

[0176] 4-N-methyl-5'-(O-4,4'-bismethoxytrityl)-2'-methoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (32) Compound 31 (975.2 mg, 1.7 mmol) was dissolved in 10 mL of dry dichloromethane, followed by the addition of N,N-diisopropylethylamine (0.89 mL, 5.1 mmol) and 2-cyanoethyl-N,N-diisopropylphosphoramidite chloride (0.57 mL, 2.55 mmol). The reaction mixture was stirred overnight at room temperature under nitrogen gas protection, quenched with water, and extracted with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product, which was then purified by column chromatography to give compound 32 (727.4 mg, 0.94 mmol, 55% yield) as a white solid. Molecular formula: C 41 H 52 N5O8P. HRMS (ESI-TOF) [M+H] + = 774.3566.

[0177] 1.8 Synthesis of 4-N-cyclopropyl-5'-(O-4,4'-bismethoxytrityl)-2'-methoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (34) [ka]

[0178] 4-N-Cyclopropyl-5'-(O-4,4'-bismethoxytrityl)-2'-methoxycytidine (33) Compound 30 (4.1 g, 7.30 mmol) was dissolved in 40 mL of dry pyridine, followed by the addition of trimethylchlorosilane (TMSCl) (1.84 mL, 14.58 mmol). The reaction was stirred at room temperature for 2 hours and then designated as Solution A and prepared for use. Another reaction flask was added, 40 mL each time, and diluted with a total of 200 mL of dry dichloromethane. The organic phase was washed with 5% aqueous NaSO (2 × 40 mL), saturated aqueous sodium carbonate (40 mL), and saturated brine (40 mL), respectively. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. Pyridine, triethylamine (1.39 mL, 72.9 mmol), and 1,2,4-triazole (1.51 g, 21.87 mmol) were added, followed by phosphorus oxychloride (3.35 g, 21.87 mmol). The reaction was stirred at room temperature for 2 hours and then designated as Solution B. Solutions A and B were mixed homogeneously and stirred for 4 hours. Cyclopropylamine (2.52 mL, 36.45 mmol) was added, and stirring was continued for 16 hours. The mixture was then diluted with 200 mL of dichloromethane. The organic phase was washed with saturated aqueous sodium carbonate (40 mL) and saturated brine (40 mL), respectively. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The resulting crude product was dissolved in 40 mL of tetrahydrofuran, and tetrabutylammonium fluoride (TBAF) (14.58 mL, 1M in THF) was added. The reaction mixture was stirred at room temperature for 16 hours, concentrated, and the crude product was separated by column chromatography to obtain compound 33 (2.5 g, 4.2 mmol, 60% yield) as a white solid. Molecular formula: C 34 H 37 N3O7. HRMS (ESI-TOF) [M+H] + = 600.2639.

[0179] 4-N-Cyclopropyl-5'-(O-4,4'-bismethoxytrityl)-2'-methoxycytidine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (34) Compound 33 (1.0 g, 1.7 mmol) was dissolved in 10 mL of dry dichloromethane, followed by the addition of N,N-diisopropylethylamine (0.89 mL, 5.1 mmol) and 2-cyanoethyl-N,N-diisopropylphosphoramidite chloride (0.57 mL, 2.55 mmol). The reaction mixture was stirred overnight at room temperature under nitrogen gas protection, quenched with water, and extracted with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product, which was then purified by column chromatography to give compound 34 (879.9 mg, 1.10 mmol, 65% yield) as a white solid. Molecular formula: C 43 H 54 N5O8P. HRMS (ESI-TOF) [M+H] + = 800.3727. [Table 2] JPEG2026503499000031.jpg197169

[0180] Example 2: Oligonucleotide synthesis and purification All oligonucleotides were synthesized using a 500 nmol Frit support (1000 Å = 100 nm) on an LK-192X synthesizer. All phosphoramidite monomers were diluted 1:40 (g / mL) with anhydrous acetonitrile and coupled twice for a total of 3 min. Deprotection was performed using 3% TCA, activation was performed using 0.3 M benzylthiotetrazole in acetonitrile, and capping and oxidation were performed using CAPA / CAPB and 50 mM I2 solutions, respectively. After trityl-off synthesis, the solid support was transferred to a 2 mL centrifuge tube, 1.2 mL of aqueous ammonia was added, and the tube was heated in an oven at 65 °C for 3 hours to remove the protecting groups. The mixture was then cooled to room temperature and concentrated under vacuum for 30 min. The solution was then filtered through a 0.22 μm membrane filter into a sample vial. Single-stranded purification was performed using a semi-preparative reverse-phase purification system with an elution gradient of 7% to 30% (ACN:100 mM TEAA) for 10 min at a flow rate of 5 mL / min. After purification, the mixture was concentrated under vacuum and then rotary dried at room temperature. Finally, the sample was dissolved in water, and each solution was desalted on a GE Hi-Trap desalting column to elute the final oligonucleotide product. All properties and purity were confirmed using ESI-MS and IEX HPLC, respectively. Concentration was measured using a microplate reader under UV light. Equimolar amounts of the sense and antisense strands were mixed, placed in a new reaction tube, heated at 95°C for 5 min, and slowly annealed to room temperature. Finally, the finished product was obtained by rotary drying at room temperature using a vacuum concentrator. [Table 3] JPEG2026503499000033.jpg242169JPEG2026503499000034.jpg242169 [Table 4]

[0181] Example 3: Anti-off-target evaluation experiment of RNA drug sequences The cells transfected with the dsRNA of the present invention were sequenced using RNA-seq technology to obtain the expression status of all mRNAs, and data analysis was performed using bioinformatics analysis methods to obtain gene regulation data by the above dsRNA. The simple steps were as follows:

[0182] Hep3B or RT4 cells were cultured in 10% FBS / DMEM-containing medium (1.5 mg / L glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin) or 10% FBS / McCoy's 5A-containing medium (100 U / mL penicillin, 100 μg / mL streptomycin) at 37°C in a 5% CO2-saturated humidity incubator. Cells were transfected in 12-well plates (200,000 cells / well) with 10 nM (PCSK9 / ANGPTL3) or 100 nM (LPA) dsRNAs listed in Table 4 using 2 μg / mL Lipo2000 (Invitrogen). Cells were harvested 48 hours post-transfection, and RNA was extracted using the TRIZOL method. A cDNA library was prepared using the TruSeq Stranded Total RNA Library Prep Kit (Illumina) and sequenced on the DNBSEQ platform. The original RNA-seq data was filtered using the Huada SOAPnuke software, with the following steps: 1) linker-containing reads (linker contamination), 2) reads with an unknown base N content greater than 5% were removed, and 3) low-mass reads were removed (we defined low-mass reads as reads with a mass value less than 15 that accounted for more than 20% of the total bases in the read). Finally, the filtered data were compared to the human genome (GCF_000001405.39_GRCh38.p13) using HISAT (RNA-seq calibrator), and uniquely aligned data were calculated using featureCounts. Differential gene expression analysis was performed using the R package DESeq2. The results were as follows:

[0183] [Table 5] Note: In the table above, VS NC indicates the change in the target sequence relative to the negative control (NC) gene, and VS blank indicates the change in the target sequence relative to the blank control (blank) gene. UP indicates up-regulated genes, and Down indicates down-regulated genes. The sum of the two is the expression level of genes that the sequence may affect under the statistical model. The higher the value, the more affected genes there are, i.e., the greater the possibility and degree of off-target effects.

[0184] The experimental data in Table 4 and Figure 1 show that adding the modified cytidine of the present invention to positions 6-8 of the AS strand of dsRNA resulted in satisfactory reduction of off-target effects, and these results were observed for multiple different sequences at different target sites, demonstrating the expected universality of the modified cytidine of the present invention in terms of reducing off-target effects at positions 6-8 of the AS strand. Furthermore, the results showed that different modifications on the sugar ring have little effect on off-target effects; for example, there was only a slight difference in the anti-off-target effect data between the 2'-F modification and the 2'-Ome modification 2M4C, and both the same sequence and the same site showed consistent anti-off-target effects.

[0185] Example 4: Experiment to evaluate the efficacy of RNA drug sequences Hep3B or RT4 cells were cultured in 10% FBS / DMEM-containing medium (1.5 mg / L glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin) or 10% FBS / MCCOY'S 5A-containing medium (100 U / mL penicillin, 100 μg / mL streptomycin) at 37°C in a 5% CO2-saturated humidity incubator. Cells were transfected with 10 nM (PCSK9 / ANGPTL3) or 100 nM (LPA) dsRNA in 24-well plates (100,000 cells / well) using 2 μg / mL Liposome 2000 (Thermo Fisher Scientific). Total cellular RNA was extracted from cell samples according to the RNA extraction kit instructions and quantitatively detected using a NanoDrop™ system. The target gene and GAPDH gene were detected in total cellular RNA using a dual RT-qPCR detection method, and the relative expression levels of the target gene in other experimental cells were analyzed using a relative quantification method based on a reference gene, with the GADPH gene as the reference gene and a blank as a control. The inventors transfected the above sequences into cells and compared the modified dsRNA of the present invention with the unmodified PC group by detecting knockout of the target gene. The results are shown in Table 5 and Figures 2 to 4. [Table 6]

[0186] The results showed that dsRNAs containing the modified nucleoside monomers of the present invention at positions 6-8 of the AS chain all achieved a "silencing" effect on target genes, and significantly reduced the expression of target genes compared to negative controls (PC-2, PC-3, PC-4) that did not contain the modified nucleoside monomers of the present invention, and that the modified nucleoside monomers of the present invention all exhibited the above effect in multiple sequences at different target sites.The above data demonstrated that the dsRNAs of the present invention have superior pharmacodynamic functions compared to dsRNAs that do not contain the modified nucleoside monomers of the present invention.

[0187] Example 5: Anti-off-target and drug efficacy evaluation experiment of targeting complement C5 gene Furthermore, the anti-off-target effect was verified using dsRNA related to the target complement C5 gene, gene bank accession number GI: 1732746243, reference sequence: NM_001735.3. Table 6 shows the sequence list, and its synthesis steps are the same as those in Example 2. [Table 7]

[0188] The cells transfected with the dsRNA of the present invention were sequenced using RNA-seq technology to obtain the expression status of all mRNAs, and data analysis was performed using bioinformatics analysis methods to obtain gene regulation data by the above dsRNA. The simple steps were as follows:

[0189] HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2, 37°C incubator. After digestion, the cell density was adjusted to 2 × 10 5The cells were adjusted to cells / mL and seeded in a 24-well plate at 1 mL of cell suspension per well. To prepare 20 μL of transfection complex, 5 μL of Opti-MEM was mixed with 5 μL of 10 nM dsRNA, followed by 8.5 μL of Opti-MEM and 1.5 μL of RNAiMax transfection reagent. The mixture was then mixed and incubated for 20 min. The transfection complex was placed in a 24-well plate and incubated for 48 h in a 5% CO2, 37°C incubator. 48 h after transfection, cells were harvested, RNA was extracted using the TRIZOL method, and a cDNA library was prepared using the TruSeq Stranded Total RNA Library Prep Kit (Illumina) and sequenced on the DNBSEQ platform. The original RNA-seq data were filtered using the Huada SOAPnuke software. Specifically, the following steps were performed: 1) remove linker-containing reads (linker contamination), 2) remove reads with an unknown base N content greater than 5%, and 3) remove low-mass reads (we defined low-mass reads as reads with a mass value less than 15 that accounted for more than 20% of the total bases in the read). Finally, the filtered data were compared with the human genome (GCF_000001405.39_GRCh38.p13) using HISAT (an RNA-seq calibrator) and uniquely aligned data were calculated using feature counts. Differential gene expression analysis was performed using the R package DESeq2. The results were as follows:

[0190] [Table 8] Note: In the table above, VS blank indicates the change in the target sequence relative to the blank control sequence (blank), where UP indicates an up-regulated gene and Down indicates a down-regulated gene. The sum of the two represents the expression level of genes that the sequence may affect in a statistical model. The higher the value, the more affected genes there are, i.e., the greater the possibility and degree of off-target effects. The off-target effect indicates the reduction efficiency of the modified group compared to the unmodified group of monomers.

[0191] The experimental data further demonstrate that adding the modified cytidine of the present invention to positions 6-8 of the AS strand of dsRNA can indeed effectively reduce the number of off-targets of the target gene (the results are shown in Table 7 and Figure 5), demonstrating that the modified cytidine of the present invention at positions 6-8 of the AS strand has the expected universality to reduce off-target effects.

[0192] Furthermore, in vitro interference efficiency was verified using E85 and FE85 as examples. Cell transfection was performed in accordance with the above transfection steps. Total cellular RNA was extracted from cell samples according to the RNA extraction kit instructions and quantitatively detected using a NanoDrop. A dual RT-qPCR detection method was used to detect the target gene and GAPDH gene in the total cellular RNA. A relative quantification method based on a reference gene was used to analyze the relative expression levels of the target gene in other experimental cells. The GADPH gene was used as the reference gene and a blank was used as a control. After transfecting the above sequences into cells, the relative activity of the target gene compared to the unmodified control was measured. The results are shown in Table 8 and Figure 6. [Table 9]

[0193] The results are shown in Table 8 and Figure 6. Adding modified cytidine to positions 6-8 of the AS strand of dsRNA effectively reduced the number of off-targets of the target gene while maintaining high inhibitory activity against the target gene. The inhibitory activity was similar before and after modification, with no statistically significant difference (P<0.05).

[0194] As described above, when the modified nucleoside monomers provided by the present invention are incorporated into positions 6-8 of the AS strand of dsRNA, they not only reduce the off-target effects of dsRNA, but also maintain and even improve to some extent the specific binding (expression inhibition) effect to the target gene. Furthermore, because the above effects were demonstrated for multiple different sequences targeting different targets, the development of multiple sequences for multiple targets is expected. Furthermore, the present invention employs simpler chemical synthesis steps and methods to modify the structure of a portion of a natural ribonucleoside monomer, which is expected to have lower physiological toxicity. [Brief explanation of the drawings]

[0195] [Figure 1] FIG. 1 shows the contrasting changes in the number of down- and up-regulated genes with modified dsRNA. [Figure 2] The effect of modified dsRNA on the percentage of target mRNA (PCSK9) remaining. [Figure 3] Effect of modified dsRNA on the percentage of target mRNA (ANGPTL3) remaining. [Figure 4] Effect of modified dsRNA on the percent survival of target mRNA (LPA). [Figure 5] FIG. 1 shows the contrasting changes in the number of down- and up-regulated genes with modified dsRNA (targeting the C5 gene). [Figure 6] Effect of modified dsRNA on the percent survival of target mRNA (C5).

Claims

1. A nucleoside monomer having the structure of formula (I): 【Chemistry 1】 Among them, the base is selected from the following structures: 【Chemistry 2】 R 3 , R 4 , R 5 , R 6 are each independently an amino protecting group, hydrogen, C 1-3 The group is selected from alkyl groups (methyl, ethyl, propyl, isopropyl), methoxyethyl, cyclopropyl, fluoromethyl, difluoromethyl, trifluoromethyl, and acetyl groups, and the condition is R 3 and R 4 At the same time, it is not hydrogen, but R 5 and R 6 but at the same time it is not hydrogen, X is selected from F or -OMe; R 1 , R 2 are each independently selected from H, a hydroxy protecting group, or a phosphoramidite group; Nucleoside monomers.

2. R 3 , R 4 , R 5 , R 6 are each independently an amino protecting group, hydrogen, C 1-3 selected from alkyl groups (methyl, ethyl, propyl, isopropyl) and cyclopropyl groups; The nucleoside monomer of claim 1.

3. Base is, 【Transformation 3】 Selected from The nucleoside monomer of claim 1.

4. R 1 , R 2 are each independently selected from DMTr or a phosphoramidite group, preferably R 1 , R 2 are each independently DMTr or 【Chemistry 4】 Among them, R 7 , R 8 are each independently C 1-3 selected from linear or branched alkyl groups, R 9 is C 1-3 Straight or branched chain alkyl groups, cyano (-CN) substituted C 1-3 A linear or branched alkyl group is preferably selected from R 1 , R 2 are each independently DMTr or 【Transformation 5】 Preferably, R 1 is selected from DMTr, and R 2 teeth, 【Transformation 6】 Preferably, R 1 , R 2 are all selected from H, The nucleoside monomer of claim 1. 【Request Item 5】 【Chemistry 7】 Selected from The nucleoside monomer of claim 1.

6. A double-stranded ribonucleic acid capable of inhibiting expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the sense strand optionally comprising a ligand, and at least one of the following nucleoside monomers at positions 6, 7, or 8 from the 5' end in the antisense strand: 【Transformation 8】 Contains Among them, R 3 , R 4 , R 5 , R 6 are each independently hydrogen, C 1-3 The alkyl group is selected from alkyl groups (methyl, ethyl, propyl, isopropyl) and cyclopropyl groups. 3 and R 4 At the same time, it is not hydrogen, but R 5 and R 6 and are not hydrogen at the same time, X is selected from F or -Ome, and the wavy line in the structure 【Chemistry 9】 represents the site where the phosphate ester bond is formed, Double-stranded ribonucleic acid.

7. At least one of the 6th, 7th, or 8th positions from the 5' end of the antisense strand contains the following nucleoside monomer: 【Chemistry 10】 Contains Among them, R 3 , R 4 , R 5 , R 6 are each independently selected from hydrogen, a methyl group, and a cyclopropyl group, and the condition is that R 3 and R 4 At the same time, it is not hydrogen, but R 5 and R 6 but at the same time not hydrogen, The double-stranded ribonucleic acid according to claim 6.

8. At least one of the 6th, 7th, or 8th positions from the 5' end of the antisense strand contains the following nucleoside monomer: 【Chemistry 11】 containing The double-stranded ribonucleic acid according to claim 6.

9. the sense strand contains a ligand; The double-stranded ribonucleic acid according to any one of claims 6 to 8.

10. The ligand is linked to the 5' or 3' end of the sense strand. The double-stranded ribonucleic acid according to claim 9.

11. The ligand is an asialoglycoprotein receptor (ASGPR) ligand. The double-stranded ribonucleic acid of claim 10.

12. The ASGPR ligand is one or more GalNAc derivatives linked by a bivalent or trivalent branched chain structure. The double-stranded ribonucleic acid of claim 11.

13. The sense strand has 21 nucleotides and the antisense strand has 23 nucleotides. The double-stranded ribonucleic acid according to any one of claims 6 to 12.

14. A method for producing a pharmaceutical composition comprising the double-stranded ribonucleic acid according to any one of claims 6 to 13 and a pharmaceutically acceptable carrier, excipient, or delivery vector. Pharmaceutical compositions.

15. Inhibiting expression of a target gene in a cell, comprising a regulatory sequence operably linked to a nucleotide sequence encoding at least one strand of the double-stranded ribonucleic acid according to any one of claims 6 to 13. vector.

16. The vector for inhibiting expression of a target gene in a cell according to claim 15, cell.

17. The double-stranded ribonucleic acid according to any one of claims 6 to 13, or the pharmaceutical composition according to claim 14, or the vector according to claim 15, in the manufacture of a drug for inhibiting or silencing expression of a target gene in a cell. use.

18. The double-stranded ribonucleic acid according to any one of claims 6 to 13, the pharmaceutical composition according to claim 14, or the vector according to claim 15, in the manufacture of a drug for suppressing off-target effects caused by an antisense strand of the double-stranded ribonucleic acid. use.

19. The double-stranded ribonucleic acid according to any one of claims 6 to 13, or the pharmaceutical composition according to claim 14, or the vector according to claim 15, in the manufacture of a medicament for delivering the double-stranded ribonucleic acid to a specific target location in a subject. use.

20. The target location is the liver.

20. The use according to claim 19.

21. The target gene is selected from PCSK9, ANGPTL3, FXII, APOB, LPA, and C5; Use according to any one of claims 17 to 20.

22. 1. A method for inhibiting expression of a target gene in a cell, the method comprising: (a) introducing the double-stranded ribonucleic acid according to any one of claims 6 to 13 into a cell, wherein the double-stranded ribonucleic acid is capable of inhibiting at least 40% of gene expression of the target gene when contacted with a cell expressing the target gene; (b) maintaining the cells obtained in step (a) for a sufficient time to inhibit expression of the target gene in the cells by degrading mRNA transcripts of the target gene; method.

23. A method for treating or preventing a disease associated with abnormal upregulation of target gene expression, the method comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of one or more double-stranded ribonucleic acids according to any one of claims 6 to 13, or the pharmaceutical composition according to claim 14, or the vector according to claim 15. method.

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