dsRNA, its uses and preparation methods

Abstract

The present invention provides dsRNA, its use and preparation methods, as well as pharmaceutical compositions, cells or reagent kits containing the dsRNA, which can disrupt the expression of APOC3 and prevent and / or treat associated diseases.

Description

[Technical field]

[0001] This disclosure claims priority to Chinese patent application No. 202210064031.8, filed on January 20, 2022, and this disclosure incorporates the entire contents of the above Chinese patent application by reference.

[0002] The present disclosure relates to the technical field of reducing expression of target RNA with dsRNA to treat associated diseases; specifically, the present disclosure relates to the preparation and use of dsRNA. [Background technology]

[0003] RNA interference (RNAi) is an efficient method for silencing gene expression. Statistics show that more than 80% of disease-related proteins in the human body cannot currently be targeted by conventional small molecule drugs and biopolymer preparations, and are therefore undruggable proteins. With RNA interference technology, appropriate dsRNA can be designed according to the mRNAs encoding these proteins, and the target mRNA can be specifically targeted and degraded to achieve inhibition of the production of the relevant protein. Therefore, dsRNA has great potential for drug development. However, to achieve therapeutic RNA interference effects in vivo, it must be delivered to specific cells in vivo.

[0004] Conjugation with a targeting ligand and endocytosis into cells using the targeting ligand and the receptor molecular structure on the cell membrane surface is an effective drug delivery method. For example, the asialoglycoprotein receptor (ASGPR) is a hepatocyte-specific receptor that has a high abundance on the hepatocyte surface and is characterized by rapid intracellular and extracellular conversion. Monosaccharide and polysaccharide molecules such as galactose, galactosamine, and N-acetylgalactosamine have high affinity for ASGPR. According to the literature (10.16476 / j.pibb.2015.0028), the use of aminogalactose molecular clusters (GalNAc) can efficiently deliver RNA to hepatocytes, and GalNAc molecules can be designed as trivalent or tetravalent molecular clusters to significantly improve the ability of monovalent or divalent GalNAc molecules to target hepatocytes.

[0005] Different molecular cluster structures and different linkage modes with RNA obviously affect the activity of dsRNA in vivo, with higher activity meaning better therapeutic effect or lower dosage, and with the same efficacy, lower dosage also means less toxic reaction.

[0006] APOC3 is mainly synthesized in the liver and plays an important role in the production, metabolism, and clearance of triglyceride-rich lipoproteins from plasma. The expression of APOC3 in the liver can promote the secretion of triglyceride-rich very low density lipoproteins (VLDL). APOC3 can also inhibit the degradation metabolism of triglyceride-rich lipoproteins by inhibiting the activity of lipoprotein lipase and hepatic lipase, further improving serum triglyceride levels. In addition, APOC3 can also inhibit the hepatic clearance of triglyceride-rich lipoproteins and their residual particles by interfering with the binding of triglyceride-rich lipoproteins and hepatic receptors.

[0007] Elevated APOC3 levels lead to elevated triglyceride levels and diseases such as cardiovascular disease, metabolic syndrome, obesity and diabetes.In recent years, APOC3 has become a promising target for treating diseases related to hypertriglyceridemia.Elevated serum triglyceride levels have been identified as an independent risk factor for cardiovascular disease and a factor promoting the progression to atherosclerosis.Individuals with severe hypertriglyceridemia are also at high risk of progressing to recurrent pancreatitis. Summary of the Invention

[0008] In a first aspect, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand comprising consecutive nucleotides in a direction from the 5' end to the 3' end, wherein the sense strand has nucleotides at positions 7, 8, and 9 modified with 2'-fluoro, the nucleotide at position 5 is independently modified with a 2'-methoxy group or modified with a 2'-fluoro, and the remaining nucleotides are modified with a 2'-methoxy group; the antisense strand has nucleotides at positions 2 and 14 modified with 2'-fluoro, the nucleotides at positions 4, 6, 8, 9, 10, 12, 16, and 18 are independently modified with a 2'-methoxy group or modified with a 2'-fluoro, and the remaining nucleotides are modified with a 2'-methoxy group; the number of nucleotides modified with 2'-fluoro in the antisense strand is 2 to 7; The antisense strand comprises, at at least one nucleotide position from 2 to 8, a chemical modification represented by formula (I), a tautomer thereof, or a pharma- ceutically acceptable salt thereof; [ka] wherein Y is selected from O, NH and S; each X is independently selected from CR4(R4'), S, NR5, and NH-CO, wherein R4, R4', and R5 are each independently H or a C1-C6 alkyl group; J2 is H or a C1-C6 alkyl group; n=0, 1 or 2, m=0, 1 or 2, s=0 or 1; R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6, wherein R6 is selected from OH, halogen, a methoxy group, an ethoxy group, N3, a C2-C6 alkenyl group, and a C2-C6 alkynyl group, and p=1, 2, or 3; Q1 is [ka] Q2 is R2, or Q1 is R2, Q2 is [ka] and Among them, R1 is H, a C1-C6 alkyl group, a C1-C6 alkoxy group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or (CH2) q R7, wherein R7 is selected from OH, halogen, methoxy, ethoxy, N3, C2-C6 alkenyl and C2-C6 alkynyl, and q=1, 2 or 3; J1 is H or a C1-C6 alkyl group; R2 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8, wherein R8 is selected from OH, halogen, methoxy, ethoxy, N3, C2-C6 alkenyl and C2-C6 alkynyl, and r=1, 2 or 3; Optionally, R1 and R2 are directly linked to form a ring; B is a base, The chemical modification as shown in the above formula (I), its tautomer or its pharma- ceutically acceptable salt, [ka] isn't it.

[0009] In some embodiments (the definitions of certain groups or features are as follows, and groups or features that are not defined are the same as those described in any one of the other embodiments, hereinafter abbreviated as "in some embodiments"), when X is NH-CO, R1 is not H.

[0010] In some embodiments, the 2'-methoxy group modification replaces the chemical modification shown in formula (I), a tautomer thereof, or a pharma- ceutically acceptable salt thereof.

[0011] In some embodiments, the antisense strand has at least one nucleotide at positions 2 to 8 modified with a 2'-methoxy group.

[0012] In some specific embodiments, the antisense strand has 2'-fluoro modified nucleotides at positions 2, 4, 6, 10, 12, 14, and 16 from the 5' end.

[0013] In some specific embodiments, the antisense strand has 2'-fluoro modified nucleotides at positions 2, 4, 6, 10, 14, and 16 from the 5' end.

[0014] In some specific embodiments, the antisense strand has 2'-fluoro modified nucleotides at positions 2, 4, 6, 12, 14, and 16 from the 5' end.

[0015] In some specific embodiments, the antisense strand has 2'-fluoro modified nucleotides at positions 2, 6, 10, 12, 14, and 16 from the 5' end.

[0016] In some specific embodiments, the antisense strand has 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 from the 5' end.

[0017] In some specific embodiments, the antisense strand has 2'-fluoro modified nucleotides at positions 2, 4, 6, 14, and 16 from the 5' end.

[0018] In some specific embodiments, the antisense strand has 2'-fluoro modified nucleotides at positions 2, 6, 10, 14, and 16 from the 5' end.

[0019] In some specific embodiments, the antisense strand has 2'-fluoro modified nucleotides at positions 2, 6, 12, 14, and 16 from the 5' end.

[0020] In some specific embodiments, the antisense strand has 2'-fluoro modified nucleotides at positions 6, 14, and 16 from the 5' end.

[0021] In some specific embodiments, the antisense strand has 2'-fluoro modified nucleotides at positions 2, 14, and 16 from the 5' end.

[0022] In some specific embodiments, the antisense strand has 2'-fluoro modified nucleotides at positions 2, 6, and 14 from the 5' end.

[0023] In some embodiments, the chemical modification according to formula (I) is selected from the chemical modifications according to formula (I-1): [ka] wherein Y is selected from O, NH and S; each X is independently selected from CR4(R4'), S, NR5, and NH-CO, wherein R4, R4', and R5 are each independently H or a C1-C6 alkyl group; Each J1, J2 is independently H or a C1-C6 alkyl group; n=0, 1 or 2, m=0, 1 or 2, s=0 or 1; R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6, wherein R6 is selected from OH, halogen, a methoxy group, an ethoxy group, N3, a C2-C6 alkenyl group, and a C2-C6 alkynyl group, and p=1, 2, or 3; R1 is H, a C1-C6 alkyl group, a C1-C6 alkoxy group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or (CH2) q R7, wherein R7 is selected from OH, halogen, methoxy, ethoxy, N3, C2-C6 alkenyl and C2-C6 alkynyl, and q=1, 2 or 3; R2 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8, wherein R8 is selected from OH, halogen, methoxy, ethoxy, N3, C2-C6 alkenyl and C2-C6 alkynyl, and r=1, 2 or 3; Optionally, R1 and R2 are directly linked to form a ring; B is as defined in formula (I).

[0024] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0025] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0026] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0027] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0028] In some embodiments, the chemical modification according to formula (I) is selected from the chemical modifications according to formula (I-2): [ka] wherein Y is selected from O, NH and S; each X is independently selected from CR4(R4'), S, NR5, and NH-CO, wherein R4, R4', and R5 are each independently H or a C1-C6 alkyl group; n=0, 1 or 2, m=0, 1 or 2, s=0 or 1; Each J1, J2 is independently H or a C1-C6 alkyl group; R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) pR6, wherein R6 is selected from OH, halogen, a methoxy group, an ethoxy group, N3, a C2-C6 alkenyl group, and a C2-C6 alkynyl group, and p=1, 2, or 3; R1 is H, a C1-C6 alkyl group, a C1-C6 alkoxy group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or (CH2) q R7, wherein R7 is selected from OH, halogen, methoxy, ethoxy, N3, C2-C6 alkenyl and C2-C6 alkynyl, and q=1, 2 or 3; R2 is H, a C1-C6 alkyl group, a C1-C6 alkoxy group, S-CH3, NCH3(CH3), OCH2CH2OCH3, an -O-alkylamino group, and (CH2) r R8, wherein R8 is selected from OH, halogen, methoxy, ethoxy, N3, C2-C6 alkenyl and C2-C6 alkynyl, and r=1, 2 or 3; Optionally, R1 and R2 are directly linked to form a ring; B is as defined in formula (I).

[0029] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0030] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0031] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0032] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0033] In some embodiments, each X is independently selected from CR4(R4'), S, NR5, and NH-CO, wherein R4, R4', and R5 are each independently H, a methyl group, an ethyl group, an n-propyl group, or an isopropyl group; n=0, 1 or 2, m=0, 1 or 2, s=0 or 1; Each of J1 and J2 is independently H or a methyl group; R3 is H, OH, F, Cl, NH2, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino, -O-ethylamino, and (CH2). p R6, wherein R6 is selected from OH, F, Cl, a methoxy group, an ethoxy group, N3, a vinyl group, an allyl group, an ethynyl group, and a propargyl group, and p=1 or 2; R1 is H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, or (CH2) q R7, wherein R7 is selected from OH, F, Cl, a methoxy group, an ethoxy group, N3, a vinyl group, an allyl group, an ethynyl group, and a propargyl group, and q=1 or 2; R2 is H, OH, F, Cl, NH2, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino, -O-ethylamino, and (CH2). r R8, wherein R8 is selected from OH, F, Cl, methoxy, ethoxy, N3, vinyl, allyl, ethynyl and propargyl, and r=1 or 2; Optionally, R1 and R2 are directly linked to form a ring; B is as defined in formula (I).

[0034] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0035] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0036] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0037] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0038] In some embodiments, each X is independently selected from CR4(R4'), S, NR5, and NH-CO, wherein R4, R4', and R5 are each independently H, a methyl group, an ethyl group, an n-propyl group, or an isopropyl group; n=0, 1 or 2, m=0, 1 or 2, s=0 or 1; Each of J1 and J2 is independently H or a methyl group; R3 is H, OH, F, Cl, NH2, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino, -O-ethylamino, and (CH2). p R6, wherein R6 is selected from OH, F, Cl, a methoxy group, an ethoxy group, N3, a vinyl group, an allyl group, an ethynyl group, and a propargyl group, and p=1 or 2; R1 is H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, or (CH2) q R7, wherein R7 is selected from OH, F, Cl, a methoxy group, an ethoxy group, N3, a vinyl group, an allyl group, an ethynyl group, and a propargyl group, and q=1 or 2; R2 is H, OH, F, Cl, NH2, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino, -O-ethylamino, and (CH2). r R8, wherein R8 is selected from OH, F, Cl, methoxy, ethoxy, N3, vinyl, allyl, ethynyl and propargyl, and r=1 or 2; Optionally, R1 and R2 are directly linked to form a ring; B is as defined in formula (I).

[0039] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0040] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0041] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0042] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0043] In some embodiments, Y is O or NH, and each X is independently selected from NH—CO, CH, and NH; n=0 or 1, m=0 or 1, s=0 or 1, Each J1 and J2 is independently H; R1 is selected from H, a methyl group, and CH2OH; R2 is selected from H, OH, NH2, a methyl group, and CH2OH; R3 is selected from H, OH, NH2, a methyl group, and CH2OH; Optionally, R1 and R2 are directly linked to form a ring; B is as defined in formula (I).

[0044] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0045] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0046] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0047] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0048] In some embodiments, Y is O or NH, and each X is independently selected from NH—CO, CH, and NH; n=0 or 1, m=0 or 1, s=0 or 1, Each J1 and J2 is independently H; R1 is selected from H, a methyl group, and CH2OH; R2 is selected from H, a methyl group, and CH2OH; R3 is selected from H, OH, NH2, a methyl group, and CH2OH; Optionally, R1 and R2 are directly linked to form a ring; B is as defined in formula (I).

[0049] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0050] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0051] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0052] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0053] In some embodiments, Y is O or NH; each X is independently selected from CR4(R4'), NR5, and NH-CO, where R4, R4', and R5 are each independently H or a C1-C6 alkyl group; J2 is H or a C1-C6 alkyl group; n=0 or 1, m=0 or 1, s=0 or 1, R3 is H, OH, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, and (CH2) p R6, where R6 is selected from OH, a methoxy group and an ethoxy group, and p=1, 2 or 3; Q1 is [ka] Q2 is R2, or Q1 is R2, Q2 is [ka] and R1 is H, OH, C1-C6 alkyl group, C1-C6 alkoxy group, and (CH2) q R7, where R7 is selected from OH, a methoxy group and an ethoxy group, and q=1, 2 or 3; J1 is H or a C1-C6 alkyl group; R2 is H, OH, C1-C6 alkyl group, C1-C6 alkoxy group, and (CH2) r R8, where R8 is selected from OH, methoxy and ethoxy groups, and r=1, 2 or 3; Optionally, R1 and R2 are directly linked to form a 3- to 6-membered ring; B is a base, The chemical modification as shown in the above formula (I), its tautomer or its pharma- ceutically acceptable salt, [ka] isn't it.

[0054] In some embodiments, X is independently selected from CR4(R4') and NH-CO.

[0055] In some embodiments, X is independently selected from CR4(R4').

[0056] In some embodiments, R3 is H, a C1-C6 alkyl group, or (CH2) p Selected from R6.

[0057] In some embodiments, R3 is selected from H and a C1-C6 alkyl group.

[0058] In some embodiments, R1 is H, a C1-C6 alkyl group, or (CH2) q Selected from R7.

[0059] In some embodiments, R1 is selected from H and a C1-C6 alkyl group.

[0060] In some embodiments, R2 is H, OH, a C1-C6 alkyl group, or (CH2) r Selected from R8.

[0061] In some embodiments, R2 is H, a C1-C6 alkyl group, or (CH2) r Selected from R8.

[0062] In some embodiments, Y is O, each X is independently selected from CR4(R4') and NH-CO, where R4 and R4' are each independently selected from H or a C1-C6 alkyl group; J2 is H or a C1-C6 alkyl group; R3 is H, C1-C6 alkyl group and (CH2) p R6 is selected from OH, and p=1, 2 or 3; Q1 is [ka] Q2 is R2, or Q1 is R2, Q2 is [ka] and R1 is H, C1-C6 alkyl group and (CH2) q R7, R7 is selected from OH, and q=1, 2 or 3; J1 is H or a C1-C6 alkyl group; R2 is H, OH, C1-C6 alkyl group and (CH2) r R8, R8 being selected from OH, r=1, 2 or 3; Optionally, R1 and R2 are directly linked to form a 5- or 6-membered ring; B is a base.

[0063] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0064] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0065] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0066] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0067] In some embodiments, Y is O, Each X is independently selected from CR(R'), where R and R' are each independently H or a C-C alkyl group; J2 is H, R3 is selected from H and C1-C6 alkyl groups; Q1 is [ka] Q2 is R2, or Q1 is R2, Q2 is [ka] and R1 is selected from H and C1-C6 alkyl groups; J1 is H or a C1-C6 alkyl group; R2 is H, C1-C6 alkyl group and (CH2) r R8, R8 being selected from OH, r=1, 2 or 3; Optionally, R1 and R2 are directly linked to form a 5- or 6-membered ring; B is a base.

[0068] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0069] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0070] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0071] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0072] In some embodiments, Y is O.

[0073] In some embodiments, X is independently selected from CR4(R4'), S, NR5, and NH-CO, and R4, R4', and R5 are each independently H, a methyl group, an ethyl group, an n-propyl group, or an isopropyl group. In some embodiments, X is independently selected from NH-CO, CH2, and NH. In some embodiments, X is independently selected from NH-CO and CH2. In some embodiments, X is CH2.

[0074] In some embodiments, J2 is H or a methyl group. In some embodiments, J2 is H.

[0075] In some embodiments, R3 is H, OH, NH2, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, and (CH2) p R6 is selected from OH, methoxy and ethoxy, and p=1 or 2. In some embodiments, R3 is selected from H, methyl, ethyl, n-propyl, isopropyl and (CH2) p R6 is selected from OH, and p=1 or 2. In some embodiments, R3 is selected from H and a methyl group.

[0076] In some embodiments, R1 is H, OH, a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a methoxy group, an ethoxy group, a n-propoxy group, an isopropoxy group, and (CH2) q R7, where R7 is selected from OH and q=1 or 2. In some embodiments, R1 is selected from H, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and (CH2) q R7, R7 is selected from OH, and q=1 or 2. In some embodiments, R1 is selected from H and a methyl group.

[0077] In some embodiments, R2 is H, OH, a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a methoxy group, an ethoxy group, a n-propoxy group, an isopropoxy group, and (CH2)r R8 is selected from OH, and r=1 or 2. In some embodiments, R2 is selected from H, OH, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and (CH2) r R8 is selected from OH, and r=1 or 2. In some embodiments, R2 is selected from H, a methyl group, and CH2OH.

[0078] In some embodiments, R1 and R2 are directly linked to form a 5- to 6-membered ring. In some embodiments, R1 and R2 are directly linked to form a 3- to 6-membered ring. In some embodiments, R1 and R2 are directly linked to form a cyclopentyl group or a cyclohexyl group.

[0079] In some embodiments, the chemical modification shown in formula (I) above is [ka] wherein B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0080] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0081] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0082] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0083] In some embodiments, the chemical modification shown in formula (I) above is [ka] wherein B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0084] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0085] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0086] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0087] In some embodiments, the chemical modification shown in formula (I) above is [ka] wherein B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0088] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0089] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0090] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0091] In some embodiments, the chemical modification shown in formula (I) above is [ka] wherein B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0092] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0093] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0094] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0095] In some embodiments, the nucleotide having the chemical modification as shown in formula (I), its tautomer, or its pharma- ceutically acceptable salt is selected from the nucleotide having the chemical modification as shown in formula (I'), its tautomer, or its pharma- ceutically acceptable salt: [ka] wherein Y is selected from O, NH and S; each X is independently selected from CR4(R4'), S, NR5, and NH-CO, wherein R4, R4', and R5 are each independently H or a C1-C6 alkyl group; J2 is H or a C1-C6 alkyl group; n=0, 1 or 2, m=0, 1 or 2, s=0 or 1; R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6, wherein R6 is selected from OH, halogen, a methoxy group, an ethoxy group, N3, a C2-C6 alkenyl group, and a C2-C6 alkynyl group, and p=1, 2, or 3; Q 1’ teeth [ka] Q 2’ is R2, or Q 1’ R2, Q 2’ teeth [ka] and Among them, R1 is H, a C1-C6 alkyl group, a C1-C6 alkoxy group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or (CH2) q R7, wherein R7 is selected from OH, halogen, methoxy, ethoxy, N3, C2-C6 alkenyl and C2-C6 alkynyl, and q=1, 2 or 3; J1 is H or a C1-C6 alkyl group; R2 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8, wherein R8 is selected from OH, halogen, methoxy, ethoxy, N3, C2-C6 alkenyl and C2-C6 alkynyl, and r=1, 2 or 3; Optionally, R1 and R2 are directly linked to form a ring; B is a base, M is O or S; The chemical modification shown in the above formula (I'), its tautomer or its pharma- ceutically acceptable salt is [ka] isn't it.

[0096] In some embodiments, when X is NH-CO, R1 is not H.

[0097] In some embodiments, the 2'-methoxy group modification replaces the chemical modification shown in formula (I'), a tautomer thereof, or a pharma- ceutically acceptable salt thereof.

[0098] In some embodiments, the chemical modification represented by formula (I') above is selected from the chemical modifications represented by formula (I'-1): [ka] wherein Y is selected from O, NH and S; each X is independently selected from CR4(R4'), S, NR5, and NH-CO, wherein R4, R4', and R5 are each independently H or a C1-C6 alkyl group; Each J1, J2 is independently H or a C1-C6 alkyl group; n=0, 1 or 2, m=0, 1 or 2, s=0 or 1; R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6, wherein R6 is selected from OH, halogen, a methoxy group, an ethoxy group, N3, a C2-C6 alkenyl group, and a C2-C6 alkynyl group, and p=1, 2, or 3; R1 is H, a C1-C6 alkyl group, a C1-C6 alkoxy group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or (CH2) q R7, wherein R7 is selected from OH, halogen, methoxy, ethoxy, N3, C2-C6 alkenyl and C2-C6 alkynyl, and q=1, 2 or 3; R2 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8, wherein R8 is selected from OH, halogen, methoxy, ethoxy, N3, C2-C6 alkenyl and C2-C6 alkynyl, and r=1, 2 or 3; M is O or S; Optionally, R1 and R2 are directly linked to form a ring; B is as defined in formula (I').

[0099] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0100] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0101] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0102] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0103] In some embodiments, the chemical modification according to formula (I') is selected from the chemical modifications according to formula (I'-2): [ka] wherein Y is selected from O, NH and S; each X is independently selected from CR4(R4'), S, NR5, and NH-CO, wherein R4, R4', and R5 are each independently H or a C1-C6 alkyl group; n=0, 1 or 2, m=0, 1 or 2, s=0 or 1; Each J1, J2 is independently H or a C1-C6 alkyl group; R3 is H, OH, halogen, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, C2-C6 alkenyl group, C2-C6 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) p R6, wherein R6 is selected from OH, halogen, a methoxy group, an ethoxy group, N3, a C2-C6 alkenyl group, and a C2-C6 alkynyl group, and p=1, 2, or 3; R1 is H, a C1-C6 alkyl group, a C1-C6 alkoxy group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or (CH2) q R7, wherein R7 is selected from OH, halogen, methoxy, ethoxy, N3, C2-C6 alkenyl and C2-C6 alkynyl, and q=1, 2 or 3; R2 is H, a C1-C6 alkyl group, a C1-C6 alkoxy group, S-CH3, NCH3(CH3), OCH2CH2OCH3, an -O-alkylamino group, and (CH2) r R8, wherein R8 is selected from OH, halogen, methoxy, ethoxy, N3, C2-C6 alkenyl and C2-C6 alkynyl, and r=1, 2 or 3; Optionally, R1 and R2 are directly linked to form a ring; M is O or S; B is as defined in formula (I').

[0104] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0105] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0106] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0107] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0108] In some embodiments, each X is independently selected from CR4(R4'), S, NR5, and NH-CO, wherein R4, R4', and R5 are each independently H or a C1-C3 alkyl group; n=0, 1 or 2, m=0, 1 or 2, s=0 or 1; Each J1 and J2 is independently H or a C1-C3 alkyl group; R3 is H, OH, halogen, NH2, C1-C3 alkyl group, C1-C3 alkoxy group, C2-C4 alkenyl group, C2-C4 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) pR6, wherein R6 is selected from OH, halogen, a methoxy group, an ethoxy group, N3, a C2-C6 alkenyl group, and a C2-C6 alkynyl group, and p=1, 2, or 3; R1 is H, a C1-C3 alkyl group, a C1-C3 alkoxy group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or (CH2) q R7, wherein R7 is selected from OH, halogen, methoxy, ethoxy, N3, C2-C4 alkenyl and C2-C4 alkynyl, and q=1, 2 or 3; R2 is H, OH, halogen, NH2, C1-C3 alkyl group, C1-C3 alkoxy group, C2-C4 alkenyl group, C2-C4 alkynyl group, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-alkylamino group and (CH2) r R8, wherein R8 is selected from OH, halogen, methoxy, ethoxy, N3, C2-C4 alkenyl and C2-C4 alkynyl, and r=1, 2 or 3; Optionally, R1 and R2 are directly linked to form a ring; B is as defined in formula (I').

[0109] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0110] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0111] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0112] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0113] In some embodiments, each X is independently selected from CR4(R4'), S, NR5, and NH-CO, wherein R4, R4', and R5 are each independently H, a methyl group, an ethyl group, an n-propyl group, or an isopropyl group; n=0, 1 or 2, m=0, 1 or 2, s=0 or 1; Each of J1 and J2 is independently H or a methyl group; R3 is H, OH, F, Cl, NH2, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino, -O-ethylamino, and (CH2). p R6, wherein R6 is selected from OH, F, Cl, a methoxy group, an ethoxy group, N3, a vinyl group, an allyl group, an ethynyl group, and a propargyl group, and p=1 or 2; R1 is H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, or (CH2) q R7, wherein R7 is selected from OH, F, Cl, a methoxy group, an ethoxy group, N3, a vinyl group, an allyl group, an ethynyl group, and a propargyl group, and q=1 or 2; R2 is H, OH, F, Cl, NH2, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH3, NCH3(CH3), OCH2CH2OCH3, -O-methylamino, -O-ethylamino, and (CH2). r R8, wherein R8 is selected from OH, F, Cl, methoxy, ethoxy, N3, vinyl, allyl, ethynyl and propargyl, and r=1 or 2; Optionally, R1 and R2 are directly linked to form a ring; B is as defined in formula (I').

[0114] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0115] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0116] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0117] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0118] In some embodiments of Formula (I'), Formula (I'-1), or Formula (I'-2), Y is O or NH; and each X is independently selected from NH-CO, CH, and NH; n=0 or 1, m=0 or 1, s=0 or 1, Each J1 and J2 is independently H; R1 is selected from H, a methyl group, and CH2OH; R2 is selected from H, OH, NH2, a methyl group, and CH2OH; R3 is selected from H, OH, NH2, a methyl group, and CH2OH; Optionally, R1 and R2 are directly linked to form a ring; B is as defined in formula (I').

[0119] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0120] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0121] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0122] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0123] In some embodiments of Formula (I'), Formula (I'-1), or Formula (I'-2), Y is O or NH; and each X is independently selected from NH-CO, CH, and NH; n=0 or 1, m=0 or 1, s=0 or 1, Each J1 and J2 is independently H; R1 is selected from H, a methyl group, and CH2OH; R2 is selected from H, a methyl group, and CH2OH; R3 is selected from H, OH, NH2, a methyl group, and CH2OH; Optionally, R1 and R2 are directly linked to form a ring; B is as defined in formula (I').

[0124] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0125] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0126] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0127] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0128] In some embodiments, Y is O or NH; each X is independently selected from CR4(R4'), NR5, and NH-CO, where R4, R4', and R5 are each independently H or a C1-C6 alkyl group; J2 is H or a C1-C6 alkyl group; n=0 or 1, m=0 or 1, s=0 or 1, R3 is H, OH, NH2, C1-C6 alkyl group, C1-C6 alkoxy group, and (CH2) p R6, where R6 is selected from OH, a methoxy group and an ethoxy group, and p=1, 2 or 3; Q 1’ teeth [ka] Q 2’ is R2, or Q 1’ R2, Q 2’ teeth [ka] and R1 is H, OH, C1-C6 alkyl group, C1-C6 alkoxy group, and (CH2) q R7, where R7 is selected from OH, a methoxy group and an ethoxy group, and q=1, 2 or 3; J1 is H or a C1-C6 alkyl group; R2 is H, OH, C1-C6 alkyl group, C1-C6 alkoxy group, and (CH2) r R8, where R8 is selected from OH, methoxy and ethoxy groups, and r=1, 2 or 3; Optionally, R1 and R2 are directly linked to form a 3- to 6-membered ring; M is O or S; B is a base, The chemical modification shown in the above formula (I'), its tautomer or its pharma- ceutically acceptable salt is [ka] isn't it.

[0129] In some embodiments, X is independently selected from CR4(R4') and NH-CO.

[0130] In some embodiments, X is independently selected from CR4(R4').

[0131] In some embodiments, R3 is H, a C1-C6 alkyl group, or (CH2) p Selected from R6.

[0132] In some embodiments, R3 is selected from H and a C1-C6 alkyl group.

[0133] In some embodiments, R1 is H, a C1-C6 alkyl group, or (CH2) q Selected from R7.

[0134] In some embodiments, R1 is selected from H and a C1-C6 alkyl group.

[0135] In some embodiments, R2 is H, OH, a C1-C6 alkyl group, or (CH2) r Selected from R8.

[0136] In some embodiments, R2 is H, a C1-C6 alkyl group, or (CH2) r Selected from R8.

[0137] In some embodiments, Y is O, each X is independently selected from CR4(R4') and NH-CO, where R4 and R4' are each independently selected from H or a C1-C6 alkyl group; J2 is H or a C1-C6 alkyl group; R3 is H, C1-C6 alkyl group and (CH2) p R6 is selected from OH, and p=1, 2 or 3; Q 1’ teeth [ka] Q 2’ is R2, or Q 1’ R2, Q 2’ teeth [ka] and R1 is H, C1-C6 alkyl group and (CH2) q R7, R7 is selected from OH, and q=1, 2 or 3; J1 is H or a C1-C6 alkyl group; R2 is H, OH, C1-C6 alkyl group and (CH2) r R8, R8 being selected from OH, r=1, 2 or 3; Optionally, R1 and R2 are directly linked to form a 5- or 6-membered ring; M is O or S; B is a base.

[0138] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0139] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0140] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0141] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0142] In some embodiments, Y is O, Each X is independently selected from CR(R'), where R and R' are each independently H or a C-C alkyl group; J2 is H, R3 is selected from H and C1-C6 alkyl groups; Q 1’ teeth [ka] Q 2’ is R2, or Q 1’ R2, Q 2’ teeth [ka] and R1 is selected from H and C1-C6 alkyl groups; J1 is H or a C1-C6 alkyl group; R2 is H, C1-C6 alkyl group and (CH2) r R8, R8 being selected from OH, r=1, 2 or 3; Optionally, R1 and R2 are directly linked to form a 5- or 6-membered ring; M is O or S; B is a base.

[0143] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0144] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0145] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0146] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0147] In some embodiments, Y is O.

[0148] In some embodiments, X is independently selected from CR4(R4'), S, NR5, and NH-CO, and R4, R4', and R5 are each independently H, a methyl group, an ethyl group, an n-propyl group, or an isopropyl group. In some embodiments, X is independently selected from NH-CO, CH2, and NH. In some embodiments, X is independently selected from NH-CO and CH2. In some embodiments, X is CH2.

[0149] In some embodiments, J2 is H or a methyl group. In some embodiments, J2 is H.

[0150] In some embodiments, R3 is H, OH, NH2, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, and (CH2) p R6 is selected from OH, methoxy and ethoxy, and p=1 or 2. In some embodiments, R3 is selected from H, methyl, ethyl, n-propyl, isopropyl and (CH2) p R6 is selected from OH, and p=1 or 2. In some embodiments, R3 is selected from H and a methyl group.

[0151] In some embodiments, R1 is H, OH, a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a methoxy group, an ethoxy group, a n-propoxy group, an isopropoxy group, and (CH2) q R7, where R7 is selected from OH and q=1 or 2. In some embodiments, R1 is selected from H, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and (CH2) q R7, R7 is selected from OH, and q=1 or 2. In some embodiments, R1 is selected from H and a methyl group.

[0152] In some embodiments, R2 is H, OH, a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a methoxy group, an ethoxy group, a n-propoxy group, an isopropoxy group, and (CH2) r R8 is selected from OH, and r=1 or 2. In some embodiments, R2 is selected from H, OH, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and (CH2) r R8 is selected from OH, and r=1 or 2. In some embodiments, R2 is selected from H, a methyl group, and CH2OH.

[0153] In some embodiments, R1 and R2 are directly linked to form a 5- to 6-membered ring. In some embodiments, R1 and R2 are directly linked to form a 3- to 6-membered ring. In some embodiments, R1 and R2 are directly linked to form a cyclopentyl group or a cyclohexyl group.

[0154] In some embodiments, the chemical modification shown in formula (I') above is [ka] [ka] wherein M is O or S; B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0155] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0156] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0157] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0158] In some embodiments, the chemical modification shown in formula (I') above is [ka] wherein M is O or S; B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0159] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0160] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0161] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0162] In some embodiments, the chemical modification shown in formula (I') above is [ka] wherein M is O or S; B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0163] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0164] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0165] In some embodiments, B is the same as the base if the nucleotide at that position in the antisense strand is unmodified.

[0166] In some embodiments, the chemical modification shown in formula (I') above is [ka] and those in which adenine is replaced by guanine, cytosine, uracil or thymine.

[0167] In some embodiments, B is selected from a purine base, a pyrimidine base, indole, 5-nitroindole, and 3-nitropyrrole.

[0168] In some embodiments, B is selected from adenine, guanine, isoguanine, hypoxanthine, xanthine, C2-modified purine, N8-modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, N6-alkyladenine, O6-alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, C5-modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0169] In some embodiments, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0170] In some embodiments, B is the same as the base when the nucleotide at that position in the antisense strand is unmodified.

[0171] In some embodiments, the chemical modification shown in formula (I) above is [ka] It is.

[0172] In a further embodiment, the present disclosure provides a dsRNA comprising a sense strand and an antisense strand.

[0173] In some embodiments, the antisense strand is at least partially reverse complementary to the target sequence to mediate RNA interference, in some embodiments there are no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 mismatch between the antisense strand and the target sequence, and in some embodiments the antisense strand is completely reverse complementary to the target sequence.

[0174] In some embodiments, the sense strand is at least partially reverse complementary to the antisense strand to form a double-stranded region, and in some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 mismatch between the sense strand and the antisense strand, and in some embodiments, the sense strand is completely reverse complementary to the antisense strand.

[0175] In some embodiments, the sense strand and the antisense strand each independently have 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides (e.g., 19, 20, 21, 22, or 23 nucleotides).

[0176] In some embodiments, the sense strand and the antisense strand are the same or different in length, the sense strand is 19-23 nucleotides in length, and the antisense strand is 19-26 nucleotides in length. In some embodiments, the length ratio of the sense strand to the antisense strand can be 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25, or 23 / 26. In some embodiments, the length ratio of the sense strand to the antisense strand is 19 / 21, 21 / 23, or 23 / 25. In some embodiments, the length ratio of the sense strand to the antisense strand is 19 / 21.

[0177] In some embodiments, the dsRNA comprises one or two blunt ends.

[0178] In some embodiments, the dsRNA comprises an overhang of 1 to 4, eg, 1, 2, 3, 4, unpaired nucleotides.

[0179] In some embodiments, the dsRNA comprises an overhang at the 3' end of the antisense strand.

[0180] In some embodiments, the nucleotide comprising the chemical modification shown in Formula (I) or Formula (I'), a tautomer thereof, or a pharma- ceutically acceptable salt thereof is located at position 5, 6, or 7 of the 5' region of the antisense strand.

[0181] In some embodiments, the nucleotide comprising the chemical modification shown in Formula (I) or Formula (I'), a tautomer thereof, or a pharma- ceutically acceptable salt thereof is at position 7 of the 5' region of the antisense strand.

[0182] In some embodiments, when the chemical modification shown in Formula (I) or Formula (I'), its tautomer, or its pharma- ceutically acceptable salt is at the 5-position of the 5' region, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0183] In some embodiments, when the chemical modification shown in Formula (I) or Formula (I'), its tautomer, or its pharma- ceutically acceptable salt is at position 6 of the 5' region, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0184] In some embodiments, when the chemical modification shown in Formula (I) or Formula (I'), its tautomer, or its pharma- ceutically acceptable salt is at position 7 of the 5' region, B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

[0185] In some embodiments, B is the same as the base when the nucleotide at position 5 of the 5' region of the antisense strand is unmodified.

[0186] In some embodiments, B is the same as the base when the nucleotide at position 6 of the 5' region of the antisense strand is unmodified.

[0187] In some embodiments, B is the same as the base when the nucleotide at position 7 of the 5' region of the antisense strand is unmodified.

[0188] In some embodiments, the nucleotide at position 5 of the sense strand is a nucleotide modified with a 2'-methoxy group.

[0189] In some embodiments, the nucleotide at position 7 of the antisense strand is a nucleotide modified with a 2'-methoxy group.

[0190] In some embodiments, the sense strand and the antisense strand are Sense strand: 5'-N a N a N a N a XN a N b N b N b N a N a N a N a N a N a N a N a N a N a-3', Antisense strand: 5'-N a 'N b 'N a 'X'N a 'X'W'X'X'X'N a 'X'N a 'N b 'N a 'X'N a 'X'N a 'N a 'N a '-3', or comprising the nucleotide sequence shown in the formula: Of which, each X is independently N a Or N b and each X' is independently N a ' or N b ' and N a and N a ' is a nucleotide modified with a 2'-methoxy group, and N b and N b ' is a 2'-fluoro modified nucleotide, W' represents a nucleotide modified with a 2'-methoxy group, or a nucleotide containing the chemical modification shown in formula (I) or formula (I'), a tautomer thereof, or a pharma- ceutically acceptable salt thereof.

[0191] In some embodiments, W' represents a nucleotide modified with a 2'-methoxy group.

[0192] In some embodiments, W' represents a nucleotide comprising a chemical modification as shown in Formula (I) or Formula (I'), a tautomer thereof, or a pharma- ceutically acceptable salt thereof.

[0193] In some embodiments, W' is [ka] It has the following structure.

[0194] In some embodiments, the sense strand and the antisense strand are Sense strand: 5'-Na N a N a N a N a N a N b N b N b N a N a N a N a N a N a N a N a N a N a -3', Antisense strand: 5'-N a 'N b 'N a 'X'N a 'X'W'X'X'X'N a 'X'N a 'N b 'N a 'X'N a 'X'N a 'N a 'N a '-3', or comprising the nucleotide sequence shown in the formula: Wherein, each X' is independently N a ' or N b ' and N a and N a ' is a nucleotide modified with a 2'-methoxy group, and N b and N b ' is a 2'-fluoro modified nucleotide, W' represents a nucleotide modified with a 2'-methoxy group, or a nucleotide containing the chemical modification shown in formula (I) or formula (I'), a tautomer thereof, or a pharma- ceutically acceptable salt thereof.

[0195] In some embodiments, W' represents a nucleotide modified with a 2'-methoxy group.

[0196] In some embodiments, W' represents a nucleotide comprising a chemical modification as shown in Formula (I) or Formula (I'), a tautomer thereof, or a pharma- ceutically acceptable salt thereof.

[0197] In some embodiments, W' is [ka] It has the following structure.

[0198] In some embodiments, the sense strand comprises: 5'-N a N a N a N a XN a N b N b N b N a N a N a N a N a N a N a N a N a N a -3', Among them, X is N a Or N b and N a is a nucleotide modified with a 2'-methoxy group, and N b is a 2'-fluoro modified nucleotide.

[0199] In some embodiments, the sense strand comprises: 5'-N a N a N a N a N a N a N b N b N b N a N a N a N a N a N a N a N a N a N a-3', Of these, N a is a nucleotide modified with a 2'-methoxy group, and N b is a 2'-fluoro modified nucleotide.

[0200] In some embodiments, the antisense strand comprises: 5'-N a 'N b 'N a 'X'N a 'X'W'X'X'X'N a 'X'N a 'N b 'N a 'X'N a 'X'N a 'N a 'N a '-3', Wherein, each X' is independently N a ' or N b ' and N a ' is a nucleotide modified with a 2'-methoxy group, and N b ' is a 2'-fluoro modified nucleotide, W' represents a nucleotide modified with a 2'-methoxy group, or a nucleotide containing the chemical modification shown in formula (I) or formula (I'), a tautomer thereof, or a pharma- ceutically acceptable salt thereof.

[0201] In some embodiments, W' represents a nucleotide modified with a 2'-methoxy group.

[0202] In some embodiments, W' represents a nucleotide comprising a chemical modification as shown in Formula (I) or Formula (I'), a tautomer thereof, or a pharma- ceutically acceptable salt thereof.

[0203] In some embodiments, W' is [ka] It has the following structure.

[0204] In some embodiments, the chemical modification according to formula (I) is [ka] wherein B is selected from guanine, adenine, cytosine and uracil; in some specific embodiments, B is the same as the base when the nucleotide at position 7 in the 5' region of the antisense strand is unmodified.

[0205] In some embodiments, the chemical modification according to formula (I) is [ka] wherein M is O or S; and wherein B is selected from guanine, adenine, cytosine, or uracil; in some specific embodiments, B is the same as the base when the nucleotide at position 7 in the 5' region of the antisense strand is unmodified.

[0206] In some specific embodiments, M is S. In some specific embodiments, M is O.

[0207] In some embodiments, the antisense strand comprises: 5'-N a 'N b 'N a 'N b 'N a 'N b 'W'N a 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N a 'N a 'N a 'N a '-3', 5'-Na ’N b ’N a ’N b ’N a ’N b ’W’N a ’N a ’N b ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N b ’N a ’N b ’W’N a ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N b ’W’N a ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’Na ’N b ’N a ’N b ’W’N a ’N a ’N a ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N b ’W’N a ’N a ’N b ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N b ’W’N a ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’Na ’N a ’W’N b ’N b ’N a ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N b ’W’N a ’N a ’N a ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N b ’W’N a ’N a ’N a ’N a ’N a ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N a ’W’Na 'N a 'N a 'N a 'N a 'N a 'N b 'N a 'N b 'N a 'N a 'N a 'N a 'N a '-3', or 5'-N a 'N b 'N a 'N a 'N a 'N a 'W'N a 'N a 'N a 'N a 'N a 'N a 'N b 'N a 'N a 'N a 'N a 'N a 'N a 'N a '-3', or comprising the nucleotide sequence shown in the formula: Of these, N a ' is a nucleotide modified with a 2'-methoxy group, and N b ' is a 2'-fluoro modified nucleotide, W' represents a nucleotide modified with a 2'-methoxy group, or a nucleotide containing the chemical modification shown in formula (I), its tautomer, or a pharma- ceutically acceptable salt thereof.

[0208] In some embodiments, W' represents a nucleotide modified with a 2'-methoxy group.

[0209] In some embodiments, W' represents a nucleotide comprising a chemical modification as shown in formula (I), a tautomer thereof, or a pharma- ceutically acceptable salt thereof.

[0210] In some embodiments, the chemical modification according to formula (I) is [ka] wherein B is selected from guanine, adenine, cytosine and uracil; in some specific embodiments, B is the same as the base when the nucleotide at position 7 in the 5' region of the antisense strand is unmodified.

[0211] In some embodiments, the chemical modification according to formula (I) is [ka] wherein M is O or S; and wherein B is selected from guanine, adenine, cytosine, or uracil; in some specific embodiments, B is the same as the base when the nucleotide at position 7 in the 5' region of the antisense strand is unmodified.

[0212] In some embodiments, W' is [ka] It has the following structure.

[0213] In some specific embodiments, M is S. In some specific embodiments, M is O.

[0214] In some embodiments, at least one phosphate group in the sense strand and / or antisense strand is a phosphate group with a modified group that improves the stability of the dsRNA in biological samples or environments, and in some embodiments, the phosphate group with the modified group is a thiophosphate group.In some embodiments, the phosphate group with the modified group is a thiophosphodiester group.

[0215] In some embodiments, the thiophosphodiester group is Between the first and second nucleotides of the 5' end of the sense strand, Between the second and third nucleotides of the 5' end of the sense strand, Between the first and second nucleotides of the 5' end of the antisense strand, Between the second and third nucleotides of the 5' end of the antisense strand, Between the first and second nucleotides at the 3' end of the antisense strand, and Between the second and third nucleotides of the 3' end of the antisense strand, at least one of the positions:

[0216] In some embodiments, the sense strand and / or the antisense strand comprises a plurality of thiophosphodiester groups, the thiophosphodiester groups being: Between the first and second nucleotides of the 5' end of the sense strand, Between the second and third nucleotides of the 5' end of the sense strand, Between the first and second nucleotides of the 5' end of the antisense strand, Between the second and third nucleotides of the 5' end of the antisense strand, Between the first and second nucleotides at the 3' end of the antisense strand, and Between the second and third nucleotides of the 3' end of the antisense strand, at least one of the positions:

[0217] In some embodiments, the sense strand comprises: 5'-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3', or 5'-NmsNmsNmNmNmNmNfNfNmNmNmNmNmNmNmNmNmNm-3', Wherein, Nm represents any nucleotide modified with a 2'-methoxy group, for example, C, G, U, A modified with a 2'-methoxy group, Nf represents any nucleotide modified with a 2'-fluoro group, for example, C, G, U, A modified with a 2'-fluoro group, The lower case letter s indicates that the two adjacent nucleotides on the left and right sides of the letter s are linked by a thiophosphodiester group, and when the lower case letter s is the first at the 3' end, it indicates that the end of the nucleotide adjacent to the left side of the letter s is a thiophosphodiester group.

[0218] In some embodiments, the sense strand comprises: 5'-NmsNmsNmNmNmNmNfNfNmNmNmNmNmNmNmNmNmNm-3', Wherein, Nm represents any nucleotide modified with a 2'-methoxy group, for example, C, G, U, A modified with a 2'-methoxy group, Nf represents any nucleotide modified with a 2'-fluoro group, for example, C, G, U, A modified with a 2'-fluoro group, The lower case letter s indicates that the two adjacent nucleotides on the left and right sides of the letter s are linked by a thiophosphodiester group, and when the lower case letter s is the first at the 3' end, it indicates that the end of the nucleotide adjacent to the left side of the letter s is a thiophosphodiester group.

[0219] In some embodiments, the antisense strand comprises: 5'-Nm'sNf'sNm'Nf'Nm'Nf'W'Nm'Nm'Nf'Nm'Nf'Nm'Nf'Nm'Nf'Nm'Nm'Nm'sNm'sNm'-3', 5'-Nm'sNf'sNm'Nf'Nm'Nf'W'Nm'Nm'Nf'Nm'Nm'Nm'Nf'Nm'Nf'Nm'Nf'Nm'sNm'sNm'-3', 5’-Nm’sNf’sNm’Nf’Nm’Nf’W’Nm’Nm’Nm’Nm’Nf’Nm’Nf’Nm’Nf’Nm’Nf’Nm’sNm’sNm’-3’, 5’-Nm’sNf’sNm’Nm’Nm’Nf’W’Nm’Nm’Nf’Nm’Nf’Nm’Nf’Nm’Nf’Nm’Nf’Nm’sNm’sNm’-3’, 5’-Nm’sNf’sNm’Nf’Nm’Nf’W’Nm’Nm’Nm’Nm’Nm’Nm’Nf’Nm’Nf’Nm’Nm’Nm’sNm’sNm’-3’, 5’-Nm’sNf’sNm’Nm’Nm’Nf’W’Nm’Nm’Nf’Nm’Nm’Nm’Nf’Nm’Nf’Nm’Nm’Nm’sNm’sNm’-3’, 5’-Nm’sNf’sNm’Nm’Nm’Nf’W’Nm’Nm’Nm’Nm’Nf’Nm’Nf’Nm’Nf’Nm’Nm’Nm’sNm’sNm’-3’, 5’-Nm’sNf’sNm’Nm’Nm’Nm’W’Nf’Nf’Nm’Nm’Nm’Nm’Nf’Nm’Nf’Nm’Nm’Nm’sNm’sNm’-3’, 5’-Nm’sNf’sNm’Nm’Nm’Nf’W’Nm’Nm’Nm’Nm’Nm’Nm’Nf’Nm’Nf’Nm’Nm’Nm’sNm’sNm’-3’, 5’-Nm’sNf’sNm’Nm’Nm’Nf’W’Nm’Nm’Nm’Nm’Nm’Nm’Nf’Nm’Nm’Nm’Nm’Nm’sNm’sNm’-3’, 5’-Nm’sNf’sNm’Nm’Nm’Nm’W’Nm’Nm’Nm’Nm’Nm’Nm’Nf’Nm’Nf’Nm’Nm’Nm’sNm’sNm’-3’, or, 5’-Nm’sNf’sNm’Nm’Nm’Nm’W’Nm’Nm’Nm’Nm’Nm’Nm’Nf’Nm’Nm’Nm’Nm’Nm’sNm’sNm’-3’, comprising or being the nucleotide sequences represented by the formula: Wherein, Nm' represents any nucleotide modified with a 2'-methoxy group, for example, C, G, U, A modified with a 2'-methoxy group, Nf' represents any nucleotide modified with a 2'-fluoro group, for example, C, G, U, A modified with a 2'-fluoro group, The lowercase letter s indicates that the two adjacent nucleotides on the left and right sides of the letter s are linked together via a thiophosphodiester group. When the lowercase letter s is the first nucleotide at the 3' end, it indicates that the end of the nucleotide adjacent to the left side of the letter s is a thiophosphodiester group. W' represents a nucleotide modified with a 2'-methoxy group, or a nucleotide containing the chemical modification shown in formula (I) or formula (I'), a tautomer thereof, or a pharma- ceutically acceptable salt thereof.

[0220] In some embodiments, W' represents a nucleotide modified with a 2'-methoxy group.

[0221] In some embodiments, W' represents a nucleotide comprising a chemical modification as shown in Formula (I) or Formula (I'), a tautomer thereof, or a pharma- ceutically acceptable salt thereof.

[0222] In some embodiments, the chemical modification according to formula (I) or formula (I') is [ka] wherein B is selected from guanine, adenine, cytosine and uracil; in some embodiments, B is the same as the base when the nucleotide at position 7 in the 5' region of the antisense strand is unmodified.

[0223] In some embodiments, the chemical modification according to formula (I) or formula (I') is [ka] wherein M is O or S; and wherein B is selected from guanine, adenine, cytosine, or uracil; in some specific embodiments, B is the same as the base when the nucleotide at position 7 in the 5' region of the antisense strand is unmodified.

[0224] In some embodiments, M is S. In some specific embodiments, M is O.

[0225] In some embodiments, the dsRNA inhibits expression of apolipoprotein C3 (APOC3).

[0226] In some embodiments, the sense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides; and / or the antisense strand comprises at least 19 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides; In some embodiments, the sense strand comprises or is selected from the nucleotide sequence of SEQ ID NO:1 and / or the antisense strand comprises or is selected from the nucleotide sequence of SEQ ID NO:2.

[0227] In some embodiments, the sense strand and the antisense strand are The sense strand is 5'-UAUUCUCAGUGCUCUCCUZ b1 -3' (SEQ ID NO:21), and the antisense strand comprises 5'-UAGGAGAGCACUGAGAAUACU-3' (SEQ ID NO:22); Alternatively, the sense strand is 5'-GCACCGUUAAGGACAAGUZ b2 -3' (SEQ ID NO: 23), and the antisense strand comprises 5'-AACUUGUCCUUAACGGUGCUC-3' (SEQ ID NO: 24); Among them, Z b1 is A or G, and Z b2 is C or U.

[0228] In some embodiments, the nucleotide sequence of the sense strand of the dsRNA comprises or is selected from SEQ ID NO: 3, and the nucleotide sequence of the antisense strand comprises or is selected from any one of SEQ ID NOs: 5 to 14; In some embodiments, the dsRNA comprises: the sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:5; the sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:6; the sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:7; the sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:8; the sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:9; the sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:10; the sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:11; the sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:12; the sense strand comprises the nucleotide sequence of SEQ ID NO:3 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:13; The sense strand comprises the nucleotide sequence of SEQ ID NO:3, and the antisense strand comprises the nucleotide sequence of SEQ ID NO:14; The solution is one of the following:

[0229] In some embodiments, the dsRNA comprises: the sense strand consists of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO:5; the sense strand consists of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO:6; the sense strand consists of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO:7; the sense strand consists of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO:8; the sense strand consists of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO:9; the sense strand consists of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO:10; the sense strand consists of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO:11; the sense strand consists of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO:12; the sense strand consists of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO:13; The sense strand consists of the nucleotide sequence shown in SEQ ID NO: 3, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO: 14. The solution is one of the following:

[0230] In some embodiments, the dsRNA comprises: The present invention is any one of the above methods, comprising a sense strand shown in SEQ ID NO: 3 and an antisense strand shown in any one of SEQ ID NOs: 5 to 14.

[0231] In some embodiments, the dsRNA comprises: The method is any one of the methods, in which the sense strand is selected from the sense strand shown in SEQ ID NO: 3 and the antisense strand shown in any one of SEQ ID NOs: 5 to 14.

[0232] In the present disclosure, in the 5'-3' direction: SEQ ID NO:3 is UmsAmsUmUmCmUmCfAfGfUmGmCmUmCmUmCmCmUmAm, SEQ ID NO:5 is UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAfCmUfGmAfGmAfAmUmAmsCmsUm, SEQ ID NO:6 is UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAfCmUmGmAfGmAfAmUfAmsCmsUm, SEQ ID NO:7 is UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAmCmUfGmAfGmAfAmUfAmsCmsUm, SEQ ID NO:8 is UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAfCmUfGmAfGmAfAmUfAmsCmsUm, SEQ ID NO:9 is UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAmCmUmGmAfGmAfAmUmAmsCmsUm, SEQ ID NO:10 is UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAmCmUmGmAfGmAfAmUmAmsCmsUm, SEQ ID NO:11 is UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAfCmUmGmAfGmAfAmUmAmsCmsUm, SEQ ID NO:12 is UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAmCmUfGmAfGmAfAmUmAmsCmsUm, SEQ ID NO:13 is UmsAfsGmGmAmGm(-)hmpNA(A)GfCfAmCmUmGmAfGmAfAmUmAmsCmsUm, SEQ ID NO:14 is UmsAfsGmGmAmGm(-)hmpNA(A)GmCmAmCmUmGmAfGmAmAmUmAmsCmsUm, Among them, Af = adenine 2'-F ribonucleoside, Cf = cytosine 2'-F ribonucleoside, Uf = uracil 2'-F ribonucleoside, Gf = guanine 2'-F ribonucleoside, Am = adenine 2'-OMe ribonucleoside, Cm = cytosine 2'-OMe ribonucleoside, Gm = guanine 2'-OMe ribonucleoside. U = uracil 2'-OMe ribonucleoside, Um = uracil 2'-OMe ribonucleoside, s indicates that the two adjacent nucleotides on the left and right sides of the letter s are linked by a thiophosphodiester group; (-)hmpNA(A) [ka] Shows.

[0233] In some embodiments, the dsRNA further comprises a conjugated ligand, the ligand being selected from a targeting ligand that targets the liver, in some embodiments, the ligand binds to the asialoglycoprotein receptor (ASGPR), and in some embodiments, the ligand comprises a galactose cluster or a galactose derivative cluster, the galactose derivative being selected from N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, Nn-butyrylgalactosamine, or N-isobutyrylgalactosamine, or a combination thereof.

[0234] In some embodiments, the dsRNA further comprises a conjugated ligand, the ligand being a compound represented by formula (II) or a pharma- ceutically acceptable salt thereof: [ka] Of these, L1 is C1-C 30 C1-C which is an alkyl chain or is interrupted by one or more oxygen, sulfur, nitrogen atoms or C=O. 30 Contains an alkyl chain, R 11 and R 12 are independently chemical bonds, NR 16 , C=O or -OC(=O)-, Q3 is, [ka] and [ka] is a single bond or a double bond, and [ka] is a single bond, R 13 is independently CR 17 R 18 , N.R. 16 , O or S; [ka] If is a double bond, R 13 is independently CR 19 or N, R 14 is independently CR 19 or N, Ring A is a cycloalkyl group, a heterocycloalkyl group, an aryl group, or a heteroaryl group, which may or may not be present, and when Ring A is present, R 15 is independently CR 19 or N, and when ring A is absent, R 15 is independently CR 17 R18 , N.R. 16 or O, R 16 and R 19 are independently hydrogen, deuterium, an alkyl group, an alkoxy group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR' or C(=O)NR'(R"), wherein the alkyl group, alkoxy group, cycloalkyl group, heterocycloalkyl group, aryl group or heteroaryl group is optionally selected from the group consisting of halogen, hydroxyl group, oxo, nitro group, cyano group, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 3-7 substituted with one or more groups selected from a cycloalkyl group, a 3- to 12-membered heterocycloalkyl group, a 6- to 12-membered aryl group, a 5- to 12-membered heteroaryl group, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR' and C(=O)NR'(R"); R 17 and R 18 are independently hydrogen, deuterium, an alkyl group, an alkoxy group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR' or C(=O)NR'(R"), wherein the alkyl group, alkoxy group, cycloalkyl group, heterocycloalkyl group, aryl group or heteroaryl group is optionally selected from the group consisting of halogen, hydroxyl group, oxo, nitro group, cyano group, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 3-7 substituted with one or more groups selected from a cycloalkyl group, a 3- to 12-membered heterocycloalkyl group, a 6- to 12-membered aryl group, a 5- to 12-membered heteroaryl group, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR' and C(=O)NR'(R"); R' and R'' are independently hydrogen, deuterium, a hydroxyl group, an alkyl group, an alkoxy group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, or a heteroaryl group, wherein the alkyl group, the alkoxy group, the cycloalkyl group, the heterocycloalkyl group, the aryl group, or the heteroaryl group is optionally substituted with one or more substituents selected from a halogen, a hydroxyl group, an oxo group, a nitro group, and a cyano group; m1, n1, p1 and q1 are independently 0, 1, 2, 3 or 4; B1 is, [ka] and R b1 , R b2 , R b3 , R b4 , R b5 , R b6 and R b7 are independently -C(=O)-, -NHC(=O)-, -C(=O)O-, -C(=O)-(CH2) z8 -O- or -NHC(=O)-(CH2) z9 -O-, z1, z2, z3, z4, z5, z6, z7, z8, and z9 are independently an integer of 0 to 10; L2 is C1-C 30 C1-C which is an alkyl chain or is interrupted by one or more oxygen, sulfur, nitrogen atoms or C=O. 30 Contains an alkyl chain, r1 is an integer from 1 to 10.

[0235] In some embodiments, L is C 30 C1-C which is an alkyl chain or is interrupted by one or more oxygen, sulfur, nitrogen atoms or C=O. 30 Contains an alkyl chain, R 11 and R 12 are independently chemical bonds, NR 16 or C=O, R 16 is hydrogen or C 1-6 is an alkyl group, Q3 is, [ka] and R 13 CR 17 R 18 , N.R. 16 , O or S; R 14 CR 19 and R 15 is independently CR 17 R 18 , N.R. 16 or O, R 17 ~R 19 are independently hydrogen, deuterium, or an alkyl group; m1, p1 and q1 are independently 0, 1, 2, 3 or 4; B1 is, [ka] and R b5 , R b6 and R b7 are independently -C(=O)-, -NHC(=O)-, -C(=O)O-, -C(=O)-(CH2) z8 -O- or -NHC(=O)-(CH2) z9 -O-, z5, z6, z7, z8, and z9 are independently an integer of 0 to 10; L2 is C1-C 30 C1-C which is an alkyl chain or is interrupted by one or more oxygen, sulfur, nitrogen atoms or C=O. 30 Contains an alkyl chain, r1 is an integer from 1 to 10.

[0236] In some embodiments, L1 is -(CH2) j11 -C(=O)-(CH2) j12 - and R 11 and R 12 are independently chemical bonds, NR16 or C=O, R 16 is hydrogen or C 1-6 is an alkyl group, Q3 is, [ka] and R 13 CR 17 R 18 or O, R 14 CR 19 and R 15 is independently CR 17 R 18 or O, R 17 ~R 19 are independently hydrogen or an alkyl group; m1, p1 and q1 are independently 0 or 1; B1 is, [ka] and R b5 , R b6 and R b7 are independently -C(=O)-(CH2) z8 -O- or -NHC(=O)-(CH2) z9 -O-, z8 and z9 are independently an integer from 0 to 10; L2 is -(CH2) j15 -(OCH2CH2) 1-4 -(CH2) j16 -or [ka] and j15 and j16 are independently an integer of 0 to 4; r1 is 3, 4, 5 or 6.

[0237] In some embodiments, L1 is L3 or L3-R 110 -R111 -L3, wherein L3 is independently C1-C 12 Alkyl chain, -(CH2) j11 -C(=O)-(CH2) j12 -or-(CH2) j13 -(CH2CH2O) 1-4 -(CH2) j14 - and R 110 and R 111 are independently chemical bonds, -NR 112 -, -C(=O)- or -OC(=O)-, R 112 is hydrogen or C1-C 12 An alkyl group, and j11, j12, j13, and j14 are independently an integer of 0 to 10. In some embodiments, j11, j12, j13, and j14 are independently an integer of 0 to 2 or 4 to 10. In some embodiments, j11, j12, j13, and j14 are independently 0, 1, 2, 6, 7, 8, 9, or 10.

[0238] In some embodiments, L1 is -(CH2) j11 -C(=O)-(CH2) j12 -, and the definitions of j11 and j12 are as described in any one of the above forms.

[0239] In some embodiments, L1 is [ka] The definition of j12 is as described in any one of the above embodiments, wherein the a1 end is linked to B1 and the b1 end is R 11 is linked to.

[0240] In some embodiments, L1 is [ka] wherein the a1 end is linked to B1 and the b1 end is R 11 is linked to.

[0241] In some embodiments, R 11 may be a chemical bond, and R 12 may be C=O.

[0242] In some embodiments, R 11 may be a chemical bond, and R 12 is NR 16 R may be 16 is as defined in any one of the above forms.

[0243] In some embodiments, R 11 may be a chemical bond, and R 12 may be -OC(=O)-.

[0244] In some embodiments, R 11 is NR 16 and R 12 may be C=O, R 16 is as defined in any one of the above forms.

[0245] In some embodiments, R is NR 16 and R 12 may be -OC(=O)-, R 16 is as defined in any one of the above forms.

[0246] In some embodiments, R 12 is NR 16 and R 11 may be C=O, R 16 is as defined in any one of the above forms.

[0247] In some embodiments, R 12 is NR 16 and R 11 may be -OC(=O)-, R 16 is as defined in any one of the above forms.

[0248] In some embodiments, R 11 may be NH, and R 12 may be C=O.

[0249] In some embodiments, R 12 may be NH, and R 11 may be C=O.

[0250] In some embodiments, R 16 is hydrogen or C 1-6 It may be an alkyl group.

[0251] In some embodiments, R 16 may be hydrogen, a methyl group, an ethyl group, a propyl group, or an isopropyl group.

[0252] In some embodiments, R 16 may be hydrogen.

[0253] In some embodiments, R 17 and R 18 may be hydrogen.

[0254] In some embodiments, R 19 may be hydrogen.

[0255] In some embodiments, when ring A is present, ring A is C 6-12 It may be an aryl group.

[0256] In some embodiments, ring A can be a phenyl group.

[0257] In some embodiments, m1 may be 0 or 1.

[0258] In some embodiments, m1 may be 3.

[0259] In some embodiments, n1 may be 0 or 1.

[0260] In some embodiments, p1 and q1 are independently 0 or 1.

[0261] In some embodiments, p1=1 and q1=1.

[0262] In some embodiments, p1=1 and q1=0.

[0263] In some embodiments, p1=0 and q1=1.

[0264] In some embodiments, p1=0 and q1=0.

[0265] In some embodiments, z1, z2, z3, z4, z5, z6, z7, z8, and z9 may independently be an integer from 0 to 4. In some embodiments, z1, z2, z3, z4, z5, z6, z7, z8, and z9 may independently be 0, 1, or 2.

[0266] In some embodiments, B1 is [ka] R may be b1 , R b2 , R b3 and R b4 are independently -C(=O)- or -NHC(=O)-, the N atom is linked to L1, and the definitions of z1, z2, z3 and z4 are as described in any one of the above forms.

[0267] In some embodiments, B1 is [ka] R may be b1 , R b2 , R b3 and R b4are independently -C(=O)- or -NHC(=O)-, the N atom is linked to L1, and R b1 , R b3 and R b4 are the same, and the definitions of z1, z2, z3, and z4 are as described in any one of the above forms.

[0268] In some embodiments, B1 is [ka] may be also possible.

[0269] In some embodiments, B1 is [ka] may be also possible.

[0270] In some embodiments, B1 is [ka] R may be b5 , R b6 and R b7 are independently -C(=O)-(CH2) z8 -O- or -NHC(=O)-(CH2) z9 -O-, the N atom is linked to L1, and the definitions of z5, z6, z7, z8 and z9 are as described in any one of the above forms.

[0271] In some embodiments, B1 is [ka] R may be b5 , R b6 and R b7 are independently -C(=O)-(CH2) z8 -O- or -NHC(=O)-(CH2) z9 -O-, the N atom is linked to L1, and R b5 , R b6 and R b7are the same, and the definitions of z5, z6, z7, z8, and z9 are as described in any one of the above forms.

[0272] In some embodiments, B1 is [ka] may be also possible.

[0273] In some embodiments, L2 is L4 or L4-R 13 -R 14 -L4, wherein L4 is independently C1-C 12 Alkyl chain or -(CH2) j15 -(OCH2CH2) 1-4 -(CH2) j16 - and R 13 and R 14 are independently chemical bonds, -NR 115 -, -C(=O)- or -OC(=O)-, R 115 are independently hydrogen or C1-C 12 An alkyl group, and j15 and j16 are independently an integer from 0 to 10. In some embodiments, j15 and j16 are independently an integer from 0 to 6. In some embodiments, j15 and j16 are independently 0, 1, 2, 3, or 4.

[0274] In some embodiments, L2 is -(CH2) j15 -(OCH2CH2) 1-4 -(CH2) j16 -, and the definitions of j15 and j16 are as described in any one of the above forms.

[0275] In some embodiments, L2 is [ka] In some embodiments, L2 may be [ka] wherein the left side is connected to the O atom and the right side is connected to B1.

[0276] In some embodiments, L2 is C1-C 12 It may also be an alkyl chain.

[0277] In some embodiments, L2 is [ka] may be also possible.

[0278] In some embodiments, L2 is [ka] In some embodiments, L2 may be [ka] In some embodiments, L2 may be [ka] In some embodiments, L2 may be [ka] wherein the a3 end is linked to an O atom and the b3 end is linked to B1.

[0279] In some embodiments, L2 is [ka] wherein the a3 end is linked to an O atom and the b3 end is linked to B1.

[0280] In some embodiments, r1 may be 3, 4, 5, or 6. In some embodiments, r1 may be 3.

[0281] In some embodiments, Q3 is [ka] In some embodiments, Q3 may be: [ka] Among them, R 13 , R 14 , R 15 and n1 is as defined in any one of the above forms.

[0282] In some embodiments, [ka] teeth, [ka] In addition, R 13 , R 14 , R 15 The definitions of p1 and q1 are as described in any one of the above forms.

[0283] In some embodiments, [ka] teeth, [ka] In addition, R 13 , R 14 , R 15 The definitions of p1 and q1 are as described in any one of the above forms.

[0284] In some embodiments, [ka] teeth, [ka] In some embodiments, [ka] teeth, [ka] In some embodiments, [ka] teeth, [ka] The definitions of p1 and q1 are as described in any one of the above forms.

[0285] In some embodiments, [ka] teeth, [ka] In some embodiments, [ka] teeth, [ka] In some embodiments, [ka] teeth, [ka] The definitions of p1 and q1 are as described in any one of the above forms.

[0286] In some embodiments, [ka] teeth, [ka] In addition, R 13 , R 14 The definitions of n1, p1 and q1 are as described in any one of the above forms.

[0287] In some embodiments, [ka] teeth, [ka] In addition, R 13 , R 14 The definitions of n1, p1 and q1 are as described in any one of the above forms.

[0288] In some embodiments, [ka] teeth, [ka] and in some embodiments, [ka] teeth, [ka] The definitions of n1, p1, and q1 are as described in any one of the above embodiments.

[0289] In some embodiments, [ka] teeth, [ka] wherein n1, p1, and q1 are as defined in any one of the above embodiments.

[0290] In some embodiments, the ligand is: [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt thereof.

[0291] In some embodiments, the ligand is: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt thereof.

[0292] In some embodiments, the ligand is: [ka] or a pharma- ceutically acceptable salt thereof.

[0293] In some embodiments, the N-acetyl-galactosamine moiety in the ligand may be replaced by N-trifluoroacetylgalactosamine, N-propionylgalactosamine, Nn-butyrylgalactosamine, or N-isobutyrylgalactosamine.

[0294] In some embodiments, the chemical modification shown in formula (I) above is [ka] and B is selected from guanine, adenine, cytosine and uracil; The ligand is [ka] [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the sense strand and the antisense strand are defined as described in any one of the above forms.

[0295] In some embodiments, the ligand is: [ka] or a pharma- ceutically acceptable salt thereof.

[0296] In some embodiments, the definitions of the sense strand and the antisense strand are as described in any one of the above embodiments.

[0297] In some embodiments, the chemical modification shown in formula (I) above is [ka] and B is selected from guanine, adenine, cytosine and uracil; The ligand is [ka] or a pharma- ceutically acceptable salt thereof.

[0298] In some embodiments, the definitions of the sense strand and the antisense strand are as described in any one of the above embodiments.

[0299] In some embodiments, the sense strand and / or the antisense strand and the ligand are covalently or non-covalently linked.

[0300] In some embodiments, the 3' and / or 5' end of the sense strand is conjugated to the ligand.

[0301] In some embodiments, the 3' end of the sense strand is conjugated to the ligand.

[0302] In some embodiments, the ligand is linked to the terminus of the sense strand and / or antisense strand via a phosphate or thiophosphate group.

[0303] In some embodiments, the ligand is linked to the end of the sense and / or antisense strand via a phosphodiester or thiophosphodiester group.

[0304] In some embodiments, the ligand is linked to the end of the sense strand and / or the antisense strand via a phosphodiester group.

[0305] In some embodiments, the ligand is indirectly linked to the terminus of the sense strand and / or antisense strand via a phosphate or thiophosphate group.

[0306] In some embodiments, the ligand is linked directly to the termini of the sense and / or antisense strands via a phosphate or thiophosphate group.

[0307] In some embodiments, the ligand is linked directly to the 3' end of the sense strand via a phosphate or thiophosphate group.

[0308] In some embodiments, the phosphate group is a phosphomonoester or a phosphodiester group. In some embodiments, the phosphate group is a phosphodiester group.

[0309] In some embodiments, the thiophosphate group is a thiophosphomonoester or a thiophosphodiester group. In some embodiments, the thiophosphate group is a thiophosphodiester group.

[0310] In some embodiments, in order to facilitate the entry of dsRNA into cells, a lipophilic group such as cholesterol can be introduced at the end of the sense strand, and the lipophilic group can be covalently bound to a small interfering nucleic acid, for example, by introducing cholesterol, lipoprotein, vitamin E, etc. at the end, it contributes to interacting with intracellular mRNA through a cell membrane made of lipid bilayer. In addition, dsRNA may be modified by non-covalent bond, for example, by binding to phospholipid molecules, polypeptides, cationic polymers, etc. by hydrophobic or ionic bond to improve stability and biological activity.

[0311] In some embodiments, the number of ligands includes, but is not limited to, 1, 2, 3, or 4. In some embodiments, the number of ligands is one.

[0312] In some embodiments, the nucleotide sequence of the sense strand of the dsRNA comprises or is selected from SEQ ID NO: 4, and the nucleotide sequence of the antisense strand comprises or is selected from any one of SEQ ID NOs: 5 to 14; In some embodiments, the dsRNA comprises: the sense strand comprises the nucleotide sequence of SEQ ID NO:4 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:5; the sense strand comprises the nucleotide sequence of SEQ ID NO:4 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:6; the sense strand comprises the nucleotide sequence of SEQ ID NO:4 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:7; the sense strand comprises the nucleotide sequence of SEQ ID NO:4 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:8; the sense strand comprises the nucleotide sequence of SEQ ID NO:4 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:9; the sense strand comprises the nucleotide sequence of SEQ ID NO:4 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:10; the sense strand comprises the nucleotide sequence of SEQ ID NO:4 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:11; the sense strand comprises the nucleotide sequence of SEQ ID NO:4 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:12; the sense strand comprises the nucleotide sequence of SEQ ID NO:4 and the antisense strand comprises the nucleotide sequence of SEQ ID NO:13; The sense strand comprises the nucleotide sequence of SEQ ID NO: 4, and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 14; The solution is one of the following:

[0313] In some embodiments, the dsRNA comprises: the sense strand consists of the nucleotide sequence shown in SEQ ID NO: 4, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO: 5; the sense strand consists of the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO:6; the sense strand consists of the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO:7; the sense strand consists of the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO:8; the sense strand consists of the nucleotide sequence shown in SEQ ID NO: 4, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO: 9; the sense strand consists of the nucleotide sequence shown in SEQ ID NO: 4, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO: 10; the sense strand consists of the nucleotide sequence shown in SEQ ID NO: 4, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO: 11; the sense strand consists of the nucleotide sequence shown in SEQ ID NO: 4, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO: 12; the sense strand consists of the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO:13; The sense strand consists of the nucleotide sequence shown in SEQ ID NO: 4, and the antisense strand consists of the nucleotide sequence shown in SEQ ID NO: 14. The solution is one of the following:

[0314] In some embodiments, the dsRNA comprises: The present invention is any one of the above methods, comprising a sense strand shown in SEQ ID NO: 4 and an antisense strand shown in any one of SEQ ID NOs: 5 to 14.

[0315] In some embodiments, the dsRNA comprises: The method is any one of the methods in which the sense strand is selected from the sense strand shown in SEQ ID NO: 4 and the antisense strand shown in any one of SEQ ID NOs: 5 to 14.

[0316] In the present disclosure, in the 5'-3' direction: SEQ ID NO:4 is UmsAmsUmUmCmUmCfAfGfUmGmCmUmCmUmCmCmUmAm-NAG0052', SEQ ID NO:5 is UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAfCmUfGmAfGmAfAmUmAmsCmsUm, SEQ ID NO:6 is UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAfCmUmGmAfGmAfAmUfAmsCmsUm, SEQ ID NO:7 is UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAmCmUfGmAfGmAfAmUfAmsCmsUm, SEQ ID NO:8 is UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAfCmUfGmAfGmAfAmUfAmsCmsUm, SEQ ID NO:9 is UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAmCmUmGmAfGmAfAmUmAmsCmsUm, SEQ ID NO:10 is UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAmCmUmGmAfGmAfAmUmAmsCmsUm, SEQ ID NO:11 is UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAfCmUmGmAfGmAfAmUmAmsCmsUm, SEQ ID NO:12 is UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAmCmUfGmAfGmAfAmUmAmsCmsUm, SEQ ID NO:13 is UmsAfsGmGmAmGm(-)hmpNA(A)GfCfAmCmUmGmAfGmAfAmUmAmsCmsUm, SEQ ID NO:14 is UmsAfsGmGmAmGm(-)hmpNA(A)GmCmAmCmUmGmAfGmAmAmUmAmsCmsUm, Among them, Af = adenine 2'-F ribonucleoside, Cf = cytosine 2'-F ribonucleoside, Uf = uracil 2'-F ribonucleoside, Gf = guanine 2'-F ribonucleoside, Am = adenine 2'-OMe ribonucleoside, Cm = cytosine 2'-OMe ribonucleoside, Gm = guanine 2'-OMe ribonucleoside. U = uracil 2'-OMe ribonucleoside, Um = uracil 2'-OMe ribonucleoside, s indicates that the two adjacent nucleotides on the left and right sides of the letter s are linked by a thiophosphodiester group; NAG0052' is [ka] indicates, (-)hmpNA(A) [ka] Shows.

[0317] In some embodiments, the dsRNA comprises: [ka] or a pharma- ceutically acceptable salt thereof, in which Af = adenine 2'-F ribonucleoside, Cf = cytosine 2'-F ribonucleoside, Uf = uracil 2'-F ribonucleoside, Am = adenine 2'-OMe ribonucleoside, Cm = cytosine 2'-OMe ribonucleoside, Gf = guanine 2'-F ribonucleoside, ribonucleoside), Gm = guanine 2'-OMe ribonucleoside, Um = uracil 2'-OMe ribonucleoside.

[0318] [ka] represents a thiophosphodiester group, [ka] indicates a phosphodiester group, NAG0052' is [ka] indicates, (-)hmpNA(A) [ka] Shows.

[0319] In some embodiments, the pharma- ceutically acceptable salts may be conventional salts in the art, including, but not limited to, sodium salts, potassium salts, ammonium salts, amine salts, and the like.

[0320] In some embodiments, the dsRNA is any one selected from TRD008043, TRD008069, TRD008042, TRD008070, TRD008071, TRD008072, TRD008073, TRD008074, TRD008075, and TRD008076.

[0321] In some embodiments, the dsRNA is TRD008043, the structure of which is as follows:

[0322] [ka] Among them, Af = adenine 2'-F ribonucleoside, Cf = cytosine 2'-F ribonucleoside, Uf = uracil 2'-F ribonucleoside, Am = adenine 2'-OMe ribonucleoside, Cm = cytosine 2'-OMe ribonucleoside, Gf = guanine 2'-F ribonucleoside, Gm = guanine 2'-OMe ribonucleoside. ribonucleoside), Um=uracil 2'-OMe ribonucleoside.

[0323] [ka] represents a thiophosphodiester group, [ka] indicates a phosphodiester group, NAG0052' is [ka] indicates, (-)hmpNA(A) [ka] Shows.

[0324] In another aspect, the present disclosure provides a pharmaceutical composition comprising the above-described dsRNA.

[0325] In some embodiments, the pharmaceutical composition further comprises one or more pharma- ceutically acceptable excipients.Various drug delivery systems are known and can be applied to the dsRNA or pharmaceutical composition of the present disclosure, such as packaging in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated cellular endocytosis, constructing the nucleic acid as part of a reverse transcription virus or other vector.

[0326] In some embodiments, the mode of administration of the dsRNA or pharmaceutical composition of the disclosure is conventional and may be local (e.g., direct injection or implantation) or systemic, or may be administered by oral, rectal, or parenteral routes, including, but not limited to, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, inhalation (e.g., aerosol), mucosal (e.g., sublingual, intranasal), intracranial, and the like.

[0327] In some embodiments, a dsRNA or pharmaceutical composition provided in this disclosure may be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, intraduodenal or intraperitoneal injection.

[0328] In some embodiments, the dsRNA or pharmaceutical compositions provided in this disclosure may be packaged into a reagent kit.

[0329] In some embodiments, the effective amount or effective dose of the dsRNA or pharmaceutical composition is about 0.001 to about 200 mg / kg body weight, about 0.01 to about 100 mg / kg body weight, or about 0.5 to about 50 mg / kg body weight.

[0330] The disclosure provides the use of the dsRNA or the pharmaceutical composition in the preparation of a medicament.

[0331] In some embodiments, the agent can be used to reduce low density lipoprotein levels in a subject or to prevent and / or treat a disease mediated by elevated triglyceride levels or elevated cholesterol levels, in some embodiments, the disease is selected from hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolic disorders, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia pancreatitis, metabolic syndrome, type 2 diabetes, familial chylomicronemia syndrome, or familial partial lipodystrophy.

[0332] In some embodiments, the agent can be used to prevent and / or treat a disease associated with expression of the APOC3 gene, in some embodiments, the disease is selected from hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolic disorders, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type 2 diabetes, familial chylomicronemia syndrome, or familial partial lipodystrophy.

[0333] The present disclosure provides a method for preventing and / or treating a disease, comprising administering to a subject an effective amount or dose of the above-described dsRNA or the above-described pharmaceutical composition.

[0334] In some embodiments, the disease may be a disease mediated by elevated triglyceride levels or elevated cholesterol levels, hi some embodiments, the disease is selected from hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolic disorders, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia pancreatitis, metabolic syndrome, type 2 diabetes, familial chylomicronemia syndrome, or familial partial lipodystrophy.

[0335] In some embodiments, the disease may be a disease associated with the expression of the APOC3 gene. In some embodiments, the disease is selected from hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolic disorders, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type 2 diabetes, familial chylomicronemia syndrome, or familial partial lipodystrophy.

[0336] The present disclosure provides a method for silencing the APOC3 gene or its mRNA in a cell in vivo or in vitro, comprising the step of introducing the dsRNA or the pharmaceutical composition into the cell.

[0337] The present disclosure provides a method for inhibiting expression of the APOC3 gene or its mRNA, comprising administering to a subject an effective amount or dose of the above-described dsRNA or the above-described pharmaceutical composition.

[0338] In some embodiments, the effective amount or effective dose of the dsRNA or pharmaceutical composition is about 0.001 to about 200 mg / kg body weight, about 0.01 to about 100 mg / kg body weight, or about 0.5 to about 50 mg / kg body weight.

[0339] The dsRNA or pharmaceutical composition of the present disclosure can reduce the expression level of a target gene or its mRNA in a subject, such as a cell, a cell population, a tissue, or a human subject, including inhibiting expression of a target gene or its mRNA in a subject by administering to the subject a therapeutically effective amount of a dsRNA or pharmaceutical composition described herein.

[0340] In some embodiments, the subject has previously been identified as having a pathological upregulation of the target gene or its mRNA in the targeted cell, cell population, tissue or subject.

[0341] The present disclosure provides a method of delivering an oligonucleotide to the liver comprising administering to a subject an effective amount or dose of the dsRNA or the pharmaceutical composition.

[0342] The present disclosure provides an RNA interference (RNAi) reagent comprising the above-mentioned dsRNA or the above-mentioned pharmaceutical composition.

[0343] In another aspect, the disclosure further provides a cell comprising said dsRNA or said pharmaceutical composition.

[0344] In another aspect, the present disclosure further provides a reagent kit comprising the above-mentioned dsRNA or the above-mentioned pharmaceutical composition.

[0345] In some embodiments, the dsRNA of the present disclosure is selected from a siRNA.

[0346] In the present disclosure, when the dsRNA or pharmaceutical composition is contacted with a cell expressing a target gene, the dsRNA or pharmaceutical composition inhibits expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as by, for example, PCR or branched DNA (bDNA) based methods, or protein based methods, for example, immunofluorescence assays such as Western Blot and flow cytometry.

[0347] In the present disclosure, when the dsRNA or pharmaceutical composition is contacted with a cell expressing a target gene, the percentage of excess expression of the target gene mRNA by the dsRNA or pharmaceutical composition is 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, as measured by, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as by, for example, PCR or branched DNA (bDNA)-based methods, or protein-based methods, for example, immunofluorescence analysis such as Western Blot or flow cytometry.

[0348] In the present disclosure, when the dsRNA or pharmaceutical composition is contacted with a cell expressing a target gene, the dsRNA retains on-target activity and reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as, for example, PCR or branched DNA (bDNA)-based methods, or protein-based methods, for example, immunofluorescence assays such as Western Blot or flow cytometry.

[0349] In the present disclosure, when the dsRNA or pharmaceutical composition is contacted with a cell expressing a target gene, the dsRNA reduces on-target activity by up to 20%, up to 19%, up to 15%, up to 10%, up to 5%, or 1% or more and reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as, for example, PCR or branched DNA (bDNA) based methods, or protein based methods, for example, immunofluorescence assays such as Western Blot or flow cytometry.

[0350] In the present disclosure, when the dsRNA or pharmaceutical composition is contacted with a cell expressing a target gene, the dsRNA enhances on-target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, and reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as, for example, PCR, branched DNA (bDNA)-based methods, or protein-based methods, for example, immunofluorescence assays such as Western Blot and flow cytometry.

[0351] The compounds of the present disclosure may have particular geometric or stereoisomeric forms. The present disclosure includes cis-trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and all such compounds, including racemic and other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, are intended to be within the scope of the present disclosure. Substituents such as alkyl groups may have other asymmetric carbon atoms. All such isomers and mixtures thereof are within the scope of the present disclosure. Compounds containing asymmetric carbon atoms of the present disclosure can be isolated in optically pure or racemic form. Optically pure forms may be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0352] Optically active (R)- and (S)-isomers and D- and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. To obtain one enantiomer of a compound of the present disclosure, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliaries, in which the resulting diastereomeric mixture is isolated and the required enantiomer is purified by cleavage of the auxiliaries. Alternatively, if the molecule contains a basic (e.g., amino) or acidic (e.g., carboxy) functional group, the diastereomeric salt can be formed with an appropriate optically active acid or base, and the diastereomeric separation can be carried out by conventional methods well known in the art, and the enantiomers can be recovered to obtain the pure enantiomers. Note that the separation of enantiomers and diastereomers is generally accomplished by using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., forming a carbamate from an amine).

[0353] In the chemical structures of the compounds described in this disclosure, [ka] indicates that the configuration is not specified, i.e., if chiral isomers are present in the chemical structure, [ka] The bond [ka] or [ka] In the chemical structure of the compound described in the present disclosure, [ka] The bond is unspecified in configuration, i.e. [ka] The bond configuration may be the E configuration or the Z configuration, or may include both E and Z configurations simultaneously.

[0354] In the chemical structures of the present disclosure: [ka] can be linked to any one or more groups according to the scope of the invention described herein, and the asterisk "*" indicates a chiral center.

[0355] Also, unless a configuration is specified, the compounds and intermediates of the present disclosure may exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible by a low energy barrier. For example, proton tautomers (also called protolytic tautomers) include interconversions by protolysis, such as keto-enol and imine-enamine, lactam-lactim isomerization. An example of lactam-lactim equilibrium is between A and B as follows:

[0356] [ka] All of the compounds in this disclosure can be depicted in Form A or Form B. All tautomeric forms are within the scope of this disclosure. The naming of a compound does not exclude any tautomeric forms.

[0357] The present disclosure further includes certain isotopically labeled compounds of the present disclosure that are the same as those described herein, but in which one or more atoms are replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Illustrative examples of isotopes that can be attached to the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, e.g., 2 H, 3 H, 11 C. 13 C.14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0358] Unless otherwise stated, when a position is specifically designated as deuterium (D), it should be understood that the position is deuterium (i.e., at least 10% deuterium incorporated) with an abundance at least 1000 times higher than the natural abundance of deuterium (which is 0.015%). In the example compounds, having an abundance higher than the natural abundance of deuterium may be at least 1000 times more abundant deuterium, at least 2000 times more abundant deuterium, at least 3000 times more abundant deuterium, at least 4000 times more abundant deuterium, at least 5000 times more abundant deuterium, at least 6000 times more abundant deuterium, or more abundant deuterium. The present disclosure further includes various deuterated forms of the compounds of formula (I), formula (I'), and formula (II). Each available hydrogen atom connected to a carbon atom may be independently replaced with a deuterium atom. Those skilled in the art can synthesize deuterated forms of the compounds of formula (I), formula (I'), and formula (II) by referring to the relevant literature. Deuterated forms of the compounds of formula (I), formula (I') and formula (II) may be prepared using commercially available deuterated starting materials or may be synthesized by conventional techniques with deuterated reagents including, but not limited to, borane deuteride, borane tritide in tetrahydrofuran, lithium aluminum deuteride, deuterated iodoethane and deuterated iodomethane.

[0359] The present disclosure further provides a method of preparing a dsRNA or pharmaceutical composition comprising synthesizing a dsRNA or pharmaceutical composition described in this disclosure.

[0360] In some embodiments, "comprising" may be replaced with "consisting of."

[0361] This disclosure incorporates WO2022028462A1, WO2023274395A, and PCT / CN2022 / 139462 in their entirety.

[0362] Explanation of terms In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless expressly defined otherwise herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0363] Unless otherwise specified, "optionally," "optionally," "optionally," or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur. For example, "optionally, R1 and R2 are directly linked to form a ring" means that R1 and R2 are directly linked to form a ring may occur but does not necessarily have to occur, and the description includes cases where R1 and R2 are directly linked to form a ring and cases where R1 and R2 do not form a ring.

[0364] The terms "about" and "approximately" mean that a numerical value is within an acceptable error range of a specific value as measured by one of ordinary skill in the art, the numerical portion being determined by how it is measured or determined (i.e., the limitations of the measurement system). For example, "about" may mean within 1 or more than 1 standard deviation. Alternatively, "about" or "essentially including" may mean a variation within a range of up to 20%, for example, between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, between 0.5% and 1%, and in this disclosure, any instance where the term "about" precedes a number or range of values ​​includes the specified number of embodiments. Unless otherwise stated, when a specific value appears in this application and the claims, the meaning of "about" or "essentially including" should be assumed to be within an acceptable error range of the specific value.

[0365] Unless otherwise specified, the "compounds", "chemical modifications", "ligands", "dsRNA", "nucleic acids" and "RNAi" disclosed herein can each independently exist in the form of a salt, a mixed salt or a non-salt (e.g., a free acid or a free base). When present in the form of a salt or mixed salt, it may be a pharma- ceutically acceptable salt.

[0366] A "pharmaceutically acceptable salt" may be selected from an inorganic salt or an organic salt, and may include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0367] "Pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic or organic acid that is capable of retaining the bioavailability of the free base without other adverse effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc., and organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, caproate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, mesylate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalenedisulfonate, etc. These salts can be prepared by methods known in the art.

[0368] "Pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that can retain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, etc. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts and magnesium salts, with sodium salts being preferred. Salts derived from organic bases include, but are not limited to, the following salts: primary amines, secondary amines, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, glycine betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamide resins, and the like. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0369] "Alkyl group" refers to a saturated aliphatic hydrocarbon group, including, for example, straight and branched chain groups containing 1 to 30 carbon atoms (C1-C 30alkyl groups), or alkyl groups containing, for example, 1 to 6 carbon atoms (C1-C6 alkyl groups), or alkyl groups containing, for example, 1 to 3 carbon atoms (C1-C3 alkyl groups). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and various branched chain isomers thereof.

[0370] The term "alkenyl group" refers to a hydrocarbon group containing at least one double bond. Non-limiting examples of alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, or 2-butenyl groups and their various branched chain isomers.

[0371] The term "alkynyl group" refers to a hydrocarbon group containing at least one triple bond. Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, or 2-butynyl groups and their various branched chain isomers.

[0372] The term "alkoxy" refers to an -O-(alkyl group), where alkyl is defined above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, and butoxy groups.

[0373] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, where the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 6 carbon atoms, and more preferably 5 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like, and polycyclic cycloalkyl groups include spiro, fused, and bridged ring cycloalkyl groups.

[0374] The term "heterocycloalkyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which may be nitrogen, oxygen or S(O). m (wherein m is an integer of 0 to 2), but does not include the ring moiety -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. It preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms, and more preferably contains 3 to 7 ring atoms. Non-limiting examples of "heterocycloalkyl groups" are: [ka] etc.

[0375] The heterocycloalkyl ring may be fused to an aryl or heteroaryl group, in which the ring connected to the parent structure is a heterocycloalkyl group, non-limiting examples of which are: [ka] etc.

[0376] The term "aryl group" refers to an all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated pi-electron system, preferably 6-14 members, such as phenyl and naphthyl groups. The aryl ring may be fused to a heteroaryl group, heterocycloalkyl group, or cycloalkyl ring, in which the ring connected to the parent structure is an aryl ring, non-limiting examples of which are: [ka] Includes.

[0377] The term "heteroaryl group" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 6 to 12 membered, more preferably 5 or 6 membered. For example, non-limiting examples thereof include imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazolyl, pyrazinyl, triazolyl, indazolyl, benzimidazolyl, [ka] etc.

[0378] The heteroaryl ring may be fused to an aryl group, a heterocycloalkyl group, or a cycloalkyl ring, in which the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which are: [ka] Includes.

[0379] The term "hydroxy" refers to an -OH group.

[0380] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0381] The term "cyano" refers to -CN.

[0382] The term "amino group" refers to -NH2.

[0383] The term "nitro group" refers to --NO.sub.2.

[0384] The term "oxo" refers to a ═O substituent.

[0385] In the present disclosure, a "phosphate group" may be a phosphomonoester group, a phosphodiester group or a phosphotriester group, and is preferably a phosphodiester group, and the "phosphate group" in a "thiophosphate group" has the same meaning.

[0386] In this disclosure, a phosphodiester group refers to a phosphodiester group modified by replacing one non-bridging oxygen atom with a sulfur atom; [ka] (M is an S atom).

[0387] "Substituted" refers to one or more hydrogen atoms in a group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, being independently replaced with a corresponding number of substituents. When a substituent is a ketone or oxo (i.e., =O), two (2) hydrogens on the atom are replaced.

[0388] In the context of this disclosure: [ka] Based on [ka] The moiety can be substituted with any group capable of linking to an adjacent nucleotide.

[0389] The term "linked," when referring to a connection between two molecules, refers to whether the two molecules are joined by a covalent bond or whether the two molecules are joined by a non-covalent bond (e.g., a hydrogen bond or an ionic bond), and includes a direct link and an indirect link.

[0390] The term "directly linked" refers to a connection between a first compound or group and a second compound or group without any intervening atoms or groups.

[0391] The term "indirectly linked" means that a first compound or group and a second compound or group are linked through an intermediate group, compound or molecule (eg, a linking group).

[0392] "Pharmaceutical composition" means a mixture of one or more compounds described herein or physiologically acceptable salts or prodrugs thereof with other chemical components, and other components such as physiologically acceptable vectors and excipients. The pharmaceutical composition is intended to facilitate administration to the living body and contribute to the absorption of the active ingredients to further exert biological activity.

[0393] A "pharmaceutically acceptable excipient" includes, but is not limited to, any auxiliary, vector, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonicity agent, solvent, or emulsifier approved by the U.S. Food and Drug Administration (FDA) for use in humans or domestic animals.

[0394] As used herein, the term "inhibition" can be used interchangeably with "reduction," "silencing," "downregulation," "suppression," and other similar terms, and includes any level of inhibition. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-administration baseline level, or a level determined from a similar untreated or control (e.g., buffer control or inactive agent only) treated subject, cell, or sample. For example, the amount of excess mRNA expression can indicate the degree of inhibition of target gene expression by dsRNA, for example, the amount of excess mRNA expression is 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less. The inhibition rate of target gene expression can be detected using the Dual-Glo (registered trademark) Luciferase Assay System. The Firefly chemiluminescence value and the Renilla chemiluminescence value are read, and the relative value Ratio = Ren / Fir, inhibition rate (%) = 1 - (Ratio + dsRNA / Ratioreporter only) × 100%, can be calculated. In the present disclosure, the excess mRNA expression ratio (or excess activity %) = 100% - inhibition rate (%).

[0395] An "effective amount" or "effective dose" includes an amount sufficient to ameliorate or prevent the symptoms or condition of a medical condition. Effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount used in a particular patient or veterinary subject can vary depending on factors such as the condition being treated, the overall health of the patient, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen in which significant side effects or toxic effects are avoided.

[0396] As used herein, the terms "subject," "patient," "subject" or "individual" can be used interchangeably and include a human or non-human animal, for example a mammal, such as a human or monkey.

[0397] As used herein, the sense strand (also referred to as SS, SS strand or sense strand) refers to the strand that contains a sequence identical or essentially identical to a target mRNA sequence, and the antisense strand (also referred to as AS or AS strand) refers to the strand that has a sequence complementary to the target mRNA sequence.

[0398] In the present disclosure, the "5' region" of the sense strand or the antisense strand, i.e., the "5' end" and the "5' end" can be used interchangeably. For example, the nucleotides at positions 2 to 8 of the 5' region of the antisense strand may be replaced with the nucleotides at positions 2 to 8 of the 5' end of the antisense strand. Similarly, the "3' region", "3' end" and the "3' end" of the sense strand or the antisense strand can be used interchangeably.

[0399] In the context of describing the dsRNA sense strand described herein, the term "at least 15 contiguous nucleotides of a sequence that differs by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1" is intended to indicate that the dsRNA sense strand described herein comprises at least 15 contiguous nucleotides of the sense strand shown in SEQ ID NO: 1, or a sequence that differs by no more than 3 nucleotides, optionally by no more than 2 nucleotides, optionally by no more than 1 nucleotide, from the at least 15 contiguous nucleotides of the sense strand shown in SEQ ID NO: 1. Optionally, the dsRNA sense strand described herein comprises at least 16 contiguous nucleotides of the sense strand shown in SEQ ID NO: 1, or a sequence that differs by no more than 3 nucleotides, optionally by no more than 2 nucleotides, optionally by no more than 1 nucleotide, from the at least 16 contiguous nucleotides of the sense strand shown in SEQ ID NO: 1; In the context of describing the dsRNA antisense strand described herein, the term "at least 15 contiguous nucleotides of a sequence which differs by no more than 3 nucleotides from the antisense strand set forth in SEQ ID NO:2" is intended to indicate including at least 15 contiguous nucleotides in SEQ ID NO:2 as described herein, or a sequence which differs by no more than 3 nucleotides, optionally a sequence which differs by no more than 2 nucleotides, optionally a sequence which differs by 1 nucleotide from at least 15 contiguous nucleotides of the antisense strand set forth in SEQ ID NO:2.

[0400] Unless otherwise specified, in the context of this disclosure, "G", "C", "A", "T" and "U" each represent a nucleotide, including guanine, cytosine, adenine, thymidine and uracil bases, respectively. Lower case m represents that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide, lower case f represents that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide, and lower case s represents that two nucleotides adjacent to the left and right of the letter s are linked by a thiophosphodiester group.

[0401] As used herein, the term "2'-fluoro (2'-F) modified nucleotide" refers to a nucleotide formed by replacing the hydroxy group at the 2' position of the ribosyl group of a nucleotide with a fluoro, and "non-fluoro modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the hydroxy group at the 2' position of the ribosyl group of a nucleotide with a non-fluoro group.

[0402] As used in this disclosure, the term "2'-methoxy (2'-OMe) modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxy group of a ribosyl group with a methoxy group.

[0403] In the context of this disclosure, a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence means that the type of nucleotide base at the same position in the former is changed compared to the latter, for example, if one nucleotide base in the latter is A and the corresponding nucleotide base at the same position in the former is U, C, G, or T, then there is considered to be a nucleotide difference at that position between the two nucleotide sequences. In some embodiments, a nucleotide difference is considered to occur at a position when an abasic nucleotide or its equivalent replaces the nucleotide at the original position.

[0404] As used herein, the terms "complementary" or "reverse complementary" can be used interchangeably and have the meaning known to those skilled in the art that in a double-stranded nucleic acid molecule, bases in one strand pair with bases in the other strand in a complementary manner. In DNA, the purine base adenine always pairs with the pyrimidine base thymine (or uracil in RNA), and the purine base guanine always pairs with the pyrimidine base cytosine. Each base pair contains one purine and one pyrimidine. If adenine in one strand always pairs with thymine (or uracil) in the other strand, and guanine always pairs with cytosine, the two strands are said to be complementary to each other, and the sequence of the strand can be deduced from the sequence of the complementary strand. Accordingly, "mismatch" is used in the art to mean that bases at corresponding positions in a double-stranded nucleic acid are not present paired in a complementary manner.

[0405] The term "dsRNA" refers to a double-stranded RNA molecule capable of RNA interference, comprising a sense strand and an antisense strand.

[0406] The term "chemical modification" or "modification" includes any alteration of a nucleotide by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0407] The term "base" includes any known DNA and RNA base, base analogs, such as purines and pyrimidines, as well as the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs. A base analog may be a universal base.

[0408] The terms "flat-ended" or "blunt-ended" can be used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of a dsRNA, i.e., the absence of nucleotide overhangs. A dsRNA with two blunt ends is often double-stranded over its entire length.

[0409] The dsRNA provided by the present disclosure can be obtained by conventional preparation methods in this field (for example, solid-phase synthesis and liquid-phase synthesis methods). Among them, solid-phase synthesis has already been commercialized as a customization service. The modified nucleotide group can be introduced into the dsRNA described in the present disclosure by the nucleoside monomer having the corresponding modification, and the method of preparing the nucleoside monomer having the corresponding modification and the method of introducing the modified nucleotide group into the dsRNA are also well known to those skilled in the art. [Brief description of the drawings]

[0410] [Figure 1] This shows the expression level of TTR mRNA 7 days after administration of TRD002218 and TRD007205. [Diagram 2] This shows the expression level of TTR mRNA 28 days after administration of TRD002218 and TRD007205. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0411] The present disclosure will be further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples of the present disclosure generally follow normal conditions or conditions recommended by the manufacturers of raw materials or products. For reagents for which a specific source is not specified, the reagents can be obtained from any molecular biology reagent supplier with the quality / purity for molecular biology applications.

[0412] Example 1: Preparation of Chemical Modifications 1.1 Synthesis of Compound 1-1a and Compound 1-1b [ka] Compound 1 (500 mg, 3.42 mmol) and triethylamine (Et3N, 692 mg, 6.84 mmol, 0.95 mL) were dissolved in dichloromethane (DCM, 10 mL), and a solution of 4-toluenesulfonyl chloride (TsCl, 717 mg, 3.76 mmol) in dichloromethane (10 mL) was added dropwise under ice bath, and after the addition was completed, the reaction was allowed to proceed with stirring at room temperature overnight. After the reaction was completed, the reaction was quenched with water, and the aqueous phase was extracted three times with dichloromethane (15 mL). The combined organic phase was first washed with saturated aqueous sodium bicarbonate solution (10 mL), and then washed with saturated saline (20 mL), and the solvent was evaporated to dryness under reduced pressure to obtain crude product 2 (820 mg, 80%), which was used directly in the next reaction. MS m / z: C 14 H 21 O5S, [M+H] + Theoretical: 301.10, Actual: 301.2.

[0413] [ka] Compound 3 (239 mg, 1.22 mmol) was dissolved in dimethylformamide (DMF, 10 mL), and a solution of NaH (60% dissolved in mineral oil, 93 mg, 2.33 mmol) was added under ice bath, and the reaction was carried out with stirring for 30 min. Compound 2 (350 mg, 1.16 mmol) was then added dropwise, and after the addition was completed, the reaction was carried out with stirring at 60° C. for 5 h. After the reaction was completed, water was added to quench the reaction, and the aqueous phase was extracted three times with ethyl acetate (15 mL). The combined organic phase was first washed three times with water (10 mL), and then washed with saturated saline (10 mL). The solvent was then evaporated to dryness under reduced pressure, and the mixture was analyzed by reversed-phase preparative HPLC (C 18 (Conditions: 5%-50% (A: HO, B: CHCN), flow rate: 70 mL / min) and freeze-drying to give 220 mg of compound 4. MS m / z: C 19 H 21 N5O3Na, [M+Na] + Theoretical: 390.16, Actual: 390.3.

[0414] [ka] Compound 4 (1.50 g, 4.08 mmol) was dissolved in 20 mL of a mixture of acetic acid and water (4:1) at room temperature and stirred at 60 °C for 30 min. After the reaction was completed, the solvent was evaporated to dryness under reduced pressure and the resulting mixture was analyzed by reverse phase preparative HPLC (C 18 (Conditions: 5-25% (A: HO, B: CHCN), flow rate: 70 mL / min) and freeze-drying to give 1.10 g of compound 5. MS m / z: C 16 H 18 N5O3, [M+H] + Theoretical: 328.13, Actual: 328.4.

[0415] [ka] Compound 5 (1.00 g, 3.05 mmol) was dissolved in pyridine (Py, 10 mL), and a solution of 4,4'-dimethoxytrityl chloride (DMTrCl, 1.50 g, 4.58 mmol) in pyridine (5 mL) was added dropwise in an ice bath. After the addition was completed, the reaction was allowed to proceed with stirring at room temperature overnight. After the reaction was completed, the reaction was quenched with water, and the solvent was evaporated to dryness under reduced pressure. The reaction was then analyzed by reversed-phase preparative HPLC (C 18 (Conditions: 5%-80% (A: HO, B: CHCN), flow rate: 70 mL / min) and freeze-drying to give 1.00 g of compound 6. MS m / z: C 37 H 36 N5O5, [MH] + Theoretical: 630.26, Found: 630.5. Racemic compound 6 was resolved by chiral column (Daicel CHIRALPAK® IE 250 mm×4.6 mm, 5 μm, A: n-hexane, B: ethanol) to give 410 mg of 6A(−) and 435 mg of 6B(+).

[0416] [ka] Compound 6A(-) (200 mg, 0.32 mmol), tetrazole (11 mg, 0.16 mmol), N-methylimidazole (5 mg, 0.06 mmol), and 3A molecular sieves (500 mg) were dissolved in 10 mL of acetonitrile, and compound 7 (144 mg, 0.48 mmol) was added at room temperature and stirred at room temperature overnight. After the reaction was completed, the molecular sieves were removed by filtration, dichloromethane (30 mL) was added, and the mixture was washed three times with saturated aqueous sodium bicarbonate solution (10 mL), and further washed with saturated saline (20 mL). The filtrate was rotary dried, and then analyzed by reverse phase preparative HPLC (C 18 (Conditions: 5% to 100% (A: water, B: CH3CN), flow rate: 70 mL / min) and freeze-drying to obtain 200 mg of compound 1-1a. MS m / z: C 40 H 39 N6O7P, [M-Diisopropyl+OH] +Theory: 747.26, Actual measurement: 747.6. 1H NMR (400 MHz, Acetonitrile-d3) δ 7.56, 7.54 (2s, 1H), 7.36-7.27 (m, 2H), 7.24-7.21 (m, 7H), 6.83-6.80 (m, 4H), 4.12-4.10 (m, 2H), 3.75-3.68 (m, 10H), 3.20-2.80 (m, 2H), 2.68-2.54 (m, 4H), 1.22-1.04 (m, 18H).

[0417] [ka] Compound 6B(+) (200 mg, 0.32 mmol), tetrazole (11 mg, 0.16 mmol), N-methylimidazole (5 mg, 0.06 mmol), and 3A molecular sieves (500 mg) were dissolved in 10 mL of acetonitrile, and compound 7 (144 mg, 0.48 mmol) was added at room temperature and stirred at room temperature overnight. After the reaction was completed, the molecular sieves were removed by filtration, dichloromethane (30 mL) was added, and the mixture was washed three times with saturated aqueous sodium bicarbonate solution (10 mL), and then with saturated saline (20 mL). The filtrate was rotovapped, and then purified by reverse phase preparative HPLC (C 18 (Conditions: 5% to 100% (A: water, B: CH3CN), flow rate: 70 mL / min) and freeze-drying to obtain 200 mg of compound 1-1b. MS m / z: C 40 H 39 N6O7P, [M-Diisopropyl+OH] + Theoretical: 747.26, Actual: 747.5.

[0418] 1.2 Synthesis of Compound 1-6a [ka] Compound 1 (10 g, 68.404 mmol), compound 2 (15 g, 62.186 mmol) and triphenylphosphine (32.62 g, 124.371 mmol) were dissolved in anhydrous THF (30 mL), and DIAD (24.656 mL, 124.371 mmol) was slowly added dropwise at 0°C. The reaction solution was reacted at 25°C for 12 h. LCMS showed that the reaction was complete. The reaction solution was extracted with ethyl acetate (200 mL) and water (200 mL), the organic phase was dried, and the filtrate was concentrated. The residue obtained was purified by normal phase column (DCM / MeOH=10 / 1) to obtain the target product 3 (20 g).

[0419] [ka] Compound 3 (20 g, 28.585 mmol) was dissolved in acetic acid (24 mL, 426.016 mmol) and HO (12 mL) and stirred at 60 °C for 1 h. The reaction was then rotovapped and THF (12 mL) and HO (12 mL) were added and stirred at 80 °C for 7 h. LCMS showed the reaction was complete. The reaction was extracted with ethyl acetate (200 mL) and water (100 mL), and solid sodium carbonate was added to the aqueous phase until a large amount of solid precipitated in the aqueous phase. The solid was filtered, washed with water, and the filter cake was dried by oil pump to give the target compound 5 (9 g).

[0420] [ka] Under nitrogen gas protection, compound 5 (6.8 g, 18.581 mmol) was dissolved in pyridine (80 mL), and TMSCl (14.250 mL, 111.489 mmol) was added slowly at 0 ° C. and stirred for 2 h. Then, isobutyryl chloride (2.044 mL, 19.511 mmol) was added at 0 ° C. and stirred at 25 ° C. for 1 h. LCMS showed that the reaction was complete. After extraction with dichloromethane (200 mL) and water (200 mL), the organic phase was spun dry, and the sample was mixed and purified by normal phase column (DCM:MeOH = 10:1) and columned, and a peak was observed at 4.8%, to obtain yellow oily compound 6 (12 g).

[0421] [ka] Under nitrogen gas protection, compound 6 (5.5 g, 12.392 mmol) was dissolved in pyridine (30 mL), MOLECULAR SIEVE 4A 1 / 16 (7 g, 12.392 mmol) was added, and DMTrCl (5.04 g, 14.870 mmol) solid was added in several portions at 0 ° C., and the reaction was carried out at 25 ° C. for 2 h. TLC (PE: EtOAc = 1: 1, Rf = 0.69) showed that the reaction was already completed. The reaction solution was treated together with TJN200879-040-P1. The reaction solution was extracted with ethyl acetate (200 mL) and water (200 mL), and the organic phase was spun dry. The sample was mixed and purified by normal phase column (PE: EtOAc was applied to the column, and a peak was obtained at 84%) to obtain yellow oily compound 7 (12 g).

[0422] [ka] Compound 7 (12 g, 15.389 mmol) was dissolved in EtOAc (140 mL), and wet palladium / carbon (Pd / C) (7 g, 15.389 mmol) was added, and the reaction solution was reacted at 25°C under hydrogen gas (15 Psi) for 2 h. TLC (PE:EtOAc=0:1, Rf=0.09) showed that the reaction was already completed. The reaction solution was filtered, and the filter cake was washed with ethyl acetate (30 mL) three times, and the filtrate was collected. After the filtrate was spun dry, 50 mL of dichloromethane and 2 mL of triethylamine were added, and the sample was mixed and purified by normal phase column (column with DCM:MeOH=10:1, peak at 0.5%) to obtain 9 g (yellow foamy solid). The obtained racemic compound was resolved by SFC to obtain the product target compound 7A(-) (3.9 g) and target compound 7B(+) (3.8 g).

[0423] [ka] Compound 7A(-) (3.30g, 5.40mmol), tetrazole (190mg, 2.70mmol), 1-methylimidazole (90mg, 1.10mmol), 3A molecular sieve (500mg) were dissolved in 30mL of acetonitrile, and compound 8 (2.50g, 8.10mmol) was added at room temperature and stirred at room temperature for 2h. After the reaction was completed, the molecular sieve was filtered off, washed with DCM (150mL), saturated aqueous sodium bicarbonate (30mL x 3), and further washed with saturated saline (30mL), the filtrate was dried by rotary evaporation, and then passed through reverse phase preparative HPLC (C18, Condition: 5%-100% (A: water, B: CH3CN), flow rate: 70mL / min) and lyophilized to obtain 1-6a (2.9g, 66%). MS m / z:C43H55N7O7P[M+H]+, theoretical: 812.38, measured: 812.5. 1H NMR (400 MHz, Acetonitrile-d3) δ 7.56, 7.54 (2s, 1H), 7.36-7.27 (m, 2H), 7.24-7.21 (m, 7H), 6.83-6.80 (m, 4H), 4.12-4.10 (m, 2H), 3.75-3.68 (m, 10H), 3.20-2.80 (m, 2H), 2.68-2.54 (m, 4H), 1.22-1.04 (m, 18H).

[0424] 1.3 Synthesis of Compound 1-7a [ka] Under nitrogen gas protection, compound 1 (5 g, 23.1272 mmol), compound 2 (6.76 g, 46.254 mmol) and triphenylphosphine (7.28 g, 27.753 mmol) were dissolved in 30 mL of dioxane, and DEAD (5.502 mL, 27.753 mmol) was slowly added dropwise at 0 ° C. After the dropwise addition was completed, the reaction was gradually heated to 25 ° C. and continued to react for 1 h. The reaction was extracted by adding 100 mL of H2O and 100 mL of EtOAc, and the organic phase was combined and dried, filtered and concentrated, and the sample was mixed and applied to a column, and purified by a normal phase column (PE: EtOAc = 1: 1, column applied to obtain the target product (4 g).

[0425] [ka] Compound 3 (3.3 g) was dissolved in HOAc (16 mL) and HO (4 mL) and heated in an oil bath at 60° C. for 0.5 h. The residue obtained by rotary evaporation of the reaction solution was purified by normal phase column (PE:EtOAc=0:1) to give the target product 4 (3 g).

[0426] [ka] Compound 4 (3 g, 8.873 mmol) was dissolved in 5 mL of pyridine and 10 mL of DMTrCl (3.91 g, 11.535 mmol) in pyridine was slowly added dropwise at 0° C. under nitrogen gas protection. After the addition was completed, the reaction was warmed to 25° C. and allowed to react for 1 h. The reaction was extracted with 50 mL of water and 100 mL of ethyl acetate. The aqueous phase was further extracted three times with 100 mL of ethyl acetate, and the combined organic phase was dried, filtered, concentrated, and purified by normal phase column (PE:EtOAc=2:1). The target product 5 (4 g) was obtained.

[0427] [ka] Compound 5 (4 g, 5.769 mmol) was dissolved in methanol (10 mL), and saturated NH3 methanol solution (40 mL) was added and reacted at 0° C. for 6 h. The reaction solution was rotovapped and purified by normal phase column (PE:EtOAc=0:1) to give 2.4 g of racemic compound, which was resolved by SFC to give target product 6A (750 mg, 100% purity) and target product 6B (400 mg, 99.16% purity).

[0428] [ka] Compound 6A(-) (700 mg, 1.40 mmol), tetrazole (50 mg, 0.70 mmol), 1-methylimidazole (23 mg, 0.28 mmol), and 3A molecular sieves (500 mg) were dissolved in 10 mL of acetonitrile, and compound 7 (630 mg, 2.10 mmol) was added at room temperature and stirred at room temperature for 2 h. After the reaction was completed, the molecular sieves were filtered off, washed with DCM (50 mL), saturated aqueous sodium bicarbonate (10 mL x 3), and further washed with saturated saline (20 mL), and the filtrate was rotovapped, and then passed through reverse phase preparative HPLC (C18, Condition: 5% to 100% (A: water, B: CH3CN), flow rate: 70 mL / min) and lyophilized to obtain 1-7a (700 mg, 72%). MS m / z: C38H47N4O7PNa[M+Na]+, theoretical: 725.32, observed: 725.5.

[0429] 1.4 Synthesis of Compound 1-8a [ka] Compound 1 (8.5 g, 76.508 mmol) and compound 2 (30.64 g, 91.809 mmol) were dissolved in DMF (150 mL), and CS2CO3 (29.91 g, 91.809 mmol) was added, and the reaction was reacted at 90 °C for 12 h under nitrogen gas protection. The reaction was detected to be complete by LCMS. The reaction solution was filtered, dried by rotation with an oil pump, and purified by normal phase column separation (80 g, DCM / MeOH = 10 / 1 to 5 / 1) to obtain the target product 3 (13.5 g, 80% purity).

[0430] [ka] Compound 3 (10.5 g, 35.105 mmol) was dissolved in pyridine (65 mL) and CH3CN (65 mL), BzCl (4.894 mL, 42.126 mmol) was added dropwise to the solution, and the mixture was reacted at 25 °C for 2 h. LCMS detected the completion of the reaction of most of the raw materials, and the mixture was quenched by adding H2O (100 mL), extracted with EtOAc (100 mL × 3), dried by rotation, and purified by column separation (combined with TJN200872-101) (80 g, PE / EtOAc = 10 / 1 to 0 / 1, DCM / MeOH = 10 / 1) to obtain the target product 4 (14 g, 90% purity).

[0431] [ka] Compound 4 (14g, 36.694mmol) was dissolved in HOAc (56mL, 314.796mmol) and HO (14mL) and reacted at 60℃ for 2h, LCMS showed the reaction was complete. It was concentrated by oil pump and separated by normal phase column (40g, DCM / MeOH=1 / 0~5 / 1) to give the target product 5 (8.4g, 90% purity & 2.4g, 80% purity).

[0432] [ka] Compound 5 (7.4g, 21.957mmol), DMAP (0.54g, 4.391mmol), and MOLECULAR SIEVE 4A (11.1g, 2.967mmol) were dissolved in pyridine (60mL) and stirred for 10min under ice bath, then DMTrCl (8.93g, 26.348mmol) was added and reacted with stirring for 1.8h. LCMS showed that about 19% of the raw material remained and about 60% of the target was MS. It was purified in combination with (TJN200872-105&106). The reaction solution was added with HO (50 mL), extracted with DCM (50 mL × 3), dried, rotovapped, and separated by column (120 g, PE / (EA:DCM:TEA = 1:1:0.05) = 1 / 0 ~ 0 / 1 to DCM / MeOH = 10 / 1) to obtain the target compound 6 (11 g, 89% purity, TJN200872-105 & 106 & 107), and the raw material (3.0 g, 70% purity) was recovered.

[0433] [ka] Compound 6 (15 g, 22.041 mmol) was separated by SFC (DAICEL CHIRALPAK AD (250 mm × 50 mm, 10 μm), 0.1% NH3HO EtOH, B: 45% to 45%, 200 mL / min) to give target product 6A(+) (5.33 g, 94.29% purity), target product 6B(-) (6.14 g, 97.91% purity), and 1.0 g of compound 6B(-) was recovered.

[0434] [ka] Compound 6B(-) (5.4g, 8.92mmol), tetrazole (312mg, 4.46mmol), 1-methylimidazole (146mg, 1.78mmol), 3A molecular sieve (500mg) were dissolved in 40mL of acetonitrile, and compound 7 (4g, 13.4mmol) was added at room temperature and stirred at room temperature for 2h. After the reaction was completed, the molecular sieve was filtered off, washed with DCM (200mL), saturated aqueous sodium bicarbonate (30mL x 3), and further washed with saturated saline (50mL), the filtrate was rotovapped, and then passed through reverse phase preparative HPLC (C18, Condition: 5%-100% (A: water, B: CH3CN), flow rate: 70mL / min) and lyophilized to obtain 1-8a (5.8g, 80%). MS m / z: C45H51N5O7P, [M+H]+, theoretical: 804.36, measured: 804.4.

[0435] Example 2: Characterization of different chemical modifications [ka] Among them, the nucleotide synthesized using 2-hydroxymethyl-1,3-propanediol as the starting material is defined as hmpNA. (+)hmpNA(A) is obtained by solid-phase synthesis from the nucleoside phosphoramidite monomer 1-1b in Example 1.1, and has the absolute configuration (S)-hmpNA(A); (-)hmpNA(A) is obtained by solid-phase synthesis from the nucleoside phosphoramidite monomer 1-1a in Example 1.1, and has the absolute configuration (R)-hmpNA(A); Similarly, the base types of hmpNA were substituted and the following structures were obtained by solid phase synthesis and the absolute configurations were confirmed: (+)hmpNA(G) has the absolute configuration of (S)-hmpNA(G), (-)hmpNA(G) has the absolute configuration of (R)-hmpNA(G); (+)hmpNA(C) has the absolute configuration of (S)-hmpNA(C); (-)hmpNA(C) has the absolute configuration of (R)-hmpNA(C); (+)hmpNA(U) has the absolute configuration of (R)-hmpNA(U), (-)hmpNA(U) has the absolute configuration of (S)-hmpNA(U).

[0436] The absolute configurations of (S)-hmpNA(G), (R)-hmpNA(G), (S)-hmpNA(C), (R)-hmpNA(C), (S)-hmpNA(U) and (R)-hmpNA(U) were confirmed by X-Ray diffraction from their intermediates or derivatives.

[0437] The structure of the intermediate or derivative is as follows:

[0438] [ka] TJ-NA067: The detected crystal is a colorless block (0.30 × 0.10 × 0.04 mm3) belonging to the monoclinic P21 space group. Cell parameters a = 16.0496(5) Å, b = 4.86260(10) Å, c = 16.4686(5) Å, α = 90°, β = 118.015(4)°, γ = 90°, V = 1134.65(7) Å3, Z = 4. Calculated density Dc = 1.389 g / cm3, number of electrons in the unit cell F(000) = 504.0, linear absorption coefficient of the unit cell μ(Cu Kα) = 0.840 mm-1, diffraction experiment temperature T = 150.00(11) K.

[0439] [ka] 6A(+): The detected crystal is a colorless block (0.30×0.20×0.10 mm3) belonging to the monoclinic P21 space group. Cell parameters a=22.6688(7)Å, b=8.5595(2)Å, c=23.3578(5)Å, α=90°, β=113.876(3)°, γ=90°, V=4144.3(2)Å3, Z=2. Calculated density Dc=0.999g / cm3, number of electrons in the unit cell F(000)=1318.0, linear absorption coefficient μ(Cu Kα)=0.570mm-1, diffraction experiment temperature T=100.01(18)K.

[0440] [ka] TJ-NA048: The detected crystal is colorless and needle-like (0.30 × 0.04 × 0.04 mm3) and belongs to the monoclinic P1 space group. Cell parameters a = 7.6165(4) Å, b = 11.3423(5) Å, c = 17.3991(8) Å, α = 85.007(4)°, β = 88.052(4)°, γ = 70.532(4)°, V = 1411.75(12) Å3, Z = 2. Calculated density Dc = 1.366 g / cm3, number of electrons in the unit cell F(000) = 620.0, linear absorption coefficient of the unit cell μ(Cu Kα) = 0.856 mm-1, diffraction experiment temperature T = 150.00(13) K.

[0441] [ka] TJ-NA092: The detected crystal is colorless and prismatic (0.30 × 0.10 × 0.10 mm3) and belongs to the monoclinic P1 space group. Cell parameters a = 5.17960(10) Å, b = 8.0667(2) Å, c = 12.4077(2) Å, α = 93.146(2)°, β = 101.266(2)°, γ = 96.134(2)°, V = 503.993(18) Å3, Z = 2. Calculated density Dc = 1.412 g / cm3, number of electrons in the unit cell F(000) = 228.0, linear absorption coefficient of the unit cell μ(Cu Kα) = 0.945 mm-1, diffraction experiment temperature T = 100.00(10) K.

[0442] Example 3: Preparation of NAG0052, L96 Compounds NAG0024 and NAG0026 were purchased from Tianjin Yaoming Kangde New Drug Development Co., Ltd. Unless otherwise stated, all reagents used in the following examples are commercially available products.

[0443] Synthesis of compound NAG0052 The starting material, compound 1, was purchased from Jiangsu Power Pharmaceutical Technology Co., Ltd. The synthetic route of compound NAG0052 is as follows:

[0444] [ka] The synthesis and characterization of specific intermediates and end products related to the above pathway are as follows.

[0445] compound 2 At 0°C and under nitrogen gas protection, NaH (12.2g, 304mmol, 60% purity) was added in portions to a solution of compound 1 (12.3mL, 101mmol) in THF (300mL). The mixture was stirred at 20°C for 1h, then cooled to 0°C again, and benzyl bromide (36.3mL, 304mmol) was added dropwise to the system, and stirred at 20°C for 12h. The reaction solution was quenched with H2O (100mL) and extracted with EtOAc (200mL x 2). The combined organic phase was washed with saturated brine (100mL), dried over Na2SO4, filtered, and concentrated to obtain a residue, which was separated by silica gel column chromatography to obtain the target compound 2 (20.0g, 51.8mmol, 51% yield). LCMS: t R = 2.615 and 2.820 min in 30-90AB_7 min_220&254_Shimadzu.lcm (Xtimate C18, 3um, 2.1*30mm), MS (ESI) m / z = 351.2 [M+Na] + . 1 H NMR: (400 MHz, CDCl3) δ ppm 7.35-7.12 (m, 10H), 5.06-4.95 (m, 1H), 4.51-4.39 (m, 4H), 4.24-3.87 (m, 2H), 3.50-3.40 (m, 2H), 3.38-3.20 (m, 3H), 2.20-1.91 (m, 2H).

[0446] Compounds 3 and 4 At 20°C and under nitrogen gas protection, TMSCN (13.5mL, 101mmol) was added to a solution of compound 2 (13.0g, 33.6mmol) in DCM (300mL) at once, and then TMSOTf (9.14mL, 50.5mmol) in DCM (30mL) was added dropwise. The reaction solution was stirred at 20°C for 15h. After the reaction was completed, the system was quenched with saturated aqueous NaHCO3 (80mL) and extracted with DCM (150mL x 2). The combined organic phase was washed with saturated brine (80mL), dried over Na2SO4, filtered and concentrated, and then separated by silica gel column chromatography to obtain the target compound 3 (3.30g, 9.18mmol, yield 27%) and pale yellow oily liquid compound 4 (8.50g, 9.18mmol, yield 70%). compound 3 1 H NMR: (400 MHz, CDCl3) δ ppm 7.42-7.29 (m, 10H), 4.81 (t, J = 7.8 Hz, 1H), 4.65-4.49 (m, 4H), 4.30-4.21 (m, 2H), 3.65-3.57 (m, 1H), 3.57-3.49 (m, 1H), 2.49-2.40 (m, 2H). compound 4 1 H NMR: (400 MHz, CDCl3) δ ppm 7.42-7.26 (m, 10H), 4.93-4.87 (m, 1H), 4.65-4.48 (m, 4H), 4.43-4.38 (m, 1H), 4.21-4.17 (m, 1H), 3.79-3.70 (m, 1H), 3.54 (d, J = 4.0 Hz, 1H), 2.45-2.37 (m, 2H).

[0447] compound 5 At 0°C and under nitrogen gas protection, a solution of compound 4 (3.00 g, 9.28 mmol) in THF (15 mL) was added dropwise to a solution of LiAlH4 (0.79 g, 20.9 mmol) in THF (15 mL), and after the completion of the addition, the system was reacted at 0°C for 1 h. The complete disappearance of the raw material was monitored by TLC (PE: EtOAc = 3: 1). Sodium sulfate decahydrate was gradually added to the reaction solution until no foaming occurred. The reaction solution was then filtered, and the filter cake was washed three times with dichloromethane (60 mL), after which the filtrate was collected and spun to dryness to obtain the target compound 5 (3.00 g, 90% yield). 1 H NMR: (400 MHz, DMSO-d6) δ ppm 7.40-7.14 (m, 10H), 4.54-4.38 (m, 4H), 4.06-3.99 (m, 2H), 3.91 (q, J = 6.4 Hz, 1H), 3.48-3.37 (m, 2H), 2.67-2.52 (m, 2H), 2.21-2.18 (m, 1H), 1.77-1.73 (m, 1H).

[0448] compound 6 Under nitrogen gas protection, compound 5 (3.00 g, 8.25 mmol) was dissolved in DCM (30 mL), TEA (3.44 mL, 24.7 mmol) and CbzCl (1.76 mL, 12.4 mmol) were added, and the mixture was reacted at 20 °C for 2 h. LCMS showed the reaction was complete. The reaction solution was extracted with dichloromethane (30 mL) and water (60 mL). The organic phase was washed three times with water (60 mL x 3), dried over anhydrous sodium sulfate, concentrated, and purified by normal phase column (PE: EtOAc = 1: 1) to give the target compound 6 (2.5 g, 90% yield). LCMS: t R = 0.810 min in 5-95AB_1min, MS (ESI) m / z =462.2 [M+H] + . 1H NMR: (400 MHz, CDCl3) δ ppm 7.39-7.29 (m, 15H), 5.35 (s, 1H), 5.15-5.01 (m, 2H), 4.72 (d, J = 6.0 Hz, 1H), 4.54-4.40 (m, 3H), 4.26 (s, 1H), 4.23-4.18 (m, 1H), 4.11-4.04 (m, 1H), 3.54-3.41 (m, 3H), 3.37-3.25 (m, 1H), 2.34-2.23 (m, 1H), 1.85-1.79 (m, 1H).

[0449] compound 7 Under nitrogen gas protection, compound 6 (2.00 g, 3.90 mmol) was dissolved in DCM (5 mL), and a solution of BCl3 in THF (1 M, 27.3 mL) was added at -78 °C and reacted for 1 h. The complete disappearance of the raw material was monitored by TLC (DCM:MeOH = 10:1). The reaction solution was quenched by adding methanol (20 mL) at -78 °C, concentrated, and purified by forward column (DCM:MeOH = 10:1) to obtain the target compound 7 (2.00 g, 60% yield). 1 H NMR: (400 MHz, CD3OD) δ ppm 7.41-7.23 (m, 5H), 5.08 (s, 2H), 4.25-4.07 (m, 2H), 3.85-3.75 (m, 1H), 3.63-3.56 (m, 1H), 3.54-3.48 (m, 1H), 3.30-3.27 (m, 2H), 2.34-2.21 (m, 1H), 1.71-1.64 (m, 1H).

[0450] compound 8 Under nitrogen gas protection, compound 7 (0.50 g, 1.78 mmol) was dissolved in pyridine (5 mL), and 4A molecular sieves (500 mg) and DMTrCl (0.66 mL, 2.13 mmol) were added at 0 ° C., and then the temperature was raised to 20 ° C. and reacted for 1.5 h. The complete disappearance of the raw material was monitored by TLC (PE: EtOAc = 2: 1). The reaction solution was added to ethyl acetate (60 mL) and water (60 mL) for extraction, and the organic phase was washed three times with water (60 mL × 3), dried over anhydrous sodium sulfate, concentrated, and purified by forward column (PE: EtOAc = 1: 1) to obtain the target compound 8 (800 mg, yield 90%). 1 H NMR: (400 MHz, CDCl3) δ ppm 7.44 (d, J = 7.6 Hz, 2H), 7.37-7.23 (m, 11H), 7.22-7.15 (m, 1H), 6.84 (d, J = 8.8 Hz, 4H), 5.09 (s, 2H), 4.31-4.17 (m, 2H), 4.02-3.91 (m, 1H), 3.84-3.73 (m, 6H), 3.33 (s, 1H), 3.28 (s, 1H), 3.19-3.01 (m, 2H), 2.34-2.25 (m, 1H), 1.70-1.62 (m, 1H).

[0451] compound 9 Compound 8 (800 mg, 1.234 mmol) was dissolved in EtOAc (5 mL), Pd / C 10% (800 mg, 7.517 mmol) was added, and the reaction was run under H2 conditions (15 Psi) at 20°C for 1 h. LCMS showed the reaction was already complete. The reaction was filtered, and the filter cake was washed three times with dichloromethane (100 mL) and methanol (100 mL), concentrated, and separated through a reverse phase column to give compound 9 (300 mg, 54%). LCMS: t R = 2.586 min in 10-80CD_3min MS (ESI) m / z = 450.2 [M+H] + .

[0452] compound 11 Compound 10 (435 mg, 1.780 mmol) was dissolved in DCM (10 mL), DIEA (0.441 mL, 2.67 mmol) and HATU (677 mg, 1.78 mmol) were added, and compound 9 (400 mg, 0.890 mmol) was further added and reacted at 20 ° C for 1 h. The reaction was monitored for completion by TLC (DCM:MeOH = 10: 1). The reaction solution was extracted with dichloromethane (60 mL) and water (60 mL), and the organic phase was washed three times with water (60 mL × 3), dried over anhydrous sodium sulfate, concentrated, and purified by normal phase column (PE: EtOAc = 0: 1, product peak appeared at 100%) to obtain the target compound 11 (600 mg, 90% yield). LCMS: t R = 2.745 min in 30-90CD_3min, MS (ESI) m / z =698.4 [M+Na] + . 1 H NMR: (400 MHz, CD3OD) δ ppm 7.46-7.38 (m, 2H), 7.35-7.24 (m, 6H), 7.22-7.16 (m, 1H), 6.90-6.78 (m, 4H), 4.29-4.21 (m, 2H), 4.02-3.95 (m, 1H), 3.77 (s, 6H), 3.66-3.62 (m, 3H), 3.41 (s, 1H), 3.18-3.04 (m, 2H), 2.36-2.17 (m, 5H), 1.71-1.50 (m, 5H), 1.39-1.25 (m, 14H).

[0453] compound 12 Compound 11 (600 mg, 0.799 mmol) was dissolved in THF (3 mL) and HO (1 mL), LiOH.HO (134 mg, 3.20 mmol) was added, and the mixture was allowed to react at 20 °C for 12 h. TLC (DCM:MeOH = 10:1) showed the reaction was complete. The reaction solution was spun dry, dissolved in water (5 mL) and methanol (5 mL), and purified by reverse phase column (HO:CHCN = 1:1, peak at about 35%) to give the target compound 12 (460 mg, 100% yield, lithium salt). LCMS: tR = 1.346 min in 10-80CD_3min, MS (ESI) m / z =684.3 [M+Na] + . HPLC: R = 1.879 min in 10-80CD_6min. 1 H NMR: (400 MHz, CD3OD) δ ppm 7.47-7.39 (m, 2H), 7.35-7.24 (m, 6H), 7.22-7.15 (m, 1H), 6.91-6.79 (m, 4H), 4.31-4.18 (m, 2H), 4.02-3.95 (m, 1H), 3.78 (s, 6H), 3.44-3.33 (m, 2H), 3.18-3.04 (m, 2H), 2.35-2.27 (m, 1H), 2.24-2.10 (m, 4H), 1.70-1.51 (m, 5H), 1.31-1.23 (m, 12H).

[0454] compound 13 At room temperature and under nitrogen gas protection, compound NAG0024 (271 mg, 0.151 mmol) was dissolved in anhydrous THF (2 mL) and anhydrous DMF (4 mL), 3A molecular sieves were added, and compound 12 (100 mg, 0.151 mmol), HOBt (25 mg, 0.181 mmol), DCC (38 mg, 0.181 mmol) and DIEA (39 mg, 0.302 mmol) were added in sequence. The reaction solution was reacted at 45 ° C for 16 h. After LC-MS showed that the reaction was complete, water was added to quench and filtered. The filtrate was concentrated and then purified (H2O / MeCN) through a C18 reverse phase column to obtain compound 13 (210 mg, 57% yield). Compound NAG0052 Compound 13 (230 mg, 0.094 mmol) was dissolved in pyridine (5 mL) at room temperature, molecular sieves were added, and DMAP (12 mg, 0.283 mmol) and succinic anhydride (28 mg, 0.283 mmol) were added. The mixture was stirred at 50 °C for 16 h under nitrogen gas protection. The reaction was detected to be complete by LCMS, filtered, and dried by rotary evaporation. After purification by C18 reverse phase column, secondary purification was performed by preparative HPLC to obtain the target compound NAG0052 (123 mg, 0.048 mmol, 51% yield). MS(ESI)m / z=2535.3[M-1] - .Theory:2536.2. 1 H NMR (400 MHz, Acetonitrile-d3) δ 7.48-7.43 (m, 2H), 7.37-7.12 (m, 11H), 7.00-6.85 (m, 10H), 6.66 (s, 1H), 5.31 (dd, J = 3.4, 1.1 Hz, 3H), 5.20-5.13 (m, 1H), 5.05 (dd, J = 11.3, 3.4 Hz, 3H), 4.56 (d, J = 8.5 Hz, 3H), 4.30 (dd, J = 7.7, 5.3 Hz, 1H), 4.18-3.93 (m, 14H), 3.79 (s, 10H), 3.65 (q, J = 4.7, 3.6 Hz, 13H), 3.56-3.07 (m, 24H), 2.56 (s, 6H), 2.37 (t, J = 5.8 Hz, 10H), 2.17 (t, J = 7.5 Hz, 9H), 2.02-1.96 (m, 20H), 1.88 (s, 8H), 1.82-1.73 (m, 2H), 1.60 (dt, J = 15.0, 7.3 Hz, 16H), 1.27 (s, 13H). Synthesis of L96 [ka] According to the method described in patent application WO2014025805A1, L96 shown in the above structural formula was prepared and obtained.

[0455] Example 4: Synthesis of dsRNA 1. Homemade vector-conjugated resin The compound NAG0052 (157 mg, 0.062 mmol) containing a carboxylic acid group was dissolved in anhydrous DMF (3 mL). After the substrate was completely dissolved, anhydrous acetonitrile (4 mL), DIEA (0.03 mL, 0.154 mmol, 2.5 eq) and HBTU (35 mg, 0.093 mmol, 1.5 eq) were added in sequence. After the reaction solution was mixed uniformly, macroporous aminomethyl resin (476 mg, blank loading 0.41 mmol / g, target loading 0.1 mmol / g) was further added. The reaction solution was placed on a shaker (temperature: 25° C., rotation speed: 200 rpm) and shaken overnight. The reaction solution was filtered, and the filter cake was washed with DCM and anhydrous acetonitrile in sequence, respectively, and the solid was collected and dried in vacuum overnight.

[0456] The solid from the previous step was dispersed in anhydrous acetonitrile (5 mL), and pyridine (0.18 mL), DMAP (3 mg), NMI (0.12 mL), and CapB1 (2.68 mL) were added in that order. The reaction solution was placed on a shaker (temperature: 25 °C, rotation speed: 200 rpm) and shaken for 2 h. The reaction solution was filtered, the filter cake was washed with anhydrous acetonitrile, and the solid was collected and dried in vacuum overnight to obtain the vector-attached resin. The loading was measured to be 0.1 mmol / g.

[0457] 2. NAG0052 already linked to resin was linked to nucleoside monomers one by one in the 3'-5' direction according to the nucleotide sequence starting from the resin. Linking to one nucleoside monomer involves four steps of deprotection, coupling, capping, and oxidation or sulfurization. The procedure is common in the field.

[0458] Compound NAG0052 was linked to the sequence by solid-phase synthesis, and then aminolysis was performed, after which some functional groups were removed from the structure of NAG0052 to obtain NAG0052'. The prepared dsRNA has the sense and antisense strands shown in Tables 1 and 2.

[0459] [Table 1]

[0460] [Table 2] In the above table, capital letters G, A, C, and U represent nucleotides containing guanine, adenine, cytosine, and uracil, respectively; lower case letter m represents a 2'-methoxy group modification; lower case letter f represents a 2'-fluoro modification; lower case letter s represents that the two adjacent nucleotides on the left and right of the letter s are linked by a thiophosphodiester group, and so on.

[0461] Example 5: Inhibition of target gene expression by dsRNA in vivo This experiment examined the inhibitory efficiency of dsRNA of the present disclosure complexed with different structures on target gene mRNA expression levels in vivo.

[0462] Male C57BL / 6 mice aged 6-8 weeks were randomly assigned to groups with 6 mice in each group and 3 mice per time point, and mice in each group were administered TRD007205, the reference positive TRD002218, or PBS, respectively.

[0463] All animals were administered a single dose by subcutaneous injection, with the dose of dsRNA (in terms of nucleotides without ligand) calculated according to body weight, and the dose was 1 mg / kg, with a volume of 5 mL / kg. After 7 and 28 days of administration, the mice were sacrificed, and the livers were collected and preserved with RNA later (Sigma Aldrich). The liver tissue was then homogenized with a tissue homogenizer, and total liver tissue RNA was extracted using a tissue RNA extraction reagent kit (Fanzhi Medical Technology, FG0412) according to the operation steps described in the instruction manual. The total RNA was reverse transcribed into cDNA, and the expression level of TTR mRNA (transthyretin mRNA) in the liver tissue was detected by real-time fluorescent quantitative PCR. In the fluorescent quantitative PCR, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference gene, and the mRNA expression levels of TTR and GAPDH were detected by Taqman probe primers for TTR and GAPDH, respectively.

[0464] [Table 3]

[0465] [Table 4] The TTR mRNA expression level was calculated by the following equation:

[0466] TTR mRNA expression level = [(TTR mRNA expression level in test group / GAPDH mRNA expression level in test group) / (TTR mRNA expression level in control group / GAPDH mRNA expression level in control group)] × 100% The inhibitory efficiencies of the dsRNA of the present disclosure complexed with different structures on the expression level of target genes in vivo after 7 and 28 days of administration are shown in Figures 1 and 2, respectively. As can be seen from the results in Figure 1, TRD007205 has good effects on the inhibition of TTR mRNA expression after 7 days of administration. As can be seen from Figure 2, after 28 days of administration, TRD007205 has better inhibitory effects on the expression level of target genes than TRD002218.

[0467] Example 6: Synthesis of dsRNA 1. We made our own vector-containing resin.

[0468] 2. Starting with the resin with NAG0052, nucleoside monomers were linked one by one in the 3'-5' direction according to the nucleotide sequence order. Linking to one nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfurization.

[0469] The prepared dsRNA has the sense and antisense strands shown in Tables 5 and 6.

[0470] [Table 5] [Table 6-1] [Table 6-2] Among them, (-)hmpNA(A) [ka] (-)hmpNA(G) [ka] indicates, The structure of NAG0052' is [ka] and The structure of A(GNA) is [ka] and The structure of L10 is as follows: [ka] and The structure of L96 is [ka] It is.

[0471] [Table 7]

[0472] Example 7: Inhibition of human APOC3 in human primary hepatocytes (PHH) - Inhibitory activity of 7 concentration points dsRNA was screened for activity in human primary hepatocytes (PHHs) at seven concentration gradients, with each dsRNA sample having an initial final concentration of 10 nM upon transfection, five-fold serial dilutions, and seven concentration points.

[0473] PHHs were cryopreserved in liquid nitrogen, and 24 h prior to transfection, human primary hepatocytes (PHHs) were seeded into 96-well plates at a seeding density of 3 × 10 4 Cells / well, medium 80μL / well. Refer to the product instruction manual, dsRNA was transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150), the final concentration of dsRNA transfection was 10nM, 2nM, 0.4nM, 0.08nM, 0.016nM, 0.0032nM and 0.00064nM. After 24h treatment, total cellular RNA was extracted using high-throughput cellular RNA extraction reagent kit, RNA reverse transcription experiment and quantitative real-time PCR detection were performed to measure the mRNA level of human APOC3, and the mRNA level of human APOC3 was corrected by the GAPDH internal reference gene level.

[0474] Among them, for real-time quantitative PCR detection, the probe Q-PCR detection experiment was adopted, and the primer information is as shown in Table 8.

[0475] [Table 8]

[0476] Results analysis method After the Q-PCR detection experiment is completed, the corresponding Ct value is obtained according to the threshold value automatically set by the system, and the expression of a certain gene can be relatively quantified by comparing the Ct values: the Ct comparison is to calculate the difference in gene expression by the difference value with the internal reference gene Ct value, and the difference is calculated by the following formula: -△△Ct Also called △△Ct = [(Ct experimental group target gene - Ct experimental group internal standard) - (Ct control group target gene - Ct control group internal standard)]. Inhibition rate (%) = (1 - excess target gene expression) x 100%.

[0477] Results are expressed as the percentage excess of human APOC3 mRNA expression over control dsRNA-treated cells. IC 50 The results are shown in Table 9. As can be seen from the experimental results in Table 9, TRD008043 is superior to the other groups.

[0478] [Table 9]

[0479] The difference in activity between TRD007972-1, TJR100385, and TJR100386 was examined using the same method, and the IC 50 The results are shown in Table 10.

[0480] As is clear from the results in Table 10, TRD007972-1 has high on-target inhibitory activity against the APOC3 gene in PHH cells, and is superior to TJR100385 and TJR100386.

[0481] [Table 10]

[0482] Example 8: Verification of off-target levels of psiCHECK antisense strand (AS strand) of dsRNA In vitro molecular level on-target and off-target screening was simulated against dsRNA at nine concentration gradients in HEK-293A cells, and the results showed that TRD008043 has high activity and low off-target properties.

[0483] An off-target sequence corresponding to the dsRNA sequence was constructed and inserted into the psiCHECK-2 plasmid. The plasmid contains Renilla luciferase gene and firefly luciferase gene. As a dual reporter gene system, the target sequence of dsRNA was inserted into the 3'UTR region of Renilla luciferase gene, and the activity of dsRNA against the target sequence can be reflected by detecting the expression status of Renilla luciferase calibrated with firefly luciferase, and the Dual-Luciferase Reporter Assay System (Promega, E2940) was used for detection.

[0484] The rules for constructing the GSSM target plasmid corresponding to the dsRNA sequence are as follows.

[0485] For the antisense strand of dsRNA, an off-target plasmid (GSSM) was constructed that is perfectly complementary to positions 1 to 8 of the 5' end of the antisense strand but does not perfectly match the bases at other positions, and the base mismatch correspondence rule is that A matches C and G matches T.

[0486] HEK293A cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 h before transfection, HEK293A cells were seeded into a 96-well plate at a seeding density of 8 × 10 3 Cells / well, 100 μL medium / well.

[0487] The cells were co-transfected with dsRNA and the corresponding plasmids by Lipofectamine2000 (ThermoFisher, 11668019) according to the instruction manual, 0.2 μL of Lipofectamine2000 was used for each well, and the transfection amount of the plasmids was 20 ng / well. For the off-target sequence plasmid, dsRNA was set at a total of 9 concentration points, with the final concentration of the highest concentration point being 20 nM, and the concentration was serially diluted 3-fold to 20 nM, 6.6667 nM, 2.2222 nM, 0.7407 nM, 0.2469 nM, 0.0823 nM, 0.0274 nM, 0.0091 nM, and 0.0030 nM. 24 h after transfection, the off-target levels were detected by Dual-Luciferase Reporter Assay System (Promega, E2940).

[0488] The results are shown in Table 11, and the results show that TRD008043 has no off-target risk compared to TJR100383 and TJR100384.

[0489] [Table 11]

[0490] Example 9: Pharmacodynamic studies in Tg mice The mice were B6, CBA-Tg(APOC3)3707Bres / J, species: #006907 (Jackson Laboratory, 11-12w), hereafter abbreviated as APOC3 Tg mice, 9 males and 9 females. After arrival, the animals were kept separately in N=2 / cage and acclimated for 10 days. Body weight was measured once a week.

[0491] At least 40 μL plasma samples (~90 μL blood) were collected 14 days before dosing for the first baseline measurement of plasma HDL, LDL, TG, TC, and ApoC3 during the adaptation period. A second baseline was performed 7 days before dosing, with at least 40 μL plasma samples (~90 μL blood) collected for the second plasma TG, TC, HDL-C, and ApoC3 detection, and 17 animals were selected and divided into 3 groups according to TG and ApoC3 (apolipoprotein C3) levels. A third baseline was performed in the morning of the day of dosing, with at least 40 μL plasma samples (~90 μL blood) collected for TG, TC, HDL-C, and ApoC3 detection, and this data set was used as the zero point (day 0) baseline.

[0492] When preparing the test samples, they were dissolved in saline (PH=7.4), 5μL of each solution was taken and diluted 10 times, and the concentration was detected three times per type by NanoDrop. The final concentration of the administration formulation was adjusted according to the results of NanoDrop. Both the saline group and the test sample were administered once by subcutaneous injection, and the injection volume was calculated from the body weight before administration.

[0493] After administration, body weight was measured once a week, and plasma samples were collected on days 7, 14, 21, 28, 35, and 42 (after overnight fasting before blood collection) for detection of ApoC3, TG, TC, and HDL-C in plasma. Blood samples were centrifuged within 30 min after collection (centrifugation conditions: 4°C, 3000g, 5 min). Plasma was collected in two polypropylene tubes with a volume of 30 μL (exact volume) and 10-15 μL (remainder), respectively, which were flash frozen on dry ice and stored in a refrigerator at -80°C before sample analysis. The results are shown in Tables 13-15.

[0494] [Table 12] [Table 13] ***p<0.001, **p<0.01, *p<0.05. p is compared with the saline group. One-way ANOVA Dunnett's test.

[0495] As is clear from the results in Table 13, after a single administration of the test sample to APOC3 Tg mice, the percentage of APOC3 in plasma of mice in the TRD008043 group relative to the baseline on day 0 was significantly lower on days 7, 14, 21, 28, 35, and 42 compared to the saline group. On the other hand, the percentage of APOC3 in plasma of mice in the TRD007972-1 group relative to the baseline on day 0 was not significantly different on days 35 and 42 compared to the saline group. Furthermore, numerically, the percentage of APOC3 in plasma relative to the baseline on day 0 in the TRD008043 group relative to the baseline on day 0 was about 3 times lower than that of TRD007972-1 on D14 to D35 after a single administration.

[0496] As a result, a comparison between TRD008043 and TRD007972-1 shows that TRD008043 has a superior effect of reducing human APOC3 to TRD007972-1.

[0497] [Table 14] ***p<0.001, **p<0.01, *p<0.05. p is in comparison with the saline group, One-way ANOVA Dunnett's test.

[0498] As is clear from the results in Table 14, even on day 28 after a single administration of the test sample to APOC3 Tg mice, TRD008043 retains the effect of significantly lowering plasma TG compared to the saline group, while TRD007972-1 loses significance compared to the saline group. Furthermore, numerically, after a single administration, the percentage of plasma TG relative to the baseline on day 0 in the TRD008043 group is approximately 2 to 4 times lower than that in the TRD007972-1 group on D14 to D35.

[0499] As a result, when TRD008043 and TRD007972-1 are compared, it is shown that TRD008043 has a superior effect of reducing TG to TRD007972-1.

[0500] [Table 15]

[0501] *p<0.05, p compared with saline group, One-way ANOVA Dunnett's test.

[0502] As is clear from the results in Table 15, even on day 28 after a single administration of the test sample to APOC3 Tg mice, TRD008043 retained the effect of significantly lowering plasma TC compared to the saline group, whereas TRD007972-1 showed no significant difference compared to the saline group. Furthermore, numerically, after a single administration, the percentage of plasma TC relative to the baseline on day 0 in the TRD008043 group was approximately two times lower than that of the TRD007972-1 group on D14 to D35.

[0503] As a result, a comparison between TRD008043 and TRD007972-1 shows that TRD008043 has a superior effect of reducing TC than TRD007972-1.

Claims

1. A double-stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand, the sense strand and the antisense strand comprising consecutive nucleotides in a 5' to 3' direction, the sense strand has nucleotides at positions 7, 8, and 9 that are 2'-fluoro modified, a nucleotide at position 5 that is independently 2'-methoxy modified or 2'-fluoro modified, and nucleotides at remaining positions that are 2'-methoxy modified; the antisense strand has nucleotides at positions 2 and 14 modified with 2'-fluoro, nucleotides at positions 4, 6, 8, 9, 10, 12, 16, and 18 independently modified with 2'-methoxy or 2'-fluoro, and nucleotides at remaining positions modified with 2'-methoxy; the number of 2'-fluoro modified nucleotides in the antisense strand is 2 to 7; The antisense strand comprises at the 7th nucleotide position a chemical modification as shown in formula (I), a tautomer thereof, or a pharma- ceutically acceptable salt thereof, i.e. The chemical modification shown in formula (I) is 【Chemistry 1】 and B is a base in the case where the nucleotide at position 7 of the 5' region in the antisense strand is not modified, and The dsRNA inhibits expression of apolipoprotein C3 (APOC3). Double-stranded ribonucleic acid.

2. The sense strand has the nucleotide sequence shown in the following formula: 5’-N ａ N ａ N ａ N ａ N ａ N ａ N ｂ N ｂ N ｂ N ａ N ａ N ａ N ａ N ａ N ａ N ａ N ａ N ａ N ａ -3’、 Including, among which, N ａ is a nucleotide modified with a 2'-methoxy group, and N ｂ is a 2'-fluoro modified nucleotide; The dsRNA described in claim 1.

3. The antisense strand has the nucleotide sequence shown in the following formula: 5’-N ａ ’N ｂ ’N ａ ’X’N ａ ’X’W’X’X’X’N ａ ’X’N ａ ’N ｂ ’N ａ ’X’N ａ ’X’N ａ ’N ａ ’N ａ ’-3’、 and preferably, the antisense strand comprises the nucleotide sequence shown in the following formula: 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’W’N ｂ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5'-N ａ 'N ｂ 'N ａ 'N ａ 'N ａ 'N ａ 'W'N ａ 'N ａ 'N ａ 'N ａ 'N ａ 'N ａ 'N ｂ 'N ａ 'N ｂ 'N ａ 'N ａ 'N ａ 'N ａ 'N ａ '-3', or 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’W’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 wherein each X' is independently N ａ ' or N ｂ ' and N ａ N' is a nucleotide modified with a 2'-methoxy group; ｂ ' is a 2'-fluoro modified nucleotide, W' represents a nucleotide containing the chemical modification shown in formula (I), a tautomer thereof, or a pharma- ceutically acceptable salt thereof; The dsRNA according to claim 1 or 2.

4. At least one phosphate group in the sense strand and / or the antisense strand is a phosphate group having a modifying group, preferably a thiophosphate group, more preferably a thiophosphodiester group. The dsRNA according to any one of claims 1 to 3.

5. The thiophosphodiester group is Between the first and second nucleotides of the 5' end of the sense strand, Between the second and third nucleotides of the 5' end of the sense strand, Between the first and second nucleotides at the 5' end of the antisense strand, Between the second and third nucleotides of the 5' end of the antisense strand, Between the first and second nucleotides at the 3' end of the antisense strand, and Between the second and third nucleotides at the 3' end of the antisense strand, and Preferably, the sense strand and / or the antisense strand comprises a plurality of thiophosphodiester groups, the thiophosphodiester groups being Between the first and second nucleotides of the 5' end of the sense strand, Between the second and third nucleotides of the 5' end of the sense strand, Between the first and second nucleotides at the 5' end of the antisense strand, Between the second and third nucleotides of the 5' end of the antisense strand, Between the first and second nucleotides at the 3' end of the antisense strand, and Between the second and third nucleotides at the 3' end of the antisense strand, The dsRNA described in claim 4.

6. the sense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides; and the antisense strand comprises at least 19 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides; Preferably, The sense strand comprises the nucleotide sequence shown in SEQ ID NO:1, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:2; The dsRNA according to any one of claims 1 to 5.

7. The antisense strand comprises a nucleotide sequence shown in any one of SEQ ID NO:12, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:14; Preferably, the sense strand comprises the nucleotide sequence shown in SEQ ID NO:

3. The dsRNA according to any one of claims 1 to 6.

8. The dsRNA further comprises a ligand complexed therewith, the ligand comprising N-acetyl-galactosamine. The dsRNA according to any one of claims 1 to 7.

9. The ligand is 【Chemistry 2】 or a pharma- ceutically acceptable salt thereof. The dsRNA described in claim 8.

10. The 3' end of the dsRNA sense strand is complexed with the ligand.

10. The dsRNA of claim 8 or 9.

11. The ligand is linked to the 3' end of the dsRNA sense strand via a phosphate or thiophosphate group, preferably via a phosphodiester or thiophosphodiester group, more preferably via a phosphodiester group; The dsRNA according to any one of claims 8 to 10.

12. The antisense strand comprises a nucleotide sequence shown in any one of SEQ ID NO:12, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:14; Preferably, the sense strand comprises the nucleotide sequence shown in SEQ ID NO:

4. The dsRNA according to any one of claims 1 to 11.

13. The dsRNA is 【Chemistry 3】 or a pharma- ceutically acceptable salt thereof, wherein Af=adenine 2'-F ribonucleoside, Cf=cytosine 2'-F ribonucleoside, Uf=uracil 2'-F ribonucleoside, Gf=guanine 2'-F ribonucleoside, Am=adenine 2'-OMe ribonucleoside, Cm=cytosine 2'-OMe ribonucleoside, Gm=guanine 2'-OMe ribonucleoside, and Um=uracil 2'-OMe ribonucleoside; 【Chemistry 4】 represents a thiophosphodiester group, 【Chemistry 5】 indicates a phosphodiester group, NAG0052' is 【Chemistry 6】 (-)hmpNA(A) 【Chemistry 7】 Showing, The dsRNA according to any one of claims 1 to 12.

14. 14. A composition comprising the dsRNA of any one of claims 1 to 13, and optionally further comprising one or more pharma- ceutically acceptable excipients. Pharmaceutical compositions.

15. The dsRNA according to any one of claims 1 to 13 is included. vector.

16. The dsRNA according to any one of claims 1 to 13 or the vector according to claim 15, cell.

17. Use of a dsRNA according to any one of claims 1 to 13, a pharmaceutical composition according to claim 14, a vector according to claim 15 or a cell according to claim 16 in the preparation of a medicament, comprising The medicament is for reducing triglyceride levels in a subject or for preventing and / or treating a disease mediated by elevated triglyceride levels or elevated cholesterol levels, preferably the disease mediated by elevated triglyceride levels or elevated cholesterol levels is selected from hypertriglyceridemia, obesity, hyperlipidemia, lipid and / or cholesterol metabolic disorders, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia pancreatitis, metabolic syndrome, type 2 diabetes, familial chylomicronemia syndrome or familial partial lipodystrophy, use.

18. A method for inhibiting expression of the APOC3 gene or its mRNA, comprising administering to a subject an effective amount or dose of the dsRNA according to any one of claims 1 to 13, the pharmaceutical composition according to claim 14, the vector according to claim 15, or the cell according to claim 16. method.

19. A method for delivering an oligonucleotide to the liver, comprising administering to a subject an effective amount or dose of the dsRNA of any one of claims 8 to 13, the pharmaceutical composition of claim 14, the vector of claim 15, or the cell of claim 16. method.

20. The composition comprises the dsRNA according to any one of claims 1 to 13, the pharmaceutical composition according to any one of claims 14, the vector according to claim 15, or the cell according to claim 16. Reagent kit.

21. The method comprises synthesizing the dsRNA according to any one of claims 1 to 13, Methods for preparing a dsRNA or pharmaceutical composition.

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