WO2023138659A1 - 一种dsRNA、其应用及制备方法 - Google Patents

一种dsRNA、其应用及制备方法 Download PDF

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Publication number
WO2023138659A1
WO2023138659A1 PCT/CN2023/073166 CN2023073166W WO2023138659A1 WO 2023138659 A1 WO2023138659 A1 WO 2023138659A1 CN 2023073166 W CN2023073166 W CN 2023073166W WO 2023138659 A1 WO2023138659 A1 WO 2023138659A1
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Prior art keywords
nucleotide
seq
dsrna
antisense strand
nucleotides
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PCT/CN2023/073166
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English (en)
French (fr)
Inventor
王艳辉
李云飞
邓永岩
林晓燕
茅松
黄敏印
周雅琴
黄龙飞
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Tuojie Biotech Shanghai Co Ltd
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Tuojie Biotech Shanghai Co Ltd
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Priority to CA3243093A priority Critical patent/CA3243093A1/en
Priority to JP2024543000A priority patent/JP2025504845A/ja
Priority to MX2024008804A priority patent/MX2024008804A/es
Priority to EP23742974.1A priority patent/EP4467645A4/en
Priority to US18/730,303 priority patent/US20260076990A1/en
Priority to KR1020247025932A priority patent/KR20240145467A/ko
Priority to CN202380015152.6A priority patent/CN118525092A/zh
Publication of WO2023138659A1 publication Critical patent/WO2023138659A1/zh
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Definitions

  • the present disclosure relates to the technical field of using dsRNA to reduce the expression of target RNA and to treat related diseases, in particular, the present disclosure relates to the preparation and application of dsRNA.
  • RNA interference is an effective way to silence gene expression. According to statistics, more than 80% of the disease-related proteins in the human body cannot be targeted by current conventional small molecule drugs and biological macromolecular preparations, and are non-druggable proteins. Using RNA interference technology, it is possible to design appropriate dsRNA based on the mRNA encoding these proteins, specifically target the target mRNA and degrade the target mRNA, thereby inhibiting the production of related proteins. Therefore, dsRNA has very important prospects for drug development. However, in order to achieve the therapeutic effect of RNA interference in vivo, it needs to be delivered to specific cells in vivo.
  • asialoglycoprotein receptor is a receptor specifically expressed in hepatocytes, which has high abundance on the surface of hepatocytes and is characterized by rapid intracellular and extracellular transitions.
  • Monosaccharide and polysaccharide molecules such as galactose, galactosamine, and N-acetylgalactosamine have high affinity for ASGPR.
  • GalNAc galactosamine molecular clusters
  • 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 lipoprotein (VLDL).
  • APOC3 can also inhibit the catabolism of triglyceride-rich lipoproteins by inhibiting the activity of lipoprotein lipase and hepatic lipase, and further increase serum triglyceride levels.
  • APOC3 can also inhibit the hepatic clearance of T-rich lipoproteins and their residual particles by interfering with T-rich lipoproteins and their binding to hepatic receptors.
  • Elevated APOC3 levels are associated with elevated triglyceride levels and diseases such as cardiovascular disease, metabolic syndrome, obesity and diabetes. In recent years, APOC3 has emerged as a promising target for the treatment of diseases associated with hypertriglyceridemia. Elevated serum triglyceride levels have been identified as an independent risk factor for cardiovascular disease and a contributory factor for the development of atherosclerosis. with severe hypertriglyceridemia Individuals are also at high risk of developing recurrent pancreatitis.
  • the present disclosure provides a double-stranded ribonucleic acid (dsRNA), which comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise continuous nucleotides according to the direction from the 5' end to the 3' end, wherein the nucleotides at the 7th, 8th and 9th positions of the sense strand are 2'-fluoro-modified nucleotides, the 5th nucleotides are independently 2'-methoxy-modified nucleotides or 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-methoxy-modified nucleotides;
  • the nucleotides at position 14 are 2'-fluoro-modified nucleotides, the nucleotides at positions 4, 6, 8, 9, 10, 12, 16, and 18 are independently 2'-methoxy or 2'-fluoro-modified nucleotides, and the nucleot
  • At least one nucleotide position from the 2nd to the 8th position of the antisense strand contains a chemical modification represented by formula (I), its tautomer or a pharmaceutically acceptable salt thereof:
  • Y is selected from O, NH and S;
  • Each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • Q 1 is Q 2 is R 2 ; or Q 1 is R 2 and Q 2 is
  • J 1 is H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is a base
  • the chemical modification represented by formula (I), its tautomer, or a pharmaceutically acceptable salt thereof is replaced with a 2'-methoxy modification.
  • At least one nucleotide in the 2nd to 8th positions of the antisense strand is a 2'-methoxy-modified nucleotide.
  • the 2'-fluoro-modified nucleotides at positions 2, 4, 6, 10, 12, 14 and 16 of the 5' end of the antisense strand.
  • the 2'-fluoro-modified nucleotides at positions 2, 4, 6, 10, 14 and 16 of the 5' end of the antisense strand are identical to each other.
  • the 2'-fluoro-modified nucleotides at positions 2, 4, 6, 12, 14 and 16 of the 5' end of the antisense strand are identical to each other.
  • the 2'-fluoro-modified nucleotides at positions 2, 6, 10, 12, 14 and 16 of the 5' end of the antisense strand are identical to each other.
  • positions 2, 6, 14 and 16 at the 5' end of the antisense strand are 2'-fluoro-modified nucleotides.
  • the 2'-fluoro-modified nucleotides at positions 2, 4, 6, 14 and 16 at the 5' end of the antisense strand are identical to each other.
  • the 2'-fluoro-modified nucleotides at positions 2, 6, 10, 14 and 16 at the 5' end of the antisense strand are identical to each other.
  • positions 2, 6, 12, 14 and 16 at the 5' end of the antisense strand are 2'-fluoro-modified nucleotides.
  • the 6th, 14th and 16th positions at the 5' end of the antisense strand are 2'-fluoro-modified nucleotides.
  • the 2, 14 and 16 positions at the 5' end of the antisense strand are 2'-fluoro modified of nucleotides.
  • positions 2, 6 and 14 at the 5' end of the antisense strand are 2'-fluoro-modified nucleotides.
  • the chemical modification shown in formula (I) is selected from the chemical modification shown in formula (I-1):
  • Y is selected from O, NH and S;
  • Each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • Each J 1 and J 2 are independently H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • the chemical modification shown in formula (I) is selected from the chemical modification shown in formula (I-2):
  • Y is selected from O, NH and S;
  • Each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • Each J 1 and J 2 are independently H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are each independently H, methyl, ethyl, n-propyl or isopropyl;
  • Each J 1 and J 2 are independently H or methyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are each independently H, methyl, ethyl, n-propyl or isopropyl;
  • Each J 1 and J 2 are independently H or methyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • J 1 and J 2 are independently H;
  • R 1 is selected from H, methyl and CH 2 OH;
  • R 2 is selected from H, OH, NH 2 , methyl and CH 2 OH;
  • R 3 is selected from H, OH, NH 2 , methyl and CH 2 OH;
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylamino Purine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • J 1 and J 2 are independently H;
  • R 1 is selected from H, methyl and CH 2 OH;
  • R 2 is selected from H, methyl and CH 2 OH;
  • R 3 is selected from H, OH, NH 2 , methyl and CH 2 OH;
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • Y is O or NH
  • Each X is independently selected from CR 4 (R 4 '), NR 5 and NH-CO, R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • R 3 is selected from H, OH, NH 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy and (CH 2 ) p R 6 ;
  • Q 1 is Q 2 is R 2 ; or Q 1 is R 2 and Q 2 is
  • R 1 is selected from H, OH, C 1 -C 6 alkyl, C 1 -C 6 alkoxy and (CH 2 ) q R 7 ;
  • J 1 is H or C 1 -C 6 alkyl
  • R 2 is selected from H, OH, C 1 -C 6 alkyl, C 1 -C 6 alkoxy and (CH 2 ) r R 8 ;
  • R 1 and R 2 are directly connected to form a 3-6 membered ring;
  • B is a base
  • X is independently selected from CR 4 (R 4 ′) and NH—CO.
  • X is independently selected from CR 4 (R 4 ′).
  • R 3 is selected from H, C 1 -C 6 alkyl, and (CH 2 ) p R 6 .
  • R 3 is selected from H and C 1 -C 6 alkyl.
  • R 1 is selected from H, C 1 -C 6 alkyl, and (CH 2 ) q R 7 .
  • R 1 is selected from H and C 1 -C 6 alkyl.
  • R 2 is selected from H, OH, C 1 -C 6 alkyl, and (CH 2 ) r R 8 .
  • R 2 is selected from H, C 1 -C 6 alkyl, and (CH 2 ) r R 8 .
  • Y is O
  • Each X is independently selected from CR 4 (R 4 ') and NH-CO, R 4 and R 4 ' are independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • R 3 is selected from H, C 1 -C 6 alkyl and (CH 2 ) p R 6 ;
  • Q 1 is Q 2 is R 2 ; or Q 1 is R 2 and Q 2 is
  • R 1 is selected from H, C 1 -C 6 alkyl and (CH 2 ) q R 7 ;
  • J 1 is H or C 1 -C 6 alkyl
  • R 2 is selected from H, OH, C 1 -C 6 alkyl and (CH 2 ) r R 8 ;
  • R 1 and R 2 are directly connected to form a 5-6 membered ring;
  • B is a base
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • Y is O
  • Each X is independently selected from CR 4 (R 4 '), R 4 and R 4 ' are independently H or C 1 -C 6 alkyl;
  • R 3 is selected from H and C 1 -C 6 alkyl
  • Q 1 is Q 2 is R 2 ; or Q 1 is R 2 and Q 2 is
  • R 1 is selected from H and C 1 -C 6 alkyl
  • J 1 is H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a 5-6 membered ring;
  • B is a base
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • Y is O.
  • X is independently selected from CR 4 (R 4 ′), NR 5 and NH-CO, and R 4 , R 4 ′, and R 5 are each independently H, methyl, ethyl, n-propyl or isopropyl.
  • X is independently selected from NH-CO, CH2 and NH.
  • X is independently selected from NH—CO and CH 2 .
  • X is CH2 .
  • J 2 is H or methyl. In some embodiments, J is H.
  • R is selected from H and methyl.
  • R2 is selected from H, methyl, and CH2OH .
  • R1 and R2 are directly linked to form a 5-6 membered ring. In some embodiments, R and R are directly connected to form a 3-6 membered cycloalkyl. In some embodiments, R 1 and R 2 are directly connected to form cyclopentyl or cyclohexyl.
  • the chemical modification represented by the formula (I) is selected from any of the following structures:
  • B is selected from purine base, pyrimidine base, indole, 5-nitroindole and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • the chemical modification represented by the formula (I) is selected from any of the following structures:
  • B is selected from purine base, pyrimidine base, indole, 5-nitroindole and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • the chemical modification represented by the formula (I) is selected from any of the following structures:
  • B is selected from purine base, pyrimidine base, indole, 5-nitroindole and 3-nitropyrrole.
  • 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 pyrimidines, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • the chemical modification represented by the formula (I) is selected from any of the following structures:
  • B is selected from purine base, pyrimidine base, indole, 5-nitroindole and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • nucleotide comprising the chemical modification represented by formula (I), its tautomer or a pharmaceutically acceptable salt thereof is selected from nucleotides comprising the chemical modification represented by formula (I'), its tautomer or its pharmaceutically acceptable salt,
  • Y is selected from O, NH and S;
  • Each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • Q 1' is Q 2' is R 2 ; or Q 1' is R 2 and Q 2' is
  • J 1 is H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is a base
  • M is O or S
  • R when X is NH-CO, R is not H.
  • the chemical modification represented by formula (I'), its tautomer, or a pharmaceutically acceptable salt thereof is replaced with a 2'-methoxy modification.
  • the chemical modification shown in the formula (I') is selected from the chemical modification shown in the formula (I'-1):
  • Y is selected from O, NH and S;
  • Each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • Each J 1 and J 2 are independently H or C 1 -C 6 alkyl
  • M is O or S
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • the chemical modification shown in formula (I') is selected from the chemical modification shown in formula (I'-2):
  • Y is selected from O, NH and S;
  • Each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • Each J 1 and J 2 are independently H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • M is O or S
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • each X is independently selected from CR4 ( R4 '), S, NR5 , and NH-CO, wherein R 4 , R 4 ', R 5 are independently H or C 1 -C 3 alkyl;
  • Each J 1 and J 2 are independently H or C 1 -C 3 alkyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • each X is independently selected from CR 4 (R 4 '), S, NR 5 and NH-CO, wherein R 4 , R 4 ', R 5 are each independently H, methyl, ethyl, n-propyl or isopropyl;
  • Each J 1 and J 2 are independently H or methyl
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • Each J 1 and J 2 are independently H;
  • R 1 is selected from H, methyl and CH 2 OH;
  • R 2 is selected from H, OH, NH 2 , methyl and CH 2 OH;
  • R 3 is selected from H, OH, NH 2 , methyl and CH 2 OH;
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • Each J 1 and J 2 are independently H;
  • R 1 is selected from H, methyl and CH 2 OH;
  • R 2 is selected from H, methyl and CH 2 OH;
  • R 3 is selected from H, OH, NH 2 , methyl and CH 2 OH;
  • R 1 and R 2 are directly connected to form a ring
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • Y is O or NH
  • Each X is independently selected from CR 4 (R 4 '), NR 5 and NH-CO, R 4 , R 4 ', R 5 are independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • R 3 is selected from H, OH, NH 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy and (CH 2 ) p R 6 ;
  • Q 1' for Q 2' is R 2 ; or Q 1' is R 2 and Q 2' is
  • R 1 is selected from H, OH, C 1 -C 6 alkyl, C 1 -C 6 alkoxy and (CH 2 ) q R 7 ;
  • J 1 is H or C 1 -C 6 alkyl
  • R 2 is selected from H, OH, C 1 -C 6 alkyl, C 1 -C 6 alkoxy and (CH 2 ) r R 8 ;
  • R 1 and R 2 are directly connected to form a 3-6 membered ring;
  • M is O or S
  • B is a base
  • X is independently selected from CR 4 (R 4 ′) and NH—CO.
  • X is independently selected from CR 4 (R 4 ′).
  • R 3 is selected from H, C 1 -C 6 alkyl, and (CH 2 ) p R 6 .
  • R 3 is selected from H and C 1 -C 6 alkyl.
  • R 1 is selected from H, C 1 -C 6 alkyl, and (CH 2 ) q R 7 .
  • R 1 is selected from H and C 1 -C 6 alkyl.
  • R 2 is selected from H, OH, C 1 -C 6 alkyl, and (CH 2 ) r R 8 .
  • R 2 is selected from H, C 1 -C 6 alkyl, and (CH 2 ) r R 8 .
  • Y is O
  • Each X is independently selected from CR 4 (R 4 ') and NH-CO, R 4 and R 4 ' are independently H or C 1 -C 6 alkyl;
  • J 2 is H or C 1 -C 6 alkyl
  • R 3 is selected from H, C 1 -C 6 alkyl and (CH 2 ) p R 6 ;
  • Q 1' for Q 2' is R 2 ; or Q 1' is R 2 and Q 2' is
  • R 1 is selected from H, C 1 -C 6 alkyl and (CH 2 ) q R 7 ;
  • J 1 is H or C 1 -C 6 alkyl
  • R 2 is selected from H, OH, C 1 -C 6 alkyl and (CH 2 ) r R 8 ;
  • R 1 and R 2 are directly connected to form a 5-6 membered ring;
  • M is O or S
  • B is a base
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • Y is O
  • Each X is independently selected from CR 4 (R 4 '), R 4 and R 4 ' are independently H or C 1 -C 6 alkyl;
  • R 3 is selected from H and C 1 -C 6 alkyl
  • Q 1' for Q 2' is R 2 ; or Q 1' is R 2 and Q 2' is
  • R 1 is selected from H and C 1 -C 6 alkyl
  • J 1 is H or C 1 -C 6 alkyl
  • R 1 and R 2 are directly connected to form a 5-6 membered ring;
  • M is O or S
  • B is a base
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • Y is O.
  • X is independently selected from CR 4 (R 4 '), NR 5 and NH-CO, R 4 , R 4 ', R 5 are independently H, methyl, ethyl, n-propyl or isopropyl.
  • X is independently selected from NH—CO, CH 2 and NH.
  • X is independently selected from NH—CO and CH 2 .
  • X is CH2 .
  • J 2 is H or methyl. In some embodiments, J is H.
  • R is selected from H and methyl.
  • R2 is selected from H, methyl, and CH2OH .
  • R1 and R2 are directly linked to form a 5-6 membered ring. In some embodiments, R and R are directly connected to form a 3-6 membered cycloalkyl. In some embodiments, R 1 and R 2 are directly connected to form cyclopentyl or cyclohexyl.
  • the chemical modification represented by the formula (I') is selected from any of the following structures:
  • M is O or S
  • B is selected from purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • the chemical modification represented by the formula (I') is selected from any of the following structures:
  • M is O or S
  • B is selected from purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • the chemical modification represented by the formula (I') is selected from any of the following structures:
  • M is O or S
  • B is selected from purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same base as if the nucleotide at that position on the antisense strand was unmodified.
  • the chemical modification represented by the formula (I') is selected from any of the following structures: and those in which the adenine in their structure is replaced by guanine, cytosine, uracil, or thymine.
  • B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.
  • 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.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.
  • B is the same as the base of the antisense strand when the nucleotide at this position is unmodified.
  • the chemical modification represented by the formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-oxidethyl
  • the present disclosure provides a dsRNA comprising a sense strand and an antisense strand:
  • 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, and no more than 1 mismatches between the antisense strand and the target sequence; The target sequence is perfectly reverse complementary.
  • the sense strand and the antisense strand are at least partially reverse complementary to form a double-stranded region; in some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, and no more than 1 mismatches between the sense strand and the antisense strand; in some embodiments, the sense strand and the antisense strand are fully reverse complementary.
  • the sense and antisense strands 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 (eg, 19, 20, 21, 22, 23 nucleotides).
  • 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.
  • the length ratio of the sense strand and the antisense strand may 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, 2 1/23, 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.
  • the sense and antisense strands have a length ratio of 19/21, 21/23, or 23/25.
  • the sense and antisense strands have a length ratio
  • the dsRNA comprises one or two blunt ends.
  • the dsRNA comprises an overhang with 1 to 4 unpaired nucleotides, eg, 1, 2, 3, 4.
  • the dsRNA comprises an overhang located 3' to the antisense strand.
  • the nucleotide comprising the chemical modification represented by formula (I) or formula (I'), its tautomer or a pharmaceutically acceptable salt thereof is located at position 5, position 6 or position 7 of the 5' region of the antisense strand.
  • the nucleotide comprising the chemical modification represented by formula (I) or formula (I'), its tautomer or a pharmaceutically acceptable salt thereof is located at position 7 of the 5' region of the antisense strand.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • B is selected from adenine, guanine, 2,6-dihydro base purine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole and 3-nitropyrrole.
  • B is the same as the base of the antisense strand when the 5th nucleotide in its 5' region is not modified.
  • B is the same as the base when the 6th nucleotide of the 5' region of the antisense strand is not modified.
  • B is the same base as when the 7th nucleotide of the 5' region of the antisense strand is not modified.
  • the nucleotide at position 5 of the sense strand is a 2'-methoxy modified nucleotide.
  • the 7th nucleotide of the antisense strand is a 2'-methoxy modified nucleotide.
  • the sense strand and antisense strand comprise or are nucleotides represented by the following formula:
  • 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 '-3';
  • each X is independently Na or Nb
  • each X' is independently Na ' or Nb '
  • Na and Na ' are 2'-methoxy-modified nucleotides
  • Nb and Nb ' are 2'-fluoro-modified nucleotides
  • W' represents a 2'-methoxy-modified nucleotide or a nucleotide comprising a chemical modification represented by formula (I) or formula (I'), a tautomer or a pharmaceutically acceptable salt thereof.
  • W' represents a 2'-methoxy modified nucleotide.
  • W' represents a nucleotide comprising a chemical modification represented by formula (I) or formula (I'), a tautomer thereof, or a pharmaceutically acceptable salt thereof.
  • W' has the following structure:
  • the sense strand and the antisense strand comprise or are nucleotide sequences represented by the following formula:
  • 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 '-3';
  • each X' is independently N a ' or N b '; N a and N a ' are 2'-methoxy-modified nucleotides, N b and N b ' are 2'-fluoro-modified nucleotides;
  • W' represents a 2'-methoxy-modified nucleotide or a nucleotide comprising a chemical modification represented by formula (I) or formula (I'), a tautomer or a pharmaceutically acceptable salt thereof.
  • W' represents a 2'-methoxy modified nucleotide.
  • W' represents a nucleotide comprising a chemical modification represented by formula (I) or formula (I'), a tautomer thereof, or a pharmaceutically acceptable salt thereof.
  • W' has the following structure:
  • the sense strand comprises or is a nucleotide sequence represented by the following formula:
  • X is Na or N b ;
  • Na is a 2'-methoxy modified nucleotide, and
  • N b is a 2' -fluoro modified nucleotide.
  • the sense strand comprises or is a nucleotide sequence represented by the following formula:
  • N a is a 2'-methoxy-modified nucleotide
  • N b is a 2'-fluoro-modified nucleotide
  • the antisense strand comprises or is a nucleotide sequence represented by the following formula:
  • each X' is independently N a ' or N b ';
  • N a ' is a 2'-methoxy-modified nucleotide, and
  • N b ' is a 2'-fluoro-modified nucleotide;
  • W' represents a 2'-methoxy-modified nucleotide or a nucleotide comprising a chemical modification represented by formula (I) or formula (I'), a tautomer or a pharmaceutically acceptable salt thereof.
  • W' represents a 2'-methoxy modified nucleotide.
  • W' represents a nucleotide comprising a chemical modification represented by formula (I) or formula (I'), a tautomer thereof, or a pharmaceutically acceptable salt thereof.
  • W' has the structure:
  • the chemical modification represented by formula (I) is selected from:
  • B is selected from guanine, adenine, cytosine and uracil; in some specific embodiments, B is the same as the base when the 7th nucleotide in the 5' region of the antisense strand is not modified.
  • the chemical modification represented by formula (I) is selected from:
  • M is O or S;
  • B is selected from guanine, adenine, cytosine or uracil; In some specific embodiments, B is the same as the base when the 7th nucleotide in the 5' region of the antisense strand is not modified.
  • M is S. In some specific embodiments, M is O.
  • the antisense strand comprises or is a nucleotide sequence represented by the following formula:
  • N a ' is a 2'-methoxy-modified nucleotide
  • N b ' is a 2'-fluoro-modified nucleotide
  • W' represents a 2'-methoxy-modified nucleotide or a nucleotide comprising a chemical modification represented by formula (I), a tautomer or a pharmaceutically acceptable salt thereof.
  • W' represents a 2'-methoxy modified nucleotide.
  • W' represents a nucleotide comprising a chemical modification represented by formula (I), a tautomer or a pharmaceutically acceptable salt thereof.
  • the chemical modification represented by formula (I) is selected from:
  • B is selected from guanine, adenine, cytosine and uracil; in some specific embodiments, B is the same as the base when the 7th nucleotide in the 5' region of the antisense strand is not modified.
  • the chemical modification represented by formula (I) is selected from:
  • M is O or S;
  • B is selected from guanine, adenine, cytosine or uracil; In some specific embodiments, B is the same as the base when the 7th nucleotide in the 5' region of the antisense strand is not modified.
  • W' has the following structure:
  • M is S. In some specific embodiments, M is O.
  • At least one phosphate group in the sense strand and/or antisense strand is a phosphate group with a modification group, which makes the dsRNA have increased stability in a biological sample or environment; in some embodiments, the phosphate group with a modification group is a phosphorothioate group. In some embodiments, the phosphate group with a modifying group is a phosphorothioate group.
  • the phosphorothioate group is present in at least one of the following positions:
  • the sense strand and/or antisense strand include multiple phosphorothioate groups present in:
  • the sense strand comprises or is a nucleotide sequence represented by the following formula:
  • Nm represents any nucleotide modified by 2'-methoxy, such as C, G, U, A modified by 2'-methoxy
  • Nf represents any nucleotide modified by 2'-fluoro, such as C, G, U, A modified by 2'-fluoro
  • the lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate group; when the lowercase letter s is the first at the 3' end, it indicates that the end of a nucleotide adjacent to the left side of the letter s is a phosphorothioate group.
  • the sense strand comprises or is a nucleotide sequence represented by the following formula:
  • Nm represents any nucleotide modified by 2'-methoxy, such as C, G, U, A modified by 2'-methoxy
  • Nf represents any nucleotide modified by 2'-fluoro, such as C, G, U, A modified by 2'-fluoro
  • the lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate group; when the lowercase letter s is the first at the 3' end, it indicates that the end of a nucleotide adjacent to the left side of the letter s is a phosphorothioate group.
  • the antisense strand comprises or is a nucleotide sequence represented by the following formula:
  • Nm' represents any nucleotide modified by 2'-methoxy, such as C, G, U, A modified by 2'-methoxy
  • Nf' represents any nucleotide modified by 2'-fluoro, such as C, G, U, A modified by 2'-fluoro
  • the lowercase letter s indicates that there is a phosphorothioate group connection between the two nucleotides adjacent to the left and right of the letter s, and when the lowercase letter s is the first at the 3' end, it indicates that the end of a nucleotide adjacent to the left side of the letter s is a phosphorothioate group;
  • W' represents a 2'-methoxy modified nucleotide or a modified nucleotide comprising a chemical modification represented by formula (I) or formula (I'), a tautomer or a pharmaceutically acceptable salt thereof.
  • W' represents a 2'-methoxy modified nucleotide.
  • W' represents a nucleotide comprising a chemical modification represented by formula (I) or formula (I'), a tautomer thereof, or a pharmaceutically acceptable salt thereof.
  • the chemical modification represented by formula (I) or formula (I') is selected from:
  • B is selected from guanine, adenine, cytosine and uracil; in some embodiments, B is the same as the base when the 7th nucleotide in the 5' region of the antisense strand is not modified.
  • the chemical modification shown in formula (I) or formula (I') is selected from:
  • M is O or S;
  • B is selected from guanine, adenine, cytosine or uracil; In some specific embodiments, B is the same as the base when the 7th nucleotide in the 5' region of the antisense strand is not modified.
  • M is S. In some specific embodiments, M is O.
  • the dsRNA inhibits expression of apolipoprotein C3 (APOC3).
  • APOC3 apolipoprotein C3
  • the sense strand comprises at least 15 consecutive 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 consecutive nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2;
  • 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.
  • the sense strand and antisense strand are any of the following:
  • the sense strand comprises 5'-UAUUCUCAGUGCUCUCCUZ b1 -3' (SEQ ID NO: 21), and the antisense strand comprises 5'-UAGGAGAGCACUGAGAAUACU-3' (SEQ ID NO: 22);
  • the sense strand comprises 5'-GCACCGUUAAGGACAAGUZ b2 -3' (SEQ ID NO: 23), and the antisense strand comprises 5'-AACUUGUCCUUAACGGUGCUC-3' (SEQ ID NO: 24);
  • Z b1 is A or G; Z b2 is C or U.
  • 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 nucleotide sequence in SEQ ID NO:5 to SEQ ID NO:14;
  • the dsRNA is any of the following:
  • 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 dsRNA is any of the following:
  • the sense strand is composed of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand is composed of the nucleotide sequence shown in SEQ ID NO:5;
  • the sense strand is composed of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand is composed of the nucleotide sequence shown in SEQ ID NO:6;
  • the sense strand is made up of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand is made up of the nucleotide sequence shown in SEQ ID NO:7;
  • the sense strand is made up of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand is made up of the nucleotide sequence shown in SEQ ID NO:8;
  • the sense strand is made up of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand is made up of the nucleotide sequence shown in SEQ ID NO:9;
  • the sense strand is composed of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand is composed of the nucleotide sequence shown in SEQ ID NO:10;
  • the sense strand is composed of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand is composed of the nucleotide sequence shown in SEQ ID NO:11;
  • the sense strand is composed of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand is composed of the nucleotide sequence shown in SEQ ID NO:12;
  • the sense strand is made up of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand is made up of the nucleotide sequence shown in SEQ ID NO:13;
  • the sense strand is made up of the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand is made up of the nucleotide sequence shown in SEQ ID NO:14.
  • the dsRNA is any of the following:
  • the dsRNA is any of the following:
  • SEQ ID NO:10 is N-(SEQ ID NO:10).
  • SEQ ID NO: 11 is N-(SEQ ID NO: 11
  • SEQ ID NO: 12 is
  • SEQ ID NO: 13 is
  • SEQ ID NO: 14 is
  • s means that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate group
  • the dsRNA further comprises a conjugated ligand selected from a liver-targeting targeting ligand; in some embodiments, the ligand binds asialoglycoprotein receptor (ASGPR); in some embodiments, the ligand comprises a galactose cluster or a galactose derivative cluster selected from N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, or N-isobutyrylgalactosamine, or a combination thereof.
  • ASGPR asialoglycoprotein receptor
  • the dsRNA further includes a conjugated ligand
  • the ligand is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof
  • R 13 is a single or double bond, and when When it is a single bond, R 13 is independently CR 17 R 18 , NR 16 , O or S, when When it is a double bond, R 13 is independently CR 19 or N;
  • R 14 is independently CR 19 or N;
  • Ring A is cycloalkyl, heterocycloalkyl, aryl or heteroaryl, present or absent, and when Ring A exists, R 15 is independently CR 19 or N, and when Ring A does not exist, R 15 is independently CR 17 R 18 , NR 16 or O;
  • R' and R" are independently hydrogen, deuterium, hydroxy, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl optionally substituted by one or more substituents selected from halogen, hydroxy, oxo, nitro and cyano;
  • n1, p1 and q1 are independently 0, 1, 2, 3 or 4;
  • z1, z2, z3, z4, z5, z6, z7, z8 and z9 are independently an integer of 0-10;
  • r1 is an integer of 1-10.
  • R 16 is hydrogen or C 1-6 alkyl
  • R 13 is CR 17 R 18 , NR 16 , O or S;
  • R 14 is CR 19 ;
  • R 15 is independently CR 17 R 18 , NR 16 or O;
  • R 17 to R 19 are independently hydrogen, deuterium or alkyl
  • m1, p1 and q1 are independently 0, 1, 2, 3 or 4;
  • z5, z6, z7, z8 and z9 are independently an integer of 0-10;
  • r1 is an integer of 1-10.
  • R 16 is hydrogen or C 1-6 alkyl
  • R 13 is CR 17 R 18 or O
  • R 14 is CR 19 ;
  • R 15 is independently CR 17 R 18 or O;
  • R 17 to R 19 are independently hydrogen or alkyl
  • n 1;
  • z8 and z9 are independently an integer of 0-10;
  • L 2 is -(CH 2 ) j15 -(OCH 2 CH 2 ) 1-4 -(CH 2 ) j16 - or
  • j15 and j16 are independently an integer of 0-4;
  • r1 is 3, 4, 5 or 6.
  • j11, j12, j13, and j14 are independently integers from 0-2 or 4-10. In some embodiments, j11, j12, j13, and j14 are independently 0, 1, 2, 6, 7, 8, 9, or 10.
  • L can be The definition of j12 is the same as that described in the previous scheme, wherein the terminal a1 is connected to B1 , and the terminal b1 is connected to R11 .
  • L can be Wherein, terminal a1 is connected to B1 , and terminal b1 is connected to R11 .
  • R 11 can be a chemical bond and R 12 can be NR 16 , and the definition of R 16 is the same as described in any of the previous schemes.
  • R 16 can be hydrogen or C 1-6 alkyl.
  • R 16 can be hydrogen, methyl, ethyl, propyl, or isopropyl.
  • R 16 can be hydrogen
  • R 17 and R 18 can be hydrogen.
  • R 19 can be hydrogen
  • ring A when present, can be a C 6-12 aryl.
  • ring A can be phenyl
  • m1 can be 0 or 1.
  • m1 can be 3.
  • n1 can be 0 or 1.
  • p1 and q1 are independently 0 or 1.
  • z1, z2, z3, z4, z5, z6, z7, z8, and z9 can independently be an integer from 0-4. In some embodiments, z1, z2, z3, z4, z5, z6, z7, z8, and z9 can be 0, 1, or 2 independently.
  • B 1 can be any organic compound
  • B 1 can be any organic compound
  • B 1 can be any organic compound
  • L 2 can be -(CH 2 ) j15 -(OCH 2 CH 2 ) 1-4 -(CH 2 ) j16 -, j15 and j16 are as defined in the previous scheme.
  • L2 can be in some embodiments, L2 can be Among them, the left side is connected with O atom, and the right side is connected with B1 .
  • L 2 can be a C 1 -C 12 alkyl chain.
  • L2 can be any organic compound
  • L2 can be In some embodiments, L2 can be In some embodiments, L2 can be In some embodiments, L2 can be In some embodiments, L2 can be Among them, the a3 end is connected to the O atom, and the b3 end is connected to the B1 .
  • L2 can be Among them, the a3 end is connected to the O atom, and the b3 end is connected to the B1 .
  • r1 can be 3, 4, 5 or 6. In some embodiments, r1 can be 3.
  • Q3 can be In some embodiments, Q3 can be Wherein, the definitions of R 13 , R 14 , R 15 and n1 are the same as those described in the previous scheme.
  • R 13 , R 14 , R 15 , p1 and q1 are the same as those described in the previous scheme.
  • R 13 , R 14 , R 15 , p1 and q1 are the same as those described in the previous scheme.
  • Can be The definitions of p1 and q1 are the same as those described in the previous scheme.
  • R 13 , R 14 , n1, p1 and q1 are the same as those described in the previous scheme.
  • R 13 , R 14 , n1, p1 and q1 are the same as those described in the previous scheme.
  • Can be The definitions of n1, p1 and q1 are the same as those described in the previous scheme.
  • the ligand can be any of the following structures or a pharmaceutically acceptable salt thereof,
  • the ligand can be any of the following structures or a pharmaceutically acceptable salt thereof,
  • the ligand can be the following structure or a pharmaceutically acceptable salt thereof,
  • the N-acetyl-galactosamine moiety in the above ligands can be replaced with N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, or N-isobutyrylgalactosamine.
  • the chemical modification represented by the formula (I) is B is selected from guanine, adenine, cytosine and uracil;
  • the ligand is any of the following structures or a pharmaceutically acceptable salt thereof, In some embodiments, The definitions of the sense strand and the antisense strand are the same as those described in the previous scheme.
  • the ligand is any of the following structures or a pharmaceutically acceptable salt thereof,
  • the definitions of the sense strand and the antisense strand are as described in any of the previous schemes.
  • the chemical modification represented by the formula (I) is B is selected from guanine, adenine, cytosine and uracil;
  • the ligand is the following structure or a pharmaceutically acceptable salt thereof,
  • the definitions of the sense strand and the antisense strand are as described in any of the previous schemes.
  • the sense strand and/or antisense strand and the ligand are covalently or non-covalently linked.
  • the 3' end and/or the 5' end of the sense strand is conjugated to the ligand.
  • the 3' end of the sense strand is conjugated to the ligand.
  • the ligand is attached to the end of the sense strand and/or the antisense strand via a phosphate group or a phosphorothioate group.
  • the ligand is attached to the end of the sense strand and/or the antisense strand via a phosphodiester group or a phosphorothioate group.
  • the ligand is attached to the sense and/or antisense strand ends via a phosphodiester group.
  • the ligand is indirectly linked to the end of the sense strand and/or antisense strand via a phosphate group or a phosphorothioate group.
  • the ligand is directly linked to the end of the sense strand and/or antisense strand via a phosphate group or a phosphorothioate group.
  • the ligand is directly attached to the 3' end of the sense strand via a phosphate group or a phosphorothioate group.
  • the phosphate group is a phosphate monoester group or a phosphodiester group. In some embodiments, the phosphate group is a phosphodiester group.
  • the phosphorothioate group is a phosphorothioate monoester group or a phosphorothioate diester group. In some embodiments, the phosphorothioate group is a phosphorothioate diester group.
  • a lipophilic group such as cholesterol can be introduced at the end of the sense strand.
  • the lipophilic group includes binding to small interfering nucleic acid with a covalent bond, such as introducing cholesterol, lipoprotein, vitamin E, etc. at the end, so as to facilitate the interaction with mRNA in the cell through the cell membrane composed of lipid bilayer.
  • dsRNA can also be modified by non-covalent bonds, such as binding phospholipid molecules, polypeptides, and cationic polymers through hydrophobic bonds or ionic bonds to increase stability and biological activity.
  • the number of ligands includes but is not limited to: 1, 2, 3 or 4. In some embodiments, the number of the ligand is 1.
  • 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 nucleotide sequence in SEQ ID NO:5 to SEQ ID NO:14;
  • the dsRNA is any of the following:
  • 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 dsRNA is any of the following:
  • the sense strand is made up of the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand is made up of the nucleotide sequence shown in SEQ ID NO:5;
  • the sense strand is made up of the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand is made up of the nucleotide sequence shown in SEQ ID NO:6;
  • the sense strand is made up of the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand is made up of the nucleotide sequence shown in SEQ ID NO:7;
  • the sense strand is composed of the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand is composed of the nucleotide sequence shown in SEQ ID NO:8;
  • the sense strand is made up of the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand is made up of the nucleotide sequence shown in SEQ ID NO:9;
  • the sense strand is made up of the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand is made up of the nucleotide sequence shown in SEQ ID NO:10;
  • the sense strand is composed of the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand is composed of the nucleotide sequence shown in SEQ ID NO:11;
  • the sense strand is made up of the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand is made up of the nucleotide sequence shown in SEQ ID NO:12;
  • the sense strand is made up of the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand is made up of the nucleotide sequence shown in SEQ ID NO:13;
  • the sense strand is made up of the nucleotide sequence shown in SEQ ID NO:4, and the antisense strand is made up of the nucleotide sequence shown in SEQ ID NO:14.
  • the dsRNA is any of the following:
  • the dsRNA is any of the following:
  • SEQ ID NO:10 is N-(SEQ ID NO:10).
  • SEQ ID NO: 11 is N-(SEQ ID NO: 11
  • SEQ ID NO: 12 is
  • SEQ ID NO: 13 is
  • SEQ ID NO: 14 is
  • s means that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate groups
  • the dsRNA is selected from the following structures or pharmaceutically acceptable salts thereof:
  • the pharmaceutically acceptable salts can be conventional salts in the art, including but not limited to: sodium salts, potassium salts, ammonium salts, amine salts and the like.
  • the dsRNA is selected from any one of TRD008043, TRD008069, TRD008042, TRD008070, TRD008071, TRD008072, TRD008073, TRD008074, TRD008075, TRD008076.
  • the dsRNA is TRD008043, which has the structure
  • the present disclosure provides a pharmaceutical composition comprising the above-mentioned dsRNA.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.
  • Various delivery systems are known and can be used with the dsRNA or pharmaceutical compositions of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, constructed nucleic acid as part of a retrovirus or other vector.
  • the administration of the dsRNA or the pharmaceutical composition of the present disclosure is conventional, and can be administered locally (for example, direct injection or implantation) or systemically, and can also be administered orally, rectally or parenterally.
  • a dsRNA or pharmaceutical composition provided herein can be administered by injection, eg, intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal injection.
  • the dsRNA or pharmaceutical compositions provided by the present disclosure can be packaged in kits.
  • the effective amount or effective dose of the dsRNA or the pharmaceutical composition is about 0.001 mg/kg body weight to about 200 mg/kg body weight, about 0.01 mg/kg body weight to about 100 mg/kg body weight, or about 0.5 mg/kg body weight to about 50 mg/kg body weight.
  • the present disclosure provides an application of the above-mentioned dsRNA or the above-mentioned pharmaceutical composition in the preparation of medicine.
  • the medicament is useful for lowering low density lipoprotein levels in a subject, or for preventing and/or treating diseases mediated by elevated triglyceride levels or elevated cholesterol levels.
  • the disease is selected from hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and/or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, or familial partial lipodystrophy.
  • the medicament can be used to prevent and/or treat diseases associated with APOC3 gene expression related diseases.
  • the disease is selected from hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and/or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, or familial partial lipodystrophy.
  • the present disclosure provides a method for preventing and/or treating a disease, which comprises administering an effective amount or an effective dose of the above-mentioned dsRNA or the above-mentioned pharmaceutical composition to a subject.
  • the disease may be a disease mediated by elevated triglyceride levels or elevated cholesterol levels.
  • the disease is selected from hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and/or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, or familial partial lipodystrophy.
  • the disease may be a disease associated with APOC3 gene expression.
  • the disease is selected from hypertriglyceridemia, obesity, hyperlipidemia, abnormal lipid and/or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, hypertriglyceridemia-induced pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome, or familial partial lipodystrophy.
  • the present disclosure provides a method for silencing APOC3 gene or its mRNA in a cell in vivo or in vitro, which includes the step of introducing the above-mentioned dsRNA or the above-mentioned pharmaceutical composition into the cell.
  • the present disclosure provides a method for inhibiting the expression of APOC3 gene or its mRNA, which comprises administering an effective amount or an effective dose of the above-mentioned dsRNA or the above-mentioned pharmaceutical composition to a subject.
  • the effective amount or effective dose of the dsRNA or the pharmaceutical composition is about 0.001 mg/kg body weight to about 200 mg/kg body weight, about 0.01 mg/kg body weight to about 100 mg/kg body weight, or about 0.5 mg/kg body weight to about 50 mg/kg body weight.
  • the dsRNA or pharmaceutical composition of the present disclosure can reduce the expression level of a target gene or its mRNA in a cell, cell group, tissue or subject, including: administering a therapeutically effective amount of the dsRNA or the pharmaceutical composition described herein to the subject, thereby inhibiting the expression of the target gene or its mRNA in the subject.
  • the subject has previously been identified as having pathological upregulation of the target gene or mRNA thereof in the targeted cell, cell population, tissue or subject.
  • the present disclosure provides a method for delivering oligonucleotides to the liver, which comprises administering to a subject an effective amount or dose of the above-mentioned dsRNA or the above-mentioned pharmaceutical composition.
  • RNA interference agent which comprises the above-mentioned dsRNA or the above-mentioned pharmaceutical composition.
  • the present disclosure also provides a cell comprising the aforementioned dsRNA or the aforementioned pharmaceutical composition.
  • the present disclosure also provides a kit comprising the above dsRNA or the above pharmaceutical composition.
  • the dsRNAs of the present disclosure are selected from siRNAs.
  • the above-mentioned dsRNA or the pharmaceutical composition when the above-mentioned dsRNA or the pharmaceutical composition contacts the cells expressing the target gene, the above-mentioned dsRNA or the pharmaceutical composition can inhibit the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, as determined by, for example, psiCHECK activity screening and luciferase reporter gene detection method, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods, such as immunofluorescence analysis, such as Western Blot or flow cytometry.
  • psiCHECK activity screening and luciferase reporter gene detection method other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods, such as immunofluorescence analysis, such as Western Blot or flow cytometry.
  • 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%.
  • the residual expression percentage of the target gene mRNA caused by the above-mentioned dsRNA or the pharmaceutical composition is not higher than 99%, not higher than 95%, not higher than 90%, not higher than 85%, not higher than 99%, not higher than 95%, not higher than 99%, not higher than 95%, not higher than 99%, not higher than 95%, not higher than 99%, not higher than 95%, not higher than 99%, not higher than 95%, not higher than 99%, not higher than 95%, not higher than 99%, not higher than 95% More than 80%, not more than 75%, not more than 70%, not more than 65%, not more than 60%, not more than 55%, not more than 50%, not more than 45%, not more than 40%, not more than 35%, not more than 30%, not more than 25%, not more than 20%, not more than 15%, or not more than 10%.
  • the dsRNA when the above-mentioned dsRNA or pharmaceutical composition contacts cells expressing the target gene, the dsRNA reduces the off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, while maintaining the target activity, as determined by, for example, psiCHECK activity screening and luciferase reporter gene detection method, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods, such as immunofluorescence analysis, such as Western Blot, or flow cytometry. At least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.
  • the dsRNA when the above-mentioned dsRNA or pharmaceutical composition is contacted with cells expressing the target gene, the dsRNA can reduce the activity of the target by at most 20%, at most 19%, at most 15%, at most 10%, at most 5%, or more than 1%, as determined by, for example, psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods, such as immunofluorescence analysis, such as Western Blot, or flow cytometry. , reducing 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%.
  • psiCHECK activity screening and luciferase reporter gene assay other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods, such
  • the dsRNA increases the activity of the target by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 3% as determined by, for example: psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or based on branched DNA (bDNA), or protein-based methods, such as immunofluorescence analysis, such as Western Blot, or flow cytometry.
  • Compounds of the present disclosure may exist in particular geometric or stereoisomeric forms. This disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of this disclosure. Compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or reagents.
  • Optically active (R)- and (S)-isomers as well as D and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, wherein the resulting mixture of diastereomers is separated and the auxiliary group cleaved to provide the pure desired enantiomer.
  • a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are resolved by conventional methods known in the art, and then the pure enantiomers are recovered.
  • separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally in combination with chemical derivatization (eg, carbamates from amines).
  • the bond Indicates unassigned configuration, i.e. if chiral isomers exist in the chemical structure, the bond can be or both Two configurations.
  • the bond configuration is not specified, i.e. the key The configuration of can be E type or Z type, or contain both E and Z configurations.
  • tautomer or "tautomeric form” refers to structural isomers of different energies that can interconvert via a low energy barrier.
  • proton tautomers also known as prototropic tautomers
  • interconversions via migration of a proton such as keto-enol and imine-enamine, lactam-lactam isomerizations.
  • An example of a lactam-lactim equilibrium is between A and B as shown below.
  • the present disclosure also includes certain isotopically labeled compounds of the disclosure that are identical to those described herein, but wherein one or more atoms are replaced by an atom of an atomic mass or mass number different from that normally found in nature.
  • isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2H , 3H , 11C , 13C , 14C , 13N , 15N, 15O , 17O , 18O, 31P , 32P , 35S , 18F , 123I , 125I , and 36Cl , respectively . wait.
  • deuterium when a position is specifically designated as deuterium (D), the position is understood to have an abundance of deuterium (i.e., at least 10% deuterium incorporation) that is at least 1000 times greater than the natural abundance of deuterium (which is 0.015%).
  • Exemplary compounds having a natural abundance greater than deuterium can 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 also includes compounds of Formula (I), Formula (I'), Formula (II) in various deuterated forms.
  • deuterated forms of formula (I), formula (I '), formula (II) compounds can be synthesized using conventional techniques using deuterated reagents, deuterated reagents include but not limited to deuterated borane, trideuterioborane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide and deuterated methyl iodide, etc.
  • the present disclosure also provides a method for preparing dsRNA or a pharmaceutical composition, which includes: synthesizing the dsRNA or the pharmaceutical composition described in the present disclosure.
  • the "compound”, “chemical modification”, “ligand”, “dsRNA”, “nucleic acid” and “RNAi” of the present disclosure can independently exist in the form of salt, mixed salt or non-salt (such as free acid or free base).
  • a salt or mixed salt it may be a pharmaceutically acceptable salt.
  • “Pharmaceutically acceptable salt” may be selected from inorganic salts or organic salts, and may also include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to a salt formed with an inorganic or organic acid that retains the biological effectiveness of the free base without other side effects.
  • Inorganic acid salts include but not limited to hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.
  • organic acid salts include but not limited to formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, caproate, caprylate, caprate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, Oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-to
  • “Pharmaceutically acceptable base addition salt” refers to a salt formed with an inorganic base or an organic base that can maintain the biological effectiveness of the free acid without other side effects.
  • Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like.
  • Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts, preferably sodium salts.
  • Salts derived from organic bases include, but are not limited to, those of primary, secondary, and tertiary amines, substituted amines, including natural 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, Lucaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resin, etc.
  • Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine,
  • Alkyl refers to a saturated aliphatic hydrocarbon group, such as a straight chain and branched chain group (C 1 -C 30 alkyl) including 1 to 30 carbon atoms, another example is an alkyl group containing 1 to 6 carbon atoms (C 1 -C 6 alkyl), and another example is an alkyl group with 1 to 3 carbon atoms (C 1 -C 3 alkyl).
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl base, 2,2-dimethylpropyl and its various branched isomers, etc.
  • alkenyl refers to a hydrocarbon group containing at least one double bond.
  • alkenyl include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, or 2-butenyl, and various branched isomers thereof.
  • alkynyl refers to a hydrocarbon group containing at least one triple bond.
  • alkynyl include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, or 2-butynyl, and various branched isomers thereof.
  • alkoxy refers to -O-(alkyl), wherein alkyl is as defined above.
  • alkoxy include: methoxy, ethoxy, propoxy, butoxy.
  • Cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring contains 3 to 20 carbon atoms, preferably contains 3 to 6 carbon atoms, more preferably contains 5-6 carbon atoms.
  • monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like; multicyclic cycloalkyls include spiro, fused, and bridged cycloalkyls.
  • Heterocycloalkyl refers to a saturated or partially unsaturated monocyclic or multicyclic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, one or more of which is a heteroatom selected from nitrogen, oxygen, or S(O) m (where m is an integer from 0 to 2), excluding the ring portion of -OO-, -OS- or -SS-, the remaining ring atoms being carbon.
  • it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably it contains 3 to 7 ring atoms.
  • Non-limiting examples of "heterocycloalkyl” include: etc.
  • the heterocycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring bonded to the parent structure is a heterocycloalkyl, non-limiting examples of which include: wait.
  • Aryl means a 6- to 14-membered all-carbon monocyclic or fused polycyclic ring with a conjugated ⁇ -electron system (that is, a shared (ring) groups adjacent to pairs of carbon atoms, preferably 6 to 12 membered, eg phenyl and naphthyl.
  • the aryl ring may be fused to a heteroaryl, heterocycloalkyl or cycloalkyl ring, where the ring bonded to the parent structure is an aryl ring, non-limiting examples of which include:
  • Heteroaryl refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen.
  • the heteroaryl group is preferably 6 to 12 membered, more preferably 5 or 6 membered.
  • Non-limiting examples thereof include: imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, triazolyl, indazolyl, benzimidazolyl, wait.
  • the heteroaryl ring may be fused to an aryl, heterocycloalkyl or cycloalkyl ring, wherein the ring bonded to the parent structure is a heteroaryl ring, non-limiting examples of which include:
  • hydroxyl refers to a -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • cyano refers to -CN.
  • amino refers to -NH2 .
  • nitro refers to -NO2 .
  • the "phosphate group” can be a phosphoric monoester group, a phosphoric diester group or a phosphoric acid triester group, preferably a phosphoric diester group; the "phosphate group” in the “thiophosphoric ester group” also has the same meaning.
  • a phosphorothioate group refers to a phosphorus modified by replacing a non-bridging oxygen atom with a sulfur atom.
  • Acid diester base, available (M is an S atom) are used interchangeably.
  • substitution means that one or more hydrogen atoms in a group, preferably at most 5, more preferably 1 to 3 hydrogen atoms are independently substituted by a corresponding number of substituents.
  • two (2) hydrogens on the atom are replaced.
  • group middle A moiety can be replaced by any group that enables linkage to adjacent nucleotides.
  • connection when referring to a link between two molecules, means that two molecules are connected by a covalent bond or two molecules are associated by a non-covalent bond (eg, a hydrogen bond or an ionic bond), including direct connection, indirect connection.
  • a non-covalent bond eg, a hydrogen bond or an ionic bond
  • directly linked means that a first compound or group is linked to a second compound or group without any intervening atoms or groups of atoms.
  • directly linked means that a first compound or group is linked to a second compound or group through an intervening group, compound or molecule (eg, a linking group).
  • “Pharmaceutical composition” means a mixture containing one or more compounds described herein or a physiologically acceptable salt or prodrug thereof and other chemical components, as well as other components such as physiologically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.
  • “Pharmaceutically acceptable excipients” include, but are not limited to, any adjuvants, carriers, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that have been approved by the U.S. Food and Drug Administration (FDA) to be acceptable for use in humans or livestock animals.
  • FDA U.S. Food and Drug Administration
  • the term “inhibit”, is used interchangeably with “reduce”, “silence”, “downregulate”, “suppress” 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 control levels.
  • the control level can be any type of control level used in the art, such as a pre-dose baseline level or a level determined from a similar untreated or control-treated subject, cell, or sample, such as a buffer-only control or an inert control.
  • the remaining expression of mRNA can be used to characterize the degree of inhibition of dsRNA on target gene expression, such as the remaining expression of mRNA is not higher than 99%, not higher than 95%, not higher than 90%, not higher than 85%, not higher Not higher than 80%, not higher than 75%, not higher than 70%, not higher than 65%, not higher than 60%, not higher than 55%, not higher than 50%, not higher than 45%, not higher than 40%, not higher than 35%, not higher than 30%, not higher than 25%, not higher than 20%, not higher than 15%, or not higher than 10%.
  • an “effective amount” or “effective dose” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • Effective amounts for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the method, route and dosage of administration, and the severity of side effects.
  • An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • subject As used herein, “subject”, “patient”, “subject” or “individual” are used interchangeably and include humans or non-human animals such as mammals such as humans or monkeys.
  • the sense strand also known as SS, SS strand or sense strand
  • the antisense strand also known as AS or AS strand
  • the "5' region” of the sense strand or the antisense strand can be used interchangeably.
  • the 2nd to 8th nucleotides in the 5' region of the antisense strand can also be replaced with the 2nd to 8th nucleotides at the 5' end of the antisense strand.
  • the "3' region”, “3' end” and “3' end” of the sense strand or the antisense strand can also be used interchangeably.
  • the term "the nucleotide sequence of SEQ ID NO: 1 differs by at least 15 consecutive nucleotides of no more than 3 nucleotide sequences" is intended to mean that the sense strand of dsRNA described herein comprises at least 15 consecutive nucleotides of the sense strand as shown in SEQ ID NO: 1, or differs from at least 15 consecutive nucleotides of the sense strand of SEQ ID NO: 1 by no more than 3 nucleotide sequences, optionally, by no more than 2 nucleotide sequences, optionally , differ by 1 nucleotide sequence.
  • the dsRNA sense strand described herein comprises at least 16 consecutive nucleotides of the sense strand shown in SEQ ID NO: 1, or differs from at least 16 consecutive nucleotides of the sense strand shown in SEQ ID NO: 1 by no more than 3 nucleotide sequences, optionally, differs by no more than 2 nucleotide sequences, optionally, differs by 1 nucleotide sequence;
  • the term "differing from the antisense strand set forth in SEQ ID NO:2 by at least 15 consecutive nucleotides of no more than 3 nucleotide sequences" is intended to mean at least 15 consecutive nucleotides of the antisense strand set forth in SEQ ID NO:2 described herein, or differing from at least 15 consecutive nucleotides of the antisense strand set forth in SEQ ID NO:2 by no more than 3 nucleotide sequences, optionally by no more than 2 nucleotide sequences, optionally by 1 Nucleotide sequence.
  • G", “C”, “A”, “T” and “U” respectively represent nucleotides, which respectively contain bases of guanine, cytosine, adenine, thymidine and uracil.
  • lowercase m table Indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; the lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate group.
  • 2'-fluoro (2'-F) modified nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribose group of the nucleotide with fluorine
  • non-fluorinated modified nucleotide refers to a nucleotide or nucleotide analogue formed by replacing the hydroxyl group at the 2' position of the ribosyl group of the nucleotide with a non-fluorine group.
  • 2'-methoxy (2'-OMe) modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is substituted with a methoxy group.
  • nucleotide difference between a nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position has changed between the former and the latter. For example, when a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G or T, it is determined that there is a nucleotide difference at this position between the two nucleotide sequences. In some embodiments, when the nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, it can also be considered that a nucleotide difference occurs at that position.
  • the terms "complementary” or “reverse complementary” are used interchangeably and have the meaning known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, bases on one strand pair with bases on the other strand in a complementary manner.
  • the purine base adenine always pairs with the pyrimidine base thymine (or, in RNA, uracil); the purine base guanine always pairs with the pyrimidine base cytosine.
  • Each base pair consists of a purine and a pyrimidine.
  • mismatch in the art means that in a double-stranded nucleic acid, the bases at the corresponding positions are not paired in a complementary form.
  • dsRNA refers to a double-stranded RNA molecule capable of RNA interference, comprising a sense strand and an antisense strand.
  • chemical modification includes all alterations of nucleotides by chemical means, such as the addition or removal of chemical moieties, or the substitution of one chemical moiety for another.
  • base includes any known DNA and RNA base, base analogues such as purine or pyrimidine, which also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogues. Base analogs can also be universal bases.
  • blunt end or blunt end are used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of a dsRNA, ie, no nucleotide overhangs. In most cases, a dsRNA with both blunt-ended ends will be double-stranded throughout its entire length.
  • the dsRNA provided in the present disclosure can be obtained by conventional preparation methods in the art (such as methods of solid-phase synthesis and liquid-phase synthesis). Among them, solid-phase synthesis has commercialized customized services.
  • Modified nucleotide groups can be introduced into the dsRNAs of the present disclosure by using correspondingly modified nucleoside monomers, and methods of preparing nucleoside monomers with corresponding modifications and methods of introducing modified nucleotide groups into dsRNA are also in the art well known to the skilled person.
  • Figure 1 shows the expression levels of TTR mRNA of TRD002218 and TRD007205 on the 7th day after administration.
  • Figure 2 shows the expression levels of TTR mRNA on day 28 after administration of TRD002218 and TRD007205.
  • Embodiment 1 Preparation of chemical modification
  • Racemate compound 6 was passed through a chiral column (Daicel IE 250*4.6mm, 5 ⁇ m, A: n-hexane, B: ethanol) resolved to obtain 410mg 6A(-) and 435mg 6B(+).
  • compound 5 (6.8 g, 18.581 mmol) was dissolved in pyridine (80 mL), and TMSCl (14.250 mL, 111.489 mmol) was slowly added 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 the reaction was complete.
  • reaction solution was extracted with ethyl acetate (200mL) and water (200mL), the organic phase was dried and spin-dried, and the mixed sample was purified by forward column (PE:EtOAc column, peak at 84%) to obtain yellow oily compound 7 (12g).
  • hmpNA from nucleotides synthesized from 2-hydroxymethyl-1,3-propanediol as the starting material
  • (+)hmpNA(A) is obtained by solid-phase synthesis of the nucleoside phosphoramidite monomer 1-1b in Example 1.1, and the absolute configuration is (S)-hmpNA(A);
  • (-)hmpNA(A) is obtained by solid-phase synthesis of nucleoside phosphoramidite monomer 1-1a in Example 1.1, and its absolute configuration is (R)-hmpNA(A);
  • (+)hmpNA(G) the absolute configuration is (S)-hmpNA(G);
  • (+)hmpNA(C) the absolute configuration is (S)-hmpNA(C);
  • (+)hmpNA(U) the absolute configuration is (R)-hmpNA(U);
  • TJ-NA067 The detection crystal is a colorless block (0.30 ⁇ 0.10 ⁇ 0.04mm3), which belongs to the monoclinic crystal system P21 space group.
  • the detection crystal is a colorless block (0.30 ⁇ 0.20 ⁇ 0.10mm3), belonging to the monoclinic crystal system P21 space group.
  • TJ-NA048 The detected crystal is colorless needle-shaped (0.30 ⁇ 0.04 ⁇ 0.04mm3), belonging to the monoclinic P1 space group.
  • Embodiment 3 preparation NAG0052, L96
  • the starting material Compound 1 was purchased from Jiangsu Beida Pharmaceutical Technology Co., Ltd.
  • the synthetic route of compound NAG0052 is as follows:
  • TMSCN (13.5 mL, 101 mmol) was added in one portion to a solution of compound 2 (13.0 g, 33.6 mmol) in DCM (300 mL), followed by dropwise addition of a solution of TMSOTf (9.14 mL, 50.5 mmol) in DCM (30 mL).
  • the reaction solution was stirred at 20°C for 15 hours. After the reaction was completed, the system was quenched with saturated NaHCO aqueous solution (80mL), and extracted with DCM (150mL x 2).
  • the compound NAG0024 (271mg, 0.151mmol) was dissolved in anhydrous THF (2mL) and anhydrous DMF (4mL), and 3A molecular sieves were added, followed by compound 12 (100mg, 0.151mmol), HOBt (25mg, 0.181mmol), DCC (38mg, 0.181mmol) and DIEA (39mg, 0.30mmol) 2 mmol).
  • the reaction solution was reacted at 45°C for 16h. After LC-MS showed that the reaction was complete, it was quenched with water and filtered. After the filtrate was concentrated, it was purified by C18 reverse phase column (H 2 O/MeCN) to obtain compound 13 (210 mg, yield 57%).
  • the compound NAG0052 (157mg, 0.062mmol) containing a carboxylic acid group was dissolved in anhydrous DMF (3mL). After the substrate was completely dissolved, anhydrous acetonitrile (4mL), DIEA (0.03mL, 0.154mmol, 2.5eq) and HBTU (35mg, 0.093mmol, 1.5eq) were added successively. After the reaction solution was mixed evenly, macroporous amine methyl resin (476mg, blank load was 0.41mmol/g, target load was 0.1mmol/g) was 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 successively with DCM and anhydrous acetonitrile, and the solid was collected and dried overnight in vacuum.
  • the above solid 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 sequentially.
  • the reaction solution was placed on a shaker (temperature: 25° C., rotation speed: 200 rpm) and shaken for 2 h.
  • the reaction liquid was filtered, and the filter cake was washed with anhydrous acetonitrile, and the solid was collected and vacuum-dried overnight to obtain a resin with a carrier.
  • the loading capacity was determined to be 0.1 mmol/g.
  • NAG0052 For the NAG0052 that has been connected to the resin, use the resin as a starting point, and connect the nucleoside monomers one by one from the 3'-5' direction according to the sequence of nucleotide arrangement. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The operation is conventional in the art.
  • the compound NAG0052 is connected to the sequence through solid-phase synthesis, and after aminolysis, the structure of NAG0052 loses some functional groups to become NAG0052'.
  • the prepared dsRNA had the sense and antisense strands shown in Table 1 and Table 2.
  • uppercase letters G, A, C, and U represent nucleotides containing guanine, adenine, cytosine, and uracil, respectively, lowercase letter m represents 2'-methoxy modification, lowercase letter f represents 2'-fluoro modification, and lowercase letter s represents a phosphorothioate group connection between two nucleotides adjacent to the left and right of the letter s; the same below.
  • Example 5 Inhibition of dsRNA on target gene mRNA expression in vivo
  • mice Male C57BL/6 mice aged 6-8 weeks were randomly divided into 6 groups, 3 at each time point, and TRD007205, reference positive TRD002218 and PBS were given to each group of mice respectively.
  • mice All animals were dosed according to their total body weight, and were administered once by subcutaneous injection.
  • the dose of dsRNA (based on the amount of ligand-free nucleotide) was 1 mg/kg, and the volume of administration was 5 mL/kg.
  • the mice were sacrificed, the liver was collected, and stored with RNA later (Sigma Aldrich); then the liver tissue was homogenized with a tissue homogenizer, and then the total RNA of the liver tissue was extracted with a tissue RNA extraction kit (Fanzhi Medical Technology, FG0412) according to the operation steps marked in the operation manual.
  • tissue RNA extraction kit Fanzhi Medical Technology, FG0412
  • the total RNA was reverse-transcribed into cDNA, and the expression of TTR mRNA (transthyretin mRNA) in liver tissue was detected by real-time fluorescent quantitative PCR method.
  • TTR mRNA transthyretin mRNA
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • Taqman probe primers for TTR and GAPDH were used to detect the mRNA expression levels of TTR and GAPDH, respectively.
  • TTR mRNA expression was calculated according to the following equation:
  • TTR mRNA expression [(TTR mRNA expression of test group/GAPDH mRNA expression of test group)/(TTR mRNA expression of control group/GAPDH mRNA expression of control group)] ⁇ 100%.
  • each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization.
  • the prepared dsRNA had the sense and antisense strands shown in Table 5 and Table 6.
  • NAG0052' The structure of NAG0052' is:
  • A(GNA) is:
  • L10 The structure of L10 is:
  • the L96 structure is a
  • Example 7 Inhibition of human APOC3 in primary human hepatocytes (PHH) - 7 concentration points inhibit activity
  • dsRNA was screened for activity in primary human hepatocytes (PHH) using 7 concentration gradients. Each dsRNA sample was transfected with a concentration of 10 nM, 5-fold serial dilution and 7 concentration point.
  • PHH was frozen and stored in liquid nitrogen. 24 hours before transfection, primary human hepatocytes (PHH) were resuscitated and inoculated in 96-well plates at a seeding density of 3 ⁇ 10 4 cells per well and 80 ⁇ L of medium per well.
  • PHH primary human hepatocytes
  • Lipofectamine RNAi MAX ThermoFisher, 13778150
  • the gradient final concentration of dsRNA transfection is 10nM, 2nM, 0.4nM, 0.08nM, 0.016nM, 0.0032nM and 0.00064nM.
  • the high-throughput cell RNA extraction kit was used to extract total cellular RNA, RNA reverse transcription experiment and quantitative real-time PCR detection to measure the mRNA level of human APOC3, and correct the mRNA level of human APOC3 according to the level of GAPDH internal reference gene.
  • Inhibition rate (%) (1-remaining amount of target gene expression)*100%.
  • Results are expressed as percent remaining human APOC3 mRNA expression relative to control dsRNA-treated cells. See Table 9 for the IC50 results of the inhibition rate. The experimental results in Table 9 show that TRD008043 is superior to the rest of the groups.
  • TRD007972-1 has a high level of on-target inhibitory activity against the APOC3 gene in PHH cells, which is superior to TJR100385 and TJR100386.
  • Example 8 psiCHECK antisense strand (AS strand) off-target level verification of dsRNA
  • the plasmid contains Renilla luciferase gene and Firefly luciferase gene.
  • the target sequence of dsRNA is inserted into the 3'UTR region of the Renilla luciferase gene, and the activity of dsRNA on the target sequence can be reflected by the detection of the expression of Renilla luciferase calibrated by firefly luciferase, and the detection uses Dual-Luciferase Reporter Assay System (Promega, E2940).
  • the GSSM target plasmid construction rules corresponding to the dsRNA sequence are as follows:
  • GSSM off-target plasmid
  • HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO 2 . 24 hours before transfection, HEK293A cells were seeded in a 96-well plate at a seeding density of 8 ⁇ 10 cells per well and 100 ⁇ L of medium per well.
  • cells were co-transfected with dsRNA and corresponding plasmids using Lipofectamine2000 (ThermoFisher, 11668019). Lipofectamine2000 was used in 0.2 ⁇ L per well, and the amount of plasmid transfection was 20 ng per well.
  • a total of 9 concentration points were set for dsRNA, and the final concentration of the highest concentration point was 20nM. 3-fold serial dilution, 20nM, 6.6667nM, 2.2222nM, 0.7407nM, 0.2469nM, 0.0823nM, 0.0274nM, 0.0091nM, 0.0030nM. 24h after transfection, the off-target level was detected by Dual-Luciferase Reporter Assay System (Promega, E2940).
  • Embodiment 9 Pharmacodynamic study of Tg mice
  • At least 40 ⁇ L of plasma samples were collected 14 days prior to dosing for the first baseline measurement of HDL, LDL, TG, TC and ApoC3 in plasma during the acclimatization period.
  • the second baseline was performed 7 days before administration, and at least 40 ⁇ L of plasma samples ( ⁇ 90 ⁇ L of blood) were collected for the second detection of TG, TC, HDL-C and ApoC3 in plasma; 17 animals were selected and divided into 3 groups according to the levels of TG and ApoC3 (apolipoprotein C3).
  • the third baseline was performed on the morning of the day of dosing, and at least 40 ⁇ L of plasma samples ( ⁇ 90 ⁇ L of blood) were collected for TG, TC, HDL-C and ApoC3 detection. This set of data will be used as the 0-point (day 0) baseline.
  • the normal saline group and the test sample were administered once, all were injected subcutaneously, and the injection volume was calculated according to the body weight before administration.
  • mice after APOC3Tg mice were given a single test sample, the percentages of APOC3 in TRD008043 group mouse plasma compared with the normal saline group on the 7th day, the 14th day, the 21st day, the 28th day, the 35th day, the 42nd day and the baseline percentage of 0 days were significantly reduced. Compared with the normal saline group, there was no significant difference in the percentages of APOC3 in the plasma of mice in the TRD007972-1 group on day 35, day 42, and day 0.
  • the percentage of APOC3 in plasma and the baseline percentage on day 0 in the TRD008043 group was about 3 times lower than that of TRD007972-1 on D14-D35 after a single administration.
  • TRD008043 has a better effect on reducing human APOC3 than TRD007972-1.
  • mice were given a single administration of the test substance and the percentage of TG in plasma and 0 day baseline ***p ⁇ 0.001, **p ⁇ 0.01, *p ⁇ 0.05, all p are compared with normal saline group, One-way ANOVA Dunnett's test
  • TRD008043 still maintains the effect of significantly reducing TG in plasma compared with the normal saline group on the 28th day, but there is no significant difference between TRD007972-1 and the normal saline group.
  • the percentage of TG in plasma in the TRD008043 group after a single administration and the baseline percentage on D14-D35 was about 2-4 times lower than that of the TRD007972-1 group.
  • TRD008043 had a better TG-reducing effect than TRD007972-1.
  • TRD008043 had a better effect on reducing TC than TRD007972-1.

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Abstract

提供一种dsRNA、其应用及制备方法。还提供包含该dsRNA的药物组合物、细胞或试剂盒。该dsRNA可以干扰APOC3的表达，预防和/或治疗相关疾病。

Description

一种dsRNA、其应用及制备方法
本公开要求申请日为2022年01月20日的中国专利申请202210064031.8的优先权，本公开引用上述中国专利申请的全文。
技术领域
本公开涉及利用dsRNA降低目的RNA表达以及治疗相关疾病的技术领域，具体地，本公开涉及dsRNA的制备和应用。
背景技术
RNA干扰(RNAi)是一种有效的沉默基因表达的方式。据统计，在人体内的疾病相关蛋白中，大约超过80％的蛋白质不能被目前常规的小分子药物以及生物大分子制剂所靶向，属于不可成药蛋白。利用RNA干扰技术，可以根据编码这些蛋白的mRNA，设计合适的dsRNA，特异性靶向目标mRNA并降解目标mRNA，从而达到抑制相关的蛋白生成。因此dsRNA具有非常重要的药物开发前景。然而要实现体内的治疗目的RNA干扰效应，需要向体内特定的细胞递送。
采用靶向配体缀合，利用靶向配体与细胞膜表面的受体分子结构，从而内吞进入到细胞内，是一种有效的药物递送方式。例如，去唾液酸糖蛋白受体(ASGPR)是肝细胞特异性表达的受体，在肝细胞表面具有高丰度，胞内外转换快速的特点。半乳糖、半乳糖胺、N-乙酰半乳糖胺等单糖和多糖分子对ASGPR有高亲和性。文献报道(10.16476/j.pibb.2015.0028)使用氨基半乳糖分子簇(GalNAc)可以有效递送RNA到肝细胞，GalNAc分子被设计成三价或四价的分子簇可以显著提高单价或二价的GalNAc分子靶向肝细胞的能力。
不同分子簇结构，和与RNA之间不同的连接方式会明显的影响dsRNA在体内的活性，更高的活性意味着更好的治疗效果，或更低的给药剂量，在同等药效下，更低的给药剂量也意味着更低的毒性反应。
APOC3主要在肝脏中合成，并在富含甘油三酯的脂蛋白的产生、代谢以及从血浆的清除中发挥重要作用。肝脏中APOC3的表达可以促进富含甘油三酯的极低密度脂蛋白(VLDL)的分泌。另外，APOC3还可以通过抑制脂蛋白脂肪酶和肝脂肪酶的活性，来抑制富含甘油三酯的脂蛋白的分解代谢，进一步提高血清甘油三酯水平。此外，APOC3也可以通过干扰富含甘油三酯的脂蛋白和与肝受体的结合来抑制富含甘油三酯的脂蛋白和其残余颗粒的肝清除。
升高的APOC3水平与升高的甘油三酯水平和诸如心血管疾病、代谢综合征、肥胖和糖尿病之类的疾病相关联。近年来，APOC3已成为用于治疗与高甘油三酯血症相关的疾病的有前景的靶标。升高的血清甘油三酯水平被鉴定为心血管疾病的独立风险因素和发展为动脉粥样硬化的促成因素。具有重度高甘油三酯血症的 个体也具有发展为复发性胰腺炎的高风险。
发明内容
第一方面，本公开提供了一种双链核糖核酸(dsRNA)，其包含有义链和反义链，所述有义链和反义链按照5’端到3’端的方向包含连续的核苷酸，其中所述有义链第7、8和9位的核苷酸为2’-氟代修饰的核苷酸，第5位的核苷酸独立地为2’-甲氧基修饰的核苷酸或2’-氟代修饰的核苷酸，其余位置的核苷酸为2’-甲氧基修饰的核苷酸；所述反义链第2和14位的核苷酸为2’-氟代修饰的核苷酸，第4、6、8、9、10、12、16和18位的核苷酸独立地为2’-甲氧基或2’-氟代修饰的核苷酸，其余位置的核苷酸为2’-甲氧基修饰的核苷酸；所述反义链中2’-氟代修饰的核苷酸的个数为2-7个；
所述反义链第2位至第8位中的至少一个核苷酸位置处包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐：
其中：Y选自O、NH和S；
每个X独立地选自CR4(R4’)、S、NR5和NH-CO，其中R4、R4’、R5分别独立地为H或C1-C6烷基；
J2为H或C1-C6烷基；
n＝0、1或2；m＝0、1或2；s＝0或1；
R3选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-烷基氨基和(CH2)pR6；其中R6选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，p＝1、2或3；
Q1为Q2为R2；或者Q1为R2，Q2为
其中：
R1选自H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基和(CH2)qR7；其中R7选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，q＝1、2或3；
J1为H或C1-C6烷基；
R2选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-烷基氨基和(CH2)rR8；其中R8选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，r＝1、2或3；
任选地，R1和R2直接相连成环；
B是碱基；
所述式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰不是
在一些实施方案中(某些基团或特征的定义如下，未定义的基团或特征同其他任一方案所述，以下简称“在一些实施方案中”)，当X为NH-CO时，R1不是H。
在一些实施方案中，以2’-甲氧基修饰替换式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐。
在一些实施方案中，所述反义链第2位至第8位中的至少一个核苷酸为2’-甲氧基修饰的核苷酸。
在一些具体的实施方案中，所述反义链5’端起第2、4、6、10、12、14和16位为2'-氟代修饰的核苷酸。
在一些具体的实施方案中，所述反义链5’端起第2、4、6、10、14和16位为2'-氟代修饰的核苷酸。
在一些具体的实施方案中，所述反义链5’端起第2、4、6、12、14和16位为2'-氟代修饰的核苷酸。
在一些具体的实施方案中，所述反义链5’端起第2、6、10、12、14和16位为2'-氟代修饰的核苷酸。
在一些具体的实施方案中，所述反义链5’端起第2、6、14和16位为2'-氟代修饰的核苷酸。
在一些具体的实施方案中，所述反义链5’端起第2、4、6、14和16位为2'-氟代修饰的核苷酸。
在一些具体的实施方案中，所述反义链5’端起第2、6、10、14和16位为2'-氟代修饰的核苷酸。
在一些具体的实施方案中，所述反义链5’端起第2、6、12、14和16位为2'-氟代修饰的核苷酸。
在一些具体的实施方案中，所述反义链5’端起第6、14和16位为2'-氟代修饰的核苷酸。
在一些具体的实施方案中，所述反义链5’端起第2、14和16位为2'-氟代修饰 的核苷酸。
在一些具体的实施方案中，所述反义链5’端起第2、6和14位为2'-氟代修饰的核苷酸。
在一些实施方案中，式(I)所示的化学修饰选自式(I-1)所示的化学修饰：
其中：Y选自O、NH和S；
每个X独立地选自CR4(R4’)、S、NR5和NH-CO，其中R4、R4’、R5分别独立地为H或C1-C6烷基；
每个J1、J2分别独立地为H或C1-C6烷基；
n＝0、1或2；m＝0、1或2；s＝0或1；
R3选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-烷基氨基和(CH2)pR6；其中R6选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，p＝1、2或3；
R1选自H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基和(CH2)qR7；其中R7选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，q＝1、2或3；
R2选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-烷基氨基和(CH2)rR8；其中R8选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，r＝1、2或3；
任选地，R1和R2直接相连成环；
B如式(I)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基 嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，式(I)所示的化学修饰选自式(I-2)所示的化学修饰：
其中Y选自O、NH和S；
每个X独立地选自CR4(R4’)、S、NR5和NH-CO，其中R4、R4’、R5分别独立地为H或C1-C6烷基；
n＝0、1或2；m＝0、1或2；s＝0或1；
每个J1、J2分别独立地为H或C1-C6烷基；
R3选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-烷基氨基和(CH2)pR6；其中R6选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，p＝1、2或3；
R1选自H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基和(CH2)qR7；其中R7选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，q＝1、2或3；
R2选自H、C1-C6烷基、C1-C6烷氧基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-烷基氨基和(CH2)rR8；其中R8选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基；r＝1、2或3；
任选地，R1和R2直接相连成环；
B如式(I)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基 嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，每个X独立地选自CR4(R4’)、S、NR5和NH-CO，其中R4、R4’、R5分别独立地为H、甲基、乙基、正丙基或异丙基；
n＝0、1或2；m＝0、1或2；s＝0或1；
每个J1、J2分别独立地为H或甲基；
R3选自H、OH、F、Cl、NH2、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、乙烯基、烯丙基、乙炔基、炔丙基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-甲基氨基、-O-乙基氨基和(CH2)pR6；其中R6选自OH、F、Cl、甲氧基、乙氧基、N3、乙烯基、烯丙基、乙炔基和炔丙基，p＝1或2；
R1选自H、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、乙烯基、烯丙基、乙炔基、炔丙基和(CH2)qR7；其中R7选自OH、F、Cl、甲氧基、乙氧基、N3、乙烯基、烯丙基、乙炔基和炔丙基，q＝1或2；
R2选自H、OH、F、Cl、NH2、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、乙烯基、烯丙基、乙炔基、炔丙基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-甲基氨基、-O-乙基氨基和(CH2)rR8；其中R8选自OH、F、Cl、甲氧基、乙氧基、N3、乙烯基、烯丙基、乙炔基和炔丙基，r＝1或2；
任选地，R1和R2直接相连成环；
B如式(I)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，每个X独立地选自CR4(R4’)、S、NR5和NH-CO，其中R4、R4’、R5分别独立地为H、甲基、乙基、正丙基或异丙基；
n＝0、1或2；m＝0、1或2；s＝0或1；
每个J1、J2分别独立地为H或甲基；
R3选自H、OH、F、Cl、NH2、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、乙烯基、烯丙基、乙炔基、炔丙基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-甲基氨基、-O-乙基氨基和(CH2)pR6；其中R6选自OH、F、 Cl、甲氧基、乙氧基、N3、乙烯基、烯丙基、乙炔基和炔丙基，p＝1或2；
R1选自H、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、乙烯基、烯丙基、乙炔基、炔丙基和(CH2)qR7；其中R7选自OH、F、Cl、甲氧基、乙氧基、N3、乙烯基、烯丙基、乙炔基和炔丙基，q＝1或2；
R2选自H、OH、F、Cl、NH2、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、乙烯基、烯丙基、乙炔基、炔丙基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-甲基氨基、-O-乙基氨基和(CH2)rR8；其中R8选自OH、F、Cl、甲氧基、乙氧基、N3、乙烯基、烯丙基、乙炔基和炔丙基，r＝1或2；
任选地，R1和R2直接相连成环；
B如式(I)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O或NH；每个X独立地选自NH-CO、CH2和NH；
n＝0或1；m＝0或1；s＝0或1；
每个J1、J2分别独立地为H；
R1选自H、甲基和CH2OH；
R2选自H、OH、NH2、甲基和CH2OH；
R3选自H、OH、NH2、甲基和CH2OH；
任选地，R1和R2直接相连成环；
B如式(I)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基 嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O或NH；每个X独立地选自NH-CO、CH2和NH；
n＝0或1；m＝0或1；s＝0或1；
每个J1、J2分别独立地为H；
R1选自H、甲基和CH2OH；
R2选自H、甲基和CH2OH；
R3选自H、OH、NH2、甲基和CH2OH；
任选地，R1和R2直接相连成环；
B如式(I)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O或NH；
每个X独立地选自CR4(R4’)、NR5和NH-CO，R4、R4’、R5分别独立地为H或C1-C6烷基；
J2为H或C1-C6烷基；
n＝0或1；m＝0或1；s＝0或1；
R3选自H、OH、NH2、C1-C6烷基、C1-C6烷氧基和(CH2)pR6；R6选自OH、甲氧基和乙氧基，p＝1、2或3；
Q1为Q2为R2；或者Q1为R2，Q2为
R1选自H、OH、C1-C6烷基、C1-C6烷氧基和(CH2)qR7；R7选自OH、甲氧基和乙氧基，q＝1、2或3；
J1为H或C1-C6烷基；
R2选自H、OH、C1-C6烷基、C1-C6烷氧基和(CH2)rR8；R8选自OH、甲氧基和乙氧基，r＝1、2或3；
任选地，R1和R2直接相连成3-6元环；
B是碱基；
所述式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰不是
在一些实施方案中，X独立地选自CR4(R4’)和NH-CO。
在一些实施方案中，X独立地选自CR4(R4’)。
在一些实施方案中，R3选自H、C1-C6烷基和(CH2)pR6。
在一些实施方案中，R3选自H和C1-C6烷基。
在一些实施方案中，R1选自H、C1-C6烷基和(CH2)qR7。
在一些实施方案中，R1选自H和C1-C6烷基。
在一些实施方案中，R2选自H、OH、C1-C6烷基和(CH2)rR8。
在一些实施方案中，R2选自H、C1-C6烷基和(CH2)rR8。
在一些实施方案中，Y为O；
每个X独立地选自CR4(R4’)和NH-CO，R4和R4’分别独立地为H或C1-C6烷基；
J2为H或C1-C6烷基；
R3选自H、C1-C6烷基和(CH2)pR6；R6选自OH，p＝1、2或3；
Q1为Q2为R2；或者Q1为R2，Q2为
R1选自H、C1-C6烷基和(CH2)qR7；R7选自OH，q＝1、2或3；
J1为H或C1-C6烷基；
R2选自H、OH、C1-C6烷基和(CH2)rR8；R8选自OH，r＝1、2或3；
任选地，R1和R2直接相连成5-6元环；
B是碱基。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O；
每个X独立地选自CR4(R4’)，R4和R4’分别独立地为H或C1-C6烷基；
J2为H；
R3选自H和C1-C6烷基；
Q1为Q2为R2；或者Q1为R2，Q2为
R1选自H和C1-C6烷基；
J1为H或C1-C6烷基；
R2选自H、C1-C6烷基和(CH2)rR8；R8选自OH，r＝1、2或3；
任选地，R1和R2直接相连成5-6元环；
B是碱基。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O。
在一些实施方案中，X独立地选自CR4(R4’)、NR5和NH-CO，R4、R4’、R5分别独立地为H、甲基、乙基、正丙基或异丙基。在一些实施方案中，X独立地 选自NH-CO、CH2和NH。在一些实施方案中，X独立地选自NH-CO和CH2。在一些实施方案中，X为CH2。
在一些实施方案中，J2为H或甲基。在一些实施方案中，J2为H。
在一些实施方案中，R3选自H、OH、NH2、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基和(CH2)pR6，R6选自OH、甲氧基和乙氧基，p＝1或2。在一些实施方案中，R3选自H、甲基、乙基、正丙基、异丙基和(CH2)pR6，R6选自OH，p＝1或2。在一些实施方案中，R3选自H和甲基。
在一些实施方案中，R1选自H、OH、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基和(CH2)qR7，R7选自OH，q＝1或2。在一些实施方案中，R1选自H、甲基、乙基、正丙基、异丙基和(CH2)qR7，R7选自OH，q＝1或2。在一些实施方案中，R1选自H和甲基。
在一些实施方案中，R2选自H、OH、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基和(CH2)rR8，R8选自OH，r＝1或2。在一些实施方案中，R2选自H、OH、甲基、乙基、正丙基、异丙基和(CH2)rR8，R8选自OH，r＝1或2。在一些实施方案中，R2选自H、甲基和CH2OH。
在一些实施方案中，R1和R2直接相连成5-6元环。在一些实施方案中，R1和R2直接相连形成3-6元环烷基。在一些实施方案中，R1和R2直接相连形成环戊基或环己基。
在一些实施方案中，所述式(I)所示的化学修饰选自以下任一结构：

其中：B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯中。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述式(I)所示的化学修饰选自以下任一结构：
其中：B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯中。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述式(I)所示的化学修饰选自以下任一结构：
其中：B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯中。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌 呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述式(I)所示的化学修饰选自以下任一结构：
其中：B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯中。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸选自包含式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸，
其中：Y选自O、NH和S；
每个X独立地选自CR4(R4’)、S、NR5和NH-CO，其中R4、R4’、R5分别独立地为H或C1-C6烷基；
J2为H或C1-C6烷基；
n＝0、1或2；m＝0、1或2；s＝0或1；
R3选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-烷基氨基和(CH2)pR6；其中R6选 自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，p＝1、2或3；
Q1’为Q2’为R2；或者Q1’为R2，Q2’为
其中：
R1选自H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基和(CH2)qR7；其中R7选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，q＝1、2或3；
J1为H或C1-C6烷基；
R2选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-烷基氨基和(CH2)rR8；其中R8选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，r＝1、2或3；
任选地，R1和R2直接相连成环；
B是碱基；
M为O或S；
所述式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐不是
在一些实施方案中，当X为NH-CO时，R1不是H。
在一些实施方案中，以2’-甲氧基修饰替换式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐。
在一些实施方案中，所述式(I’)所示的化学修饰选自式(I’-1)所示的化学修饰：
其中：Y选自O、NH和S；
每个X独立地选自CR4(R4’)、S、NR5和NH-CO，其中R4、R4’、R5分别独立地为H或C1-C6烷基；
每个J1、J2分别独立地为H或C1-C6烷基；
n＝0、1或2；m＝0、1或2；s＝0或1；
R3选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-烷基氨基和(CH2)pR6；其中R6选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，p＝1、2或3；
R1选自H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基和(CH2)qR7；其中R7选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，q＝1、2或3；
R2选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-烷基氨基和(CH2)rR8；其中R8选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，r＝1、2或3；
M为O或S；
任选地，R1和R2直接相连成环；
B如式(I’)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，式(I’)所示的化学修饰选自式(I’-2)所示的化学修饰：
其中，Y选自O、NH和S；
每个X独立地选自CR4(R4’)、S、NR5和NH-CO，其中R4、R4’、R5分别独立地为H或C1-C6烷基；
n＝0、1或2；m＝0、1或2；s＝0或1；
每个J1、J2分别独立地为H或C1-C6烷基；
R3选自H、OH、卤素、NH2、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-烷基氨基和(CH2)pR6；其中R6选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，p＝1、2或3；
R1选自H、C1-C6烷基、C1-C6烷氧基、C2-C6烯基、C2-C6炔基和(CH2)qR7；其中R7选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，q＝1、2或3；
R2选自H、C1-C6烷基、C1-C6烷氧基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-烷基氨基和(CH2)rR8；其中R8选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基；r＝1、2或3；
任选地，R1和R2直接相连成环；
M为O或S；
B如式(I’)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，每个X独立地选自CR4(R4’)、S、NR5和NH-CO，其中 R4、R4’、R5分别独立地为H或C1-C3烷基；
n＝0、1或2；m＝0、1或2；s＝0或1；
每个J1、J2分别独立地为H或C1-C3烷基；
R3选自H、OH、卤素、NH2、C1-C3烷基、C1-C3烷氧基、C2-C4烯基、C2-C4炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-烷基氨基和(CH2)pR6；其中R6选自OH、卤素、甲氧基、乙氧基、N3、C2-C6烯基和C2-C6炔基，p＝1、2或3；
R1选自H、C1-C3烷基、C1-C3烷氧基、C2-C4烯基、C2-C4炔基和(CH2)qR7；其中R7选自OH、卤素、甲氧基、乙氧基、N3、C2-C4烯基和C2-C4炔基，q＝1、2或3；
R2选自H、OH、卤素、NH2、C1-C3烷基、C1-C3烷氧基、C2-C4烯基、C2-C4炔基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-烷基氨基和(CH2)rR8；其中R8选自OH、卤素、甲氧基、乙氧基、N3、C2-C4烯基和C2-C4炔基，r＝1、2或3；
任选地，R1和R2直接相连成环；
B如式(I’)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，每个X独立地选自CR4(R4’)、S、NR5和NH-CO，其中R4、R4’、R5分别独立地为H、甲基、乙基、正丙基或异丙基；
n＝0、1或2；m＝0、1或2；s＝0或1；
每个J1、J2分别独立地为H或甲基；
R3选自H、OH、F、Cl、NH2、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、乙烯基、烯丙基、乙炔基、炔丙基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-甲基氨基、-O-乙基氨基和(CH2)pR6；其中R6选自OH、F、Cl、甲氧基、乙氧基、N3、乙烯基、烯丙基、乙炔基和炔丙基，p＝1或2；
R1选自H、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基、乙烯基、烯丙基、乙炔基、炔丙基和(CH2)qR7；其中R7选自OH、F、Cl、甲氧基、乙氧基、N3、乙烯基、烯丙基、乙炔基和炔丙基，q＝1或2；
R2选自H、OH、F、Cl、NH2、甲基、乙基、正丙基、异丙基、甲氧基、乙氧 基、正丙氧基、异丙氧基、乙烯基、烯丙基、乙炔基、炔丙基、S-CH3、NCH3(CH3)、OCH2CH2OCH3、-O-甲基氨基、-O-乙基氨基和(CH2)rR8；其中R8选自OH、F、Cl、甲氧基、乙氧基、N3、乙烯基、烯丙基、乙炔基和炔丙基，r＝1或2；
任选地，R1和R2直接相连成环；
B如式(I’)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在式(I’)、式(I’-1)或式(I’-2)的一些实施方案中，Y为O或NH；每个X独立地选自NH-CO、CH2和NH；
n＝0或1；m＝0或1；s＝0或1；
每个J1、J2分别独立地为H；
R1选自H、甲基和CH2OH；
R2选自H、OH、NH2、甲基和CH2OH；
R3选自H、OH、NH2、甲基和CH2OH；
任选地，R1和R2直接相连成环；
B如式(I’)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在式(I’)、式(I’-1)或式(I’-2)的一些实施方案中，Y为O或NH；每个X独立地选自NH-CO、CH2和NH；
n＝0或1；m＝0或1；s＝0或1；
每个J1、J2分别独立地为H；
R1选自H、甲基和CH2OH；
R2选自H、甲基和CH2OH；
R3选自H、OH、NH2、甲基和CH2OH；
任选地，R1和R2直接相连成环；
B如式(I’)中所定义。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O或NH；
每个X独立地选自CR4(R4’)、NR5和NH-CO，R4、R4’、R5分别独立地为H或C1-C6烷基；
J2为H或C1-C6烷基；
n＝0或1；m＝0或1；s＝0或1；
R3选自H、OH、NH2、C1-C6烷基、C1-C6烷氧基和(CH2)pR6；R6选自OH、甲氧基和乙氧基，p＝1、2或3；
Q1’为Q2’为R2；或者Q1’为R2，Q2’为
R1选自H、OH、C1-C6烷基、C1-C6烷氧基和(CH2)qR7；R7选自OH、甲氧基和乙氧基，q＝1、2或3；
J1为H或C1-C6烷基；
R2选自H、OH、C1-C6烷基、C1-C6烷氧基和(CH2)rR8；R8选自OH、甲氧基和乙氧基，r＝1、2或3；
任选地，R1和R2直接相连成3-6元环；
M为O或S；
B是碱基；
所述式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐不是
在一些实施方案中，X独立地选自CR4(R4’)和NH-CO。
在一些实施方案中，X独立地选自CR4(R4’)。
在一些实施方案中，R3选自H、C1-C6烷基和(CH2)pR6。
在一些实施方案中，R3选自H和C1-C6烷基。
在一些实施方案中，R1选自H、C1-C6烷基和(CH2)qR7。
在一些实施方案中，R1选自H和C1-C6烷基。
在一些实施方案中，R2选自H、OH、C1-C6烷基和(CH2)rR8。
在一些实施方案中，R2选自H、C1-C6烷基和(CH2)rR8。
在一些实施方案中，Y为O；
每个X独立地选自CR4(R4’)和NH-CO，R4和R4’分别独立地为H或C1-C6烷基；
J2为H或C1-C6烷基；
R3选自H、C1-C6烷基和(CH2)pR6；R6选自OH，p＝1、2或3；
Q1’为Q2’为R2；或者Q1’为R2，Q2’为
R1选自H、C1-C6烷基和(CH2)qR7；R7选自OH，q＝1、2或3；
J1为H或C1-C6烷基；
R2选自H、OH、C1-C6烷基和(CH2)rR8；R8选自OH，r＝1、2或3；
任选地，R1和R2直接相连成5-6元环；
M为O或S；
B是碱基。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O；
每个X独立地选自CR4(R4’)，R4和R4’分别独立地为H或C1-C6烷基；
J2为H；
R3选自H和C1-C6烷基；
Q1’为Q2’为R2；或者Q1’为R2，Q2’为
R1选自H和C1-C6烷基；
J1为H或C1-C6烷基；
R2选自H、C1-C6烷基和(CH2)rR8；R8选自OH，r＝1、2或3；
任选地，R1和R2直接相连成5-6元环；
M为O或S；
B是碱基。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，Y为O。
在一些实施方案中，X独立地选自CR4(R4’)、NR5和NH-CO，R4、R4’、R5 分别独立地为H、甲基、乙基、正丙基或异丙基。在一些实施方案中，X独立地选自NH-CO、CH2和NH。在一些实施方案中，X独立地选自NH-CO和CH2。在一些实施方案中，X为CH2。
在一些实施方案中，J2为H或甲基。在一些实施方案中，J2为H。
在一些实施方案中，R3选自H、OH、NH2、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基和(CH2)pR6，R6选自OH、甲氧基和乙氧基，p＝1或2。在一些实施方案中，R3选自H、甲基、乙基、正丙基、异丙基和(CH2)pR6，R6选自OH，p＝1或2。在一些实施方案中，R3选自H和甲基。
在一些实施方案中，R1选自H、OH、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基和(CH2)qR7，R7选自OH，q＝1或2。在一些实施方案中，R1选自H、甲基、乙基、正丙基、异丙基和(CH2)qR7，R7选自OH，q＝1或2。在一些实施方案中，R1选自H和甲基。
在一些实施方案中，R2选自H、OH、甲基、乙基、正丙基、异丙基、甲氧基、乙氧基、正丙氧基、异丙氧基和(CH2)rR8，R8选自OH，r＝1或2。在一些实施方案中，R2选自H、OH、甲基、乙基、正丙基、异丙基和(CH2)rR8，R8选自OH，r＝1或2。在一些实施方案中，R2选自H、甲基和CH2OH。
在一些实施方案中，R1和R2直接相连成5-6元环。在一些实施方案中，R1和R2直接相连形成3-6元环烷基。在一些实施方案中，R1和R2直接相连形成环戊基或环己基。
在一些实施方案中，所述式(I’)所示的化学修饰选自以下任一结构：

其中：M为O或S；
B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述式(I’)所示的化学修饰选自以下任一结构：
其中：M为O或S；
B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述式(I’)所示的化学修饰选自以下任一结构：
其中：M为O或S；
B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述式(I’)所示的化学修饰选自以下任一结构：
以及它们结构中的腺嘌呤被置换为鸟嘌呤、胞嘧啶、尿嘧啶或胸腺嘧啶的那些。
在一些实施方案中，B选自嘌呤碱基、嘧啶碱基、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、异鸟嘌呤、次黄嘌呤、黄嘌呤、C2修饰的嘌呤、N8修饰的嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、N6-烷基腺嘌呤、O6-烷基鸟嘌呤、7-脱氮嘌呤、胞嘧啶、5-甲基胞嘧啶、异胞嘧啶、假胞嘧啶、尿嘧啶、假尿嘧啶、2-硫代尿苷、4-硫代尿苷、C5修饰的嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与所述反义链该位置处核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述式(I)所示的化学修饰为
再一方面，本公开提供了一种dsRNA，其包含有义链和反义链：
在一些实施方案中，所述反义链与靶序列至少部分地反向互补以介导RNA干扰；在一些实施方案中，所述反义链与靶序列之间存在不多于5个、不多于4个、不多于3个、不多于2个、不多于1个错配；在一些实施方案中，所述反义链与 靶序列完全反向互补。
在一些实施方案中，所述有义链与反义链至少部分地反向互补以形成双链区；在一些实施方案中，所述有义链与反义链之间存在不多于5个、不多于4个、不多于3个、不多于2个、不多于1个错配；在一些实施方案中，所述有义链与反义链完全反向互补。
在一些实施方案中，所述有义链和反义链各自独立地具有16至35个、16至34个、17至34个、17至33个、18至33个、18至32个、18至31个、18至30个、18至29个、18至28个、18至27个、18至26个、18至25个、18至24个、18至23个、19至25个、19至24个、或19至23个核苷酸(例如19、20、21、22、23个核苷酸)。
在一些实施方案中，所述有义链和反义链长度相同或不同，所述有义链的长度为19-23个核苷酸，所述反义链的长度为19-26个核苷酸。在一些实施方案中，所述有义链和反义链的长度比可以是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或23/26。在一些实施方案中，所述有义链和反义链的长度比为19/21、21/23或23/25。在一些实施方案中，所述有义链和反义链的长度比为19/21。
在一些实施方案中，所述dsRNA包含一个或两个平端。
在一些实施方案中，所述dsRNA包含具有1至4个未配对核苷酸的突出端，例如1个、2个、3个、4个。
在一些实施方案中，所述dsRNA包含位于所述反义链3’端的突出端。
在一些实施方案中，包含式(I)或式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸位于反义链5’区域的第5位、第6位或第7位。
在一些实施方案中，包含式(I)或式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸位于反义链5’区域的位于第7位。
在一些实施方案中，式(I)或式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰在其5’区域的第5位时，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，式(I)或式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰在其5’区域的第6位时，B选自腺嘌呤、鸟嘌呤、2,6-二氨基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，式(I)或式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰在其5’区域的第7位时，B选自腺嘌呤、鸟嘌呤、2,6-二氨 基嘌呤、6-二甲基氨基嘌呤、2-氨基嘌呤、胞嘧啶、尿嘧啶、胸腺嘧啶、吲哚、5-硝基吲哚和3-硝基吡咯。
在一些实施方案中，B与所述反义链在其5’区域的第5位核苷酸未被修饰时的碱基相同。
在一些实施方案中，B与所述反义链在其5’区域的第6位核苷酸未被修饰时的碱基相同。
在一些实施方案中，B与所述反义链在其5’区域的第7位核苷酸未被修饰时的碱基相同。
在一些实施方案中，所述有义链第5位的核苷酸为2'-甲氧基修饰的核苷酸。
在一些实施方案中，所述反义链第7位的核苷酸为2'-甲氧基修饰的核苷酸。
在一些实施方案中，所述有义链和反义链包含或为如下式所示的核苷酸：
有义链：5’-NaNaNaNaXNaNbNbNbNaNaNaNaNaNaNaNaNaNa-3’；
反义链：5’-Na’Nb’Na’X’Na’X’W’X’X’X’Na’X’Na’Nb’Na’X’Na’X’Na’Na’Na’-3’；
其中，每个X独立地为Na或Nb，每个X’独立地为Na’或Nb’；Na和Na’为2'-甲氧基修饰的核苷酸，Nb和Nb’为2'-氟代修饰的核苷酸；
W’表示2'-甲氧基修饰的核苷酸或包含式(I)或式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸。
在一些实施方案中，W’表示2'-甲氧基修饰的核苷酸。
在一些实施方案中，W’表示包含式(I)或式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸。
在一些实施方案中，W’具有以下的结构：
在一些实施方案中，所述有义链和反义链包含或为如下式所示的核苷酸序列：
有义链：5’-NaNaNaNaNaNaNbNbNbNaNaNaNaNaNaNaNaNaNa-3’；
反义链：5’-Na’Nb’Na’X’Na’X’W’X’X’X’Na’X’Na’Nb’Na’X’Na’X’Na’Na’Na’-3’；
其中，每个X’独立地为Na’或Nb’；Na和Na’为2'-甲氧基修饰的核苷酸，Nb和Nb’为2'-氟代修饰的核苷酸；
W’表示2'-甲氧基修饰的核苷酸或包含式(I)或式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸。
在一些实施方案中，W’表示2'-甲氧基修饰的核苷酸。
在一些实施方案中，W’表示包含式(I)或式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸。
在一些实施方案中，W’具有以下的结构：
在一些实施方案中，所述有义链包含或为如下式所示的核苷酸序列：
5’-NaNaNaNaXNaNbNbNbNaNaNaNaNaNaNaNaNaNa-3’；
其中，X为Na或Nb；Na为2'-甲氧基修饰的核苷酸，Nb为2'-氟代修饰的核苷酸。
在一些实施方案中，所述有义链包含或为如下式所示的核苷酸序列：
5’-NaNaNaNaNaNaNbNbNbNaNaNaNaNaNaNaNaNaNa-3’；
其中，Na为2'-甲氧基修饰的核苷酸，Nb为2'-氟代修饰的核苷酸。
在一些实施方案中，所述反义链包含或为如下式所示的核苷酸序列：
5’-Na’Nb’Na’X’Na’X’W’X’X’X’Na’X’Na’Nb’Na’X’Na’X’Na’Na’Na’-3’；
其中，每个X’独立地为Na’或Nb’；Na’为2'-甲氧基修饰的核苷酸，Nb’为2'-氟代修饰的核苷酸；
W’表示2'-甲氧基修饰的核苷酸或包含式(I)或式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸。
在一些实施方案中，W’表示2'-甲氧基修饰的核苷酸。
在一些实施方案中，W’表示包含式(I)或式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸。
在一些实施方案中，W’具有以下的结构：
在一些实施方案中，式(I)所示的化学修饰选自：
其中：B选自鸟嘌呤、腺嘌呤、胞嘧啶和尿嘧啶；在一些具体的实施方案中，B与所述反义链在其5’区域的第7位核苷酸未被修饰时的碱基相同。
在一些实施方案中，式(I)所示的化学修饰选自：
其中：M为O或S；其中：B选自鸟嘌呤、腺嘌呤、胞嘧啶或尿嘧啶；在一些具体的实施方案中，B与所述反义链在其5’区域的第7位核苷酸未被修饰时的碱基相同。
在一些具体的实施方案中，M为S。一些具体的实施方案中，M为O。
在一些实施方案中，所述反义链包含或为如下式所示的核苷酸序列：
5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’；
5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’；
5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’；
5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’；
5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’；
5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’；
5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’；
5’-Na’Nb’Na’Na’Na’Na’W’Nb’Nb’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’；
5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’；
5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Na’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’；
5’-Na’Nb’Na’Na’Na’Na’W’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’；或，
5’-Na’Nb’Na’Na’Na’Na’W’Na’Na’Na’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’；
其中，Na’为2'-甲氧基修饰的核苷酸，Nb’为2'-氟代修饰的核苷酸；
W’表示2'-甲氧基修饰的核苷酸或包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸。
在一些实施方案中，W’表示2'-甲氧基修饰的核苷酸。
在一些实施方案中，W’表示包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸。
在一些实施方案中，式(I)所示的化学修饰选自：
其中：B选自鸟嘌呤、腺嘌呤、胞嘧啶和尿嘧啶；在一些具体的实施方案中，B与所述反义链在其5’区域的第7位核苷酸未被修饰时的碱基相同。
在一些实施方案中，式(I)所示的化学修饰选自：
其中：M为O或S；其中：B选自鸟嘌呤、腺嘌呤、胞嘧啶或尿嘧啶；在一些具体的实施方案中，B与所述反义链在其5’区域的第7位核苷酸未被修饰时的碱基相同。
在一些实施方案中，W’具有以下的结构：
在一些具体的实施方案中，M为S。一些具体的实施方案中，M为O。
在一些实施方案中，所述有义链和/或反义链中至少一个磷酸酯基为具有修饰基团的磷酸酯基，所述修饰基团使得所述dsRNA在生物样品或环境中具有增加的稳定性；在一些实施方案中，所述具有修饰基团的磷酸酯基为硫代磷酸酯基。在一些实施方案中，所述具有修饰基团的磷酸酯基为硫代磷酸二酯基。
在一些实施方案中，所述硫代磷酸二酯基存在于以下位置中的至少一处：
所述有义链的5'端第1个核苷酸和第2个核苷酸之间；
所述有义链的5'端第2个核苷酸和第3个核苷酸之间；
所述反义链的5'端第1个核苷酸和第2个核苷酸之间；
所述反义链的5'端第2个核苷酸和第3个核苷酸之间；
所述反义链的3'端第1个核苷酸和第2个核苷酸之间；以及
所述反义链的3'端第2个核苷酸和第3个核苷酸之间。
在一些实施方案中，所述有义链和/或反义链中包括多个硫代磷酸二酯基，所述硫代磷酸二酯基存在于：
所述有义链的5'端第1个核苷酸和第2个核苷酸之间；和，
所述有义链的5'端第2个核苷酸和第3个核苷酸之间；和，
所述反义链的5'端第1个核苷酸和第2个核苷酸之间；和，
所述反义链的5'端第2个核苷酸和第3个核苷酸之间；和，
所述反义链的3'端第1个核苷酸和第2个核苷酸之间；和，
所述反义链的3'端第2个核苷酸和第3个核苷酸之间。
在一些实施方案中，所述有义链包含或为如下式所示的核苷酸序列：
5’-NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’，或，
5’-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’，
其中，Nm表示2'-甲氧基修饰的任意核苷酸，例如2'-甲氧基修饰的C、G、U、A；Nf表示2'-氟代修饰的任意核苷酸，例如2'-氟代修饰的C、G、U、A；
小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸二酯基连接；小写字母s在3’端第一个时表示与该字母s左侧相邻的一个核苷酸末端为硫代磷酸二酯基。
在一些实施方案中，所述有义链包含或为如下式所示的核苷酸序列：
5’-NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm-3’，
其中，Nm表示2'-甲氧基修饰的任意核苷酸，例如2'-甲氧基修饰的C、G、U、A；Nf表示2'-氟代修饰的任意核苷酸，例如2'-氟代修饰的C、G、U、A；
小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸二酯基连接；小写字母s在3’端第一个时表示与该字母s左侧相邻的一个核苷酸末端为硫代磷酸二酯基。
在一些实施方案中，所述反义链包含或为如下式所示的核苷酸序列：
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’；或，
5’-Nm’sNf’sNm’Nm’Nm’Nm’W’Nm’Nm’Nm’Nm’Nm’Nm’Nf’Nm’Nm’Nm’Nm’Nm’sNm’sNm’-3’；
其中，Nm’表示2'-甲氧基修饰的任意核苷酸，例如2'-甲氧基修饰的C、G、U、A；Nf’表示2'-氟代修饰的任意核苷酸，例如2'-氟代修饰的C、G、U、A；
小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸二酯基连接，小写字母s在3’端第一个时表示与该字母s左侧相邻的一个核苷酸末端为硫代磷酸二酯基；
W’表示2'-甲氧基修饰的核苷酸或包含式(I)或式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐修饰的核苷酸。
在一些实施方案中，W’表示2'-甲氧基修饰的核苷酸。
在一些实施方案中，W’表示包含式(I)或式(I’)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸。
在一些实施方案中，式(I)或式(I’)所示的化学修饰选自：
其中：B选自鸟嘌呤、腺嘌呤、胞嘧啶和尿嘧啶；在一些实施方案中，B与所述反义链在其5’区域的第7位核苷酸未被修饰时的碱基相同。
一些实施方案中，式(I)或式(I’)所示的化学修饰选自：
其中：M为O或S；其中：B选自鸟嘌呤、腺嘌呤、胞嘧啶或尿嘧啶；在一些具体的实施方案中，B与所述反义链在其5’区域的第7位核苷酸未被修饰时的碱基相同。
在一些实施方案中，M为S。在一些具体的实施方案中，M为O。
在一些实施方案中，所述dsRNA抑制载脂蛋白C3(APOC3)的表达。
在一些实施方案中，所述有义链包含与SEQ ID NO:1的核苷酸序列相差不超过3个核苷酸的至少15个连续核苷酸，和/或，
所述反义链包含与SEQ ID NO:2的核苷酸序列相差不超过3个核苷酸的至少19个连续核苷酸；
在一些实施方案中，所述有义链包含或选自SEQ ID NO:1的核苷酸序列，和/或，所述反义链包含或选自SEQ ID NO:2的核苷酸序列。
在一些实施方案中，所述有义链和反义链为以下任一方案：
有义链包含5’-UAUUCUCAGUGCUCUCCUZb1-3’(SEQ ID NO：21)，反义链包含5’-UAGGAGAGCACUGAGAAUACU-3’(SEQ ID NO:22)；
或者，有义链包含5’-GCACCGUUAAGGACAAGUZb2-3’(SEQ ID NO：23)，反义链包含5’-AACUUGUCCUUAACGGUGCUC-3’(SEQ ID NO:24)；
其中，Zb1为A或G；Zb2为C或U。
在一些实施方案中，所述dsRNA的有义链的核苷酸序列包含或选自SEQ ID NO:3，反义链的核苷酸序列包含或选自SEQ ID NO:5至SEQ ID NO:14中任一的核苷酸序列；
在一些实施方案中，所述dsRNA为以下任一方案：
有义链包含SEQ ID NO:3的核苷酸序列，反义链包含SEQ ID NO:5的核苷酸序列；
有义链包含SEQ ID NO:3的核苷酸序列，反义链包含SEQ ID NO:6的核苷酸序列；
有义链包含SEQ ID NO:3的核苷酸序列，反义链包含SEQ ID NO:7的核苷酸序列；
有义链包含SEQ ID NO:3的核苷酸序列，反义链包含SEQ ID NO:8的核苷酸序列；
有义链包含SEQ ID NO:3的核苷酸序列，反义链包含SEQ ID NO:9的核苷酸序列；
有义链包含SEQ ID NO:3的核苷酸序列，反义链包含SEQ ID NO:10的核苷酸序列；
有义链包含SEQ ID NO:3的核苷酸序列，反义链包含SEQ ID NO:11的核苷酸序列；
有义链包含SEQ ID NO:3的核苷酸序列，反义链包含SEQ ID NO:12的核苷酸序列；
有义链包含SEQ ID NO:3的核苷酸序列，反义链包含SEQ ID NO:13的核苷酸序列；
有义链包含SEQ ID NO:3的核苷酸序列，反义链包含SEQ ID NO:14的核苷酸序列。
在一些实施方案中，所述dsRNA为以下任一方案：
有义链由SEQ ID NO:3所示的核苷酸序列组成，反义链由SEQ ID NO:5所示的核苷酸序列组成；
有义链由SEQ ID NO:3所示的核苷酸序列组成，反义链由SEQ ID NO:6所示的核苷酸序列组成；
有义链由SEQ ID NO:3所示的核苷酸序列组成，反义链由SEQ ID NO:7所示的核苷酸序列组成；
有义链由SEQ ID NO:3所示的核苷酸序列组成，反义链由SEQ ID NO:8所示的核苷酸序列组成；
有义链由SEQ ID NO:3所示的核苷酸序列组成，反义链由SEQ ID NO:9所示的核苷酸序列组成；
有义链由SEQ ID NO:3所示的核苷酸序列组成，反义链由SEQ ID NO:10所示的核苷酸序列组成；
有义链由SEQ ID NO:3所示的核苷酸序列组成，反义链由SEQ ID NO:11所示的核苷酸序列组成；
有义链由SEQ ID NO:3所示的核苷酸序列组成，反义链由SEQ ID NO:12所示的核苷酸序列组成；
有义链由SEQ ID NO:3所示的核苷酸序列组成，反义链由SEQ ID NO:13所示的核苷酸序列组成；
有义链由SEQ ID NO:3所示的核苷酸序列组成，反义链由SEQ ID NO:14所示的核苷酸序列组成。
在一些实施方案中，所述dsRNA为以下任一方案：
包含SEQ ID NO:3所示的有义链和SEQ ID NO:5至SEQ ID NO:14中任一项所示的反义链。
在一些实施方案中，所述dsRNA为以下任一方案：
选自SEQ ID NO:3所示的有义链和SEQ ID NO:5至SEQ ID NO:14中任一项所示的反义链。
本公开中，按照5’-3’方向，
SEQ ID NO:3是
UmsAmsUmUmCmUmCfAfGfUmGmCmUmCmUmCmCmUmAm；
SEQ ID NO:5是
UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAfCmUfGmAfGmAfAmUmAmsCmsUm；
SEQ ID NO:6是
UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAfCmUmGmAfGmAfAmUfAmsCmsUm；
SEQ ID NO:7是
UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAmCmUfGmAfGmAfAmUfAmsCmsUm；
SEQ ID NO:8是
UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAfCmUfGmAfGmAfAmUfAmsCmsUm；
SEQ ID NO:9是
UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAmCmUmGmAfGmAfAmUmAmsCmsUm；
SEQ ID NO:10是
UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAmCmUmGmAfGmAfAmUmAmsCmsUm；
SEQ ID NO:11是
UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAfCmUmGmAfGmAfAmUmAmsCmsUm；
SEQ ID NO:12是
UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAmCmUfGmAfGmAfAmUmAmsCmsUm；
SEQ ID NO:13是
UmsAfsGmGmAmGm(-)hmpNA(A)GfCfAmCmUmGmAfGmAfAmUmAmsCmsUm；
SEQ ID NO:14是
UmsAfsGmGmAmGm(-)hmpNA(A)GmCmAmCmUmGmAfGmAmAmUmAmsCmsUm；
其中，Af＝腺嘌呤2'-F核糖核苷(adenine 2'-F ribonucleoside)；Cf＝胞嘧啶2'-F核糖核苷(cytosine 2'-F ribonucleoside)；Uf＝尿嘧啶2'-F核糖核苷(uracil 2'-F ribonucleoside)；Gf＝鸟嘌呤2'-F核糖核苷(guanine 2'-F ribonucleoside)；Am＝腺嘌呤2'-OMe核糖核苷(adenine 2'-OMe ribonucleoside)；Cm＝胞嘧啶2'-OMe核糖核苷(cytosine 2'-OMe ribonucleoside)；Gm＝鸟嘌呤2'-OMe核糖核苷(guanine 2'-OMe ribonucleoside)；Um＝尿嘧啶2'-OMe核糖核苷(uracil 2'-OMe ribonucleoside)；
s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸二酯基连接；
(-)hmpNA(A)表示
在一些实施方案中，所述dsRNA还包括缀合的配体，所述配体选自靶向肝脏的靶向配体；在一些实施方案中，所述配体结合脱唾液酸糖蛋白受体(ASGPR)；在一些实施方案中，所述配体包括半乳糖簇或半乳糖衍生物簇，所述半乳糖衍生物选自N‐乙酰基‐半乳糖胺、N‐三氟乙酰基半乳糖胺、N‐丙酰基半乳糖胺、N‐正丁酰基半乳糖胺或N‐异丁酰基半乳糖胺、或其组合。
在一些实施方案中，所述dsRNA还包括缀合的配体，所述配体为如式(II)所示化合物或其药学上可接受的盐，
其中，L1为C1-C30烷基链、或包含被一个或多个氧、硫、氮原子或C＝O间断的C1-C30烷基链；
R11和R12独立地为化学键、NR16、C＝O或-OC(＝O)-；
Q3为
为单键或双键，且当为单键时，R13独立地为CR17R18、NR16、O或S，当为双键时，R13独立地为CR19或N；
R14独立地为CR19或N；
环A为存在或不存在的环烷基、杂环烷基、芳基或杂芳基，且当环A存在时，R15独立地为CR19或N，当环A不存在时，R15独立地为CR17R18、NR16或O；
R16和R19独立地为氢、氘、烷基、烷氧基、环烷基、杂环烷基、芳基、杂芳基、SR'、S(＝O)R'、S(＝O)2R'、S(＝O)2NR'(R”)、NR'(R”)、C(＝O)R'、C(＝O)OR'或C(＝O)NR'(R”)，所述烷基、烷氧基、环烷基、杂环烷基、芳基或杂芳基任选被一个或多个选自卤素、羟基、氧代、硝基、氰基、C1-6烷基、C1-6烷氧基、C3-7环烷基、3-12元杂环烷基、6-12元芳基、5-12元杂芳基、SR'、S(＝O)R'、S(＝O)2R'、S(＝O)2NR'(R”)、 NR'(R”)、C(＝O)R'、C(＝O)OR'和C(＝O)NR'(R”)中的基团所取代；
R17和R18独立地为氢、氘、烷基、烷氧基、环烷基、杂环烷基、芳基、杂芳基、SR'、S(＝O)R'、S(＝O)2R'、S(＝O)2NR'(R”)、NR'(R”)、C(＝O)R'、C(＝O)OR'或C(＝O)NR'(R”)，所述烷基、烷氧基、环烷基、杂环烷基、芳基或杂芳基任选被一个或多个选自卤素、羟基、氧代、硝基、氰基、C1-6烷基、C1-6烷氧基、C3-7环烷基、3-12元杂环烷基、6-12元芳基、5-12元杂芳基、SR'、S(＝O)R'、S(＝O)2R'、S(＝O)2NR'(R”)、NR'(R”)、C(＝O)R'、C(＝O)OR'和C(＝O)NR'(R”)中的基团所取代；
R'和R”独立地为氢、氘、羟基、烷基、烷氧基、环烷基、杂环烷基、芳基或杂芳基，所述烷基、烷氧基、环烷基、杂环烷基、芳基或杂芳基任选被一个或多个选自卤素、羟基、氧代、硝基和氰基中的取代基所取代；
m1、n1、p1和q1独立地为0、1、2、3或4；
B1为
Rb1、Rb2、Rb3、Rb4、Rb5、Rb6和Rb7独立地为-C(＝O)-、-NHC(＝O)-、-C(＝O)O-、-C(＝O)-(CH2)z8-O-或-NHC(＝O)-(CH2)z9-O-；
z1、z2、z3、z4、z5、z6、z7、z8和z9独立地为0-10的整数；
L2为C1-C30烷基链、或包含被一个或多个氧、硫、氮原子或C＝O间断的C1-C30烷基链；
r1为1-10的整数。
在一些实施方案中，L1为C1-C30烷基链、或包含被一个或多个氧、硫、氮原子或C＝O间断的C1-C30烷基链；
R11和R12独立地为化学键、NR16或C＝O；
R16为氢或C1-6烷基；
Q3为
R13为CR17R18、NR16、O或S；
R14为CR19；
R15独立地为CR17R18、NR16或O；
R17至R19独立地为氢、氘或烷基；
m1、p1和q1独立地为0、1、2、3或4；
B1为
Rb5、Rb6和Rb7独立地为-C(＝O)-、-NHC(＝O)-、-C(＝O)O-、-C(＝O)-(CH2)z8-O-或-NHC(＝O)-(CH2)z9-O-；
z5、z6、z7、z8和z9独立地为0-10的整数；
L2为C1-C30烷基链、或包含被一个或多个氧、硫、氮原子或C＝O间断的C1-C30烷基链；
r1为1-10的整数。
在一些实施方案中，L1为-(CH2)j11-C(＝O)-(CH2)j12-；
R11和R12独立地为化学键、NR16或C＝O；
R16为氢或C1-6烷基；
Q3为
R13为CR17R18或O；
R14为CR19；
R15独立地为CR17R18或O；
R17至R19独立地为氢或烷基；
m1、p1和q1独立地为0或1；
B1为
Rb5、Rb6和Rb7独立地为-C(＝O)-(CH2)z8-O-或-NHC(＝O)-(CH2)z9-O-；
z8和z9独立地为0-10的整数；
L2为-(CH2)j15-(OCH2CH2)1-4-(CH2)j16-或
j15和j16独立地为0-4的整数；
r1为3、4、5或6。
在一些实施方案中，L1可为L3或L3-R110-R111-L3，其中，L3独立地为C1-C12烷基链、-(CH2)j11-C(＝O)-(CH2)j12-或-(CH2)j13-(CH2CH2O)1-4-(CH2)j14-，R110和R111独立地为化学键、-NR112-、-C(＝O)-或-OC(＝O)-，R112为氢或C1-C12烷基，j11、j12、 j13和j14独立地为0-10的整数。在一些实施方案中，j11、j12、j13和j14独立地为0-2或4-10的整数。在一些实施方案中，j11、j12、j13和j14独立地为0、1、2、6、7、8、9或10。
在一些实施方案中，L1可为-(CH2)j11-C(＝O)-(CH2)j12-，j11和j12的定义同前任一方案所述。
在一些实施方案中，L1可为j12的定义同前任一方案所述，其中，a1端与B1相连，b1端与R11相连。
在一些实施方案中，L1可为 其中，a1端与B1相连，b1端与R11相连。
在一些实施方案中，R11可为化学键且R12可为C＝O。
在一些实施方案中，R11可为化学键且R12可为NR16，R16的定义同前任一方案所述。
在一些实施方案中，R11可为化学键且R12可为-OC(＝O)-。
在一些实施方案中，R11可为NR16且R12可为C＝O，R16的定义同前任一方案所述。
在一些实施方案中，R1可为NR16且R12可为-OC(＝O)-，R16的定义同前任一方案所述。
在一些实施方案中，R12可为NR16且R11可为C＝O，R16的定义同前任一方案所述。
在一些实施方案中，R12可为NR16且R11可为-OC(＝O)-，R16的定义同前任一方案所述。
在一些实施方案中，R11可为NH且R12可为C＝O。
在一些实施方案中，R12可为NH且R11可为C＝O。
在一些实施方案中，R16可为氢或C1-6烷基。
在一些实施方案中，R16可为氢、甲基、乙基、丙基或异丙基。
在一些实施方案中，R16可为氢。
在一些实施方案中，R17和R18可为氢。
在一些实施方案中，R19可为氢。
在一些实施方案中，环A存在时，环A可为C6-12芳基。
在一些实施方案中，环A可为苯基。
在一些实施方案中，m1可为0或1。
在一些实施方案中，m1可为3。
在一些实施方案中，n1可为0或1。
在一些实施方案中，p1和q1独立地为0或1。
在一些实施方案中，p1＝1且q1＝1。
在一些实施方案中，p1＝1且q1＝0。
在一些实施方案中，p1＝0且q1＝1。
在一些实施方案中，p1＝0且q1＝0。
在一些实施方案中，z1、z2、z3、z4、z5、z6、z7、z8和z9可独立地为0-4的整数。在一些实施方案中，z1、z2、z3、z4、z5、z6、z7、z8和z9可独立地为0、1或2。
在一些实施方案中，B1可为Rb1、Rb2、Rb3和Rb4独立地为-C(＝O)-或-NHC(＝O)-，N原子与L1相连，z1、z2、z3和z4的定义同前任一方案所述。
在一些实施方案中，B1可为Rb1、Rb2、Rb3和Rb4独立地为-C(＝O)-或-NHC(＝O)-，N原子与L1相连，Rb1、Rb3和Rb4相同，z1、z2、z3和z4的定义同前任一方案所述。
在一些实施方案中，B1可为
在一些实施方案中，B1可为
在一些实施方案中，B1可为Rb5、Rb6和Rb7独立地为-C(＝O)-(CH2)z8-O-或-NHC(＝O)-(CH2)z9-O-，N原子与L1相连，z5、z6、z7、z8和z9的定义同前任一方案所述。
在一些实施方案中，B1可为Rb5、Rb6和Rb7独立地为-C(＝O)-(CH2)z8-O-或-NHC(＝O)-(CH2)z9-O-，N原子与L1相连，Rb5、Rb6和Rb7相同，z5、z6、z7、z8和z9的定义同前任一方案所述。
在一些实施方案中，B1可为
在一些实施方案中，L2可为L4或L4-R13-R14-L4，其中，L4独立地为C1-C12烷基链或-(CH2)j15-(OCH2CH2)1-4-(CH2)j16-，R13和R14独立地为化学键、-NR115-、-C(＝O)-或-OC(＝O)-，R115独立地为氢或C1-C12烷基，j15和j16独立地为0-10的整数。在一些实施方案中，j15和j16独立地为0-6的整数。在一些实施方案中，j15和j16独立地为0、1、2、3或4。
在一些实施方案中，L2可为-(CH2)j15-(OCH2CH2)1-4-(CH2)j16-，j15和j16的定义同前任一方案所述。
在一些实施方案中，L2可为在一些 实施方案中，L2可为其中，左侧与O原子相连，右侧与B1相连。
在一些实施方案中，L2可为C1-C12烷基链。
在一些实施方案中，L2可为
在一些实施方案中，L2可为在一些实施方案中，L2可为在一些实施方案中，L2可为在一些实施方案中，L2可为其中，a3端与O原子相连，b3端与B1相连。
在一些实施方案中，L2可为其中，a3端与O原子相连，b3端与B1相连。
在一些实施方案中，r1可为3、4、5或6。在一些实施方案中，r1可为3。
在一些实施方案中，Q3可为在一些实施方案中，Q3可为其中，R13、R14、R15和n1的定义同前任一方案所述。
在一些实施方案中，可为其中，R13、R14、R15、p1和q1的定义同前任一方案所述。
在一些实施方案中，可为 其中，R13、R14、R15、p1和q1的定义同前任一方案所述。
在一些实施方案中，可为在一些实施方案中，可为在一些实施方案中，可为p1和q1的定义同前任一方案所述。
在一些实施方案中，可为 在一些实施方案中，可为
在一些实施方案中，可为 p1和q1的定义同前任一方案所述。
在一些实施方案中，可为其中，R13、R14、n1、p1和q1的定义同前任一方案所述。
在一些实施方案中，可为其中，R13、R14、n1、p1和q1的定义同前任一方案所述。
在一些实施方案中，可为在一些实施方案中，可为n1、p1和q1的定义同前任一方案所 述。
在一些实施方案中，可为n1、p1和q1的定义同前任一方案所述。
在一些实施方案中，所述的配体可为以下任一结构或其药学上可接受的盐，




在一些实施方案中，所述的配体可为以下任一结构或其药学上可接受的盐，















在一些实施方案中，所述的配体可为以下结构或其药学上可接受的盐，
在一些实施方案中，可以N-三氟乙酰基半乳糖胺、N-丙酰基半乳糖胺、N-正丁酰基半乳糖胺或N-异丁酰基半乳糖胺替换以上配体中的N-乙酰基-半乳糖胺部分。
在一些实施方案中，所述式(I)所示的化学修饰为 B选自鸟嘌呤、腺嘌呤、胞嘧啶和尿嘧啶；
所述配体为如下任一结构或其药学上可接受的盐，


在一些实施方案中，
所述有义链和反义链的定义同前任一方案所述。
在一些实施方案中，所述配体为如下任一结构或其药学上可接受的盐，

在一些实施方案中，所述有义链和反义链的定义同前任一方案所述。
在一些实施方案中，所述式(I)所示的化学修饰为 B选自鸟嘌呤、腺嘌呤、胞嘧啶和尿嘧啶；
所述配体为如下结构或其药学上可接受的盐，
在一些实施方案中，所述有义链和反义链的定义同前任一方案所述。
在一些实施方案中，所述有义链和/或反义链和所述配体共价或非共价连接。
在一些实施方案中，所述有义链的3’端和/或5’端与所述配体缀合。
在一些实施方案中，所述有义链的3’端与所述配体缀合。
在一些实施方案中，所述配体通过磷酸酯基团或硫代磷酸酯基团与所述有义链和/或反义链末端连接。
在一些实施方案中，所述配体通过磷酸二酯基团或硫代磷酸二酯基团与所述有义链和/或反义链末端连接。
在一些实施方案中，所述配体通过磷酸二酯基团与所述有义链和/或反义链末端连接。
在一些实施方案中，所述配体通过磷酸酯基团或硫代磷酸酯基团与所述有义链和/或反义链末端间接连接。
在一些实施方案中，所述配体通过磷酸酯基团或硫代磷酸酯基团与所述有义链和/或反义链末端直接连接。
在一些实施方案中，所述配体通过磷酸酯基团或硫代磷酸酯基团与所述有义链3’末端直接连接。
在一些实施方案中，所述磷酸酯基团为磷酸一酯基团或磷酸二酯基团。在一些实施方案中，所述磷酸酯基团为磷酸二酯基团。
在一些实施方案中，所述硫代磷酸酯基团为硫代磷酸一酯基团或硫代磷酸二酯基团。在一些实施方案中，所述硫代磷酸酯基团为硫代磷酸二酯基团。
在一些实施方案中，为了促进dsRNA进入细胞，可以在有义链的末端引入胆固醇等亲脂性的基团，亲脂性的基团包括以共价键与小干扰核酸结合，如末端引入胆固醇、脂蛋白、维生素E等，以利于通过由脂质双分子层构成的细胞膜与细胞内的mRNA发生作用。同时，dsRNA也可以进行非共价键修饰，如通过疏水键或离子键结合磷脂分子、多肽、阳离子聚合物等增加稳定性和生物学活性。
在一些实施方案中，所述配体的个数包括但不限于：1、2、3或4个。在一些实施方案中，所述配体的个数为1个。
在一些实施方案中，所述dsRNA的有义链的核苷酸序列包含或选自SEQ ID NO:4，反义链的核苷酸序列包含或选自SEQ ID NO:5至SEQ ID NO:14中任一的核苷酸序列；
在一些实施方案中，所述dsRNA为以下任一方案：
有义链包含SEQ ID NO:4的核苷酸序列，反义链包含SEQ ID NO:5的核苷酸序列；
有义链包含SEQ ID NO:4的核苷酸序列，反义链包含SEQ ID NO:6的核苷酸序列；
有义链包含SEQ ID NO:4的核苷酸序列，反义链包含SEQ ID NO:7的核苷酸序列；
有义链包含SEQ ID NO:4的核苷酸序列，反义链包含SEQ ID NO:8的核苷酸序列；
有义链包含SEQ ID NO:4的核苷酸序列，反义链包含SEQ ID NO:9的核苷酸序列；
有义链包含SEQ ID NO:4的核苷酸序列，反义链包含SEQ ID NO:10的核苷酸序列；
有义链包含SEQ ID NO:4的核苷酸序列，反义链包含SEQ ID NO:11的核苷酸序列；
有义链包含SEQ ID NO:4的核苷酸序列，反义链包含SEQ ID NO:12的核苷酸序列；
有义链包含SEQ ID NO:4的核苷酸序列，反义链包含SEQ ID NO:13的核苷酸序列；
有义链包含SEQ ID NO:4的核苷酸序列，反义链包含SEQ ID NO:14的核苷酸序列。
在一些实施方案中，所述dsRNA为以下任一方案：
有义链由SEQ ID NO:4所示的核苷酸序列组成，反义链由SEQ ID NO:5所示的核苷酸序列组成；
有义链由SEQ ID NO:4所示的核苷酸序列组成，反义链由SEQ ID NO:6所示的核苷酸序列组成；
有义链由SEQ ID NO:4所示的核苷酸序列组成，反义链由SEQ ID NO:7所示的核苷酸序列组成；
有义链由SEQ ID NO:4所示的核苷酸序列组成，反义链由SEQ ID NO:8所示的核苷酸序列组成；
有义链由SEQ ID NO:4所示的核苷酸序列组成，反义链由SEQ ID NO:9所示的核苷酸序列组成；
有义链由SEQ ID NO:4所示的核苷酸序列组成，反义链由SEQ ID NO:10所示的核苷酸序列组成；
有义链由SEQ ID NO:4所示的核苷酸序列组成，反义链由SEQ ID NO:11所示的核苷酸序列组成；
有义链由SEQ ID NO:4所示的核苷酸序列组成，反义链由SEQ ID NO:12所示的核苷酸序列组成；
有义链由SEQ ID NO:4所示的核苷酸序列组成，反义链由SEQ ID NO:13所示的核苷酸序列组成；
有义链由SEQ ID NO:4所示的核苷酸序列组成，反义链由SEQ ID NO:14所示的核苷酸序列组成。
在一些实施方案中，所述dsRNA为以下任一方案：
包含SEQ ID NO:4所示的有义链和SEQ ID NO:5至SEQ ID NO:14中任一项所示的反义链。
在一些实施方案中，所述dsRNA为以下任一方案：
选自SEQ ID NO:4所示的有义链和SEQ ID NO:5至SEQ ID NO:14中任一项所示的反义链。
本公开中，按照5’-3’方向，
SEQ ID NO:4是
UmsAmsUmUmCmUmCfAfGfUmGmCmUmCmUmCmCmUmAm-NAG0052’；
SEQ ID NO:5是
UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAfCmUfGmAfGmAfAmUmAmsCmsUm；
SEQ ID NO:6是
UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAfCmUmGmAfGmAfAmUfAmsCmsUm；
SEQ ID NO:7是
UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAmCmUfGmAfGmAfAmUfAmsCmsUm；
SEQ ID NO:8是
UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAfCmUfGmAfGmAfAmUfAmsCmsUm；
SEQ ID NO:9是
UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAmCmUmGmAfGmAfAmUmAmsCmsUm；
SEQ ID NO:10是
UmsAfsGmGfAmGf(-)hmpNA(A)GmCmAmCmUmGmAfGmAfAmUmAmsCmsUm；
SEQ ID NO:11是
UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAfCmUmGmAfGmAfAmUmAmsCmsUm；
SEQ ID NO:12是
UmsAfsGmGmAmGf(-)hmpNA(A)GmCmAmCmUfGmAfGmAfAmUmAmsCmsUm；
SEQ ID NO:13是
UmsAfsGmGmAmGm(-)hmpNA(A)GfCfAmCmUmGmAfGmAfAmUmAmsCmsUm；
SEQ ID NO:14是
UmsAfsGmGmAmGm(-)hmpNA(A)GmCmAmCmUmGmAfGmAmAmUmAmsCmsUm；
其中，Af＝腺嘌呤2'-F核糖核苷(adenine 2'-F ribonucleoside)；Cf＝胞嘧啶2'-F核糖核苷(cytosine 2'-F ribonucleoside)；Uf＝尿嘧啶2'-F核糖核苷(uracil 2'-F ribonucleoside)；Gf＝鸟嘌呤2'-F核糖核苷(guanine 2'-F ribonucleoside)；Am＝腺嘌呤2'-OMe核糖核苷(adenine 2'-OMe ribonucleoside)；Cm＝胞嘧啶2'-OMe核糖核苷(cytosine 2'-OMe ribonucleoside)；Gm＝鸟嘌呤2'-OMe核糖核苷(guanine 2'-OMe ribonucleoside)；Um＝尿嘧啶2'-OMe核糖核苷(uracil 2'-OMe ribonucleoside)；
s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸二酯基连接；
NAG0052’表示
(-)hmpNA(A)表示
在一些实施方案中，所述dsRNA选自如下结构或其药学上可接受的盐：
其中，Af＝腺嘌呤2'-F核糖核苷(adenine 2'-F ribonucleoside)；Cf＝胞嘧啶2'-F核糖核苷(cytosine 2'-F ribonucleoside)；Uf＝尿嘧啶2'-F核糖核苷(uracil 2'-F ribonucleoside)；Am＝腺嘌呤2'-OMe核糖核苷(adenine 2'-OMe ribonucleoside)；Cm＝胞嘧啶2'-OMe核糖核苷(cytosine 2'-OMe ribonucleoside)；Gf＝鸟嘌呤2'-F核糖核苷(guanine 2'-F ribonucleoside)；Gm＝鸟嘌呤2'-OMe核糖核苷(guanine 2'-OMe ribonucleoside)；Um＝尿嘧啶2'-OMe核糖核苷(uracil 2'-OMe ribonucleoside)。表示硫代磷酸二酯基，表示磷酸二酯基，
NAG0052’表示
(-)hmpNA(A)表示
在一些实施方案中，所述药学上可接受的盐可为本领域常规的盐，包括但不限于：钠盐、钾盐、铵盐、胺盐等。
在一些实施方案中，所述dsRNA选自TRD008043、TRD008069、TRD008042、TRD008070、TRD008071、TRD008072、TRD008073、TRD008074、TRD008075、TRD008076中的任一项。
在一些实施方案中，所述dsRNA为TRD008043，其为如下结构
其中，Af＝腺嘌呤2'-F核糖核苷(adenine 2'-F ribonucleoside)；Cf＝胞嘧啶2'-F核糖核苷(cytosine 2'-F ribonucleoside)；Uf＝尿嘧啶2'-F核糖核苷(uracil 2'-F ribonucleoside)；Am＝腺嘌呤2'-OMe核糖核苷(adenine 2'-OMe ribonucleoside)；Cm＝胞嘧啶2'-OMe核糖核苷(cytosine 2'-OMe ribonucleoside)；Gf＝鸟嘌呤2'-F核糖核苷(guanine 2'-F ribonucleoside)；Gm＝鸟嘌呤2'-OMe核糖核苷(guanine 2'-OMe ribonucleoside)；Um＝尿嘧啶2'-OMe核糖核苷(uracil 2'-OMe ribonucleoside)。表示硫代磷酸二酯基，表示磷酸二酯基，
NAG0052’表示
(-)hmpNA(A)表示
另一方面，本公开提供了一种药物组合物，其包含上述的dsRNA。
在一些实施方案中，所述的药物组合物还包含一种或多种药学上可接受的赋形剂。各种递药系统是已知的并且可以用于本公开的dsRNA或药物组合物，例如封装在脂质体中、微粒、微囊、能够表达该化合物的重组细胞、受体介导的细胞内吞作用、构建核酸作为逆转录病毒或其他载体的一部分。
在一些实施方案中，本公开的dsRNA或药物组合物的给药方式是常规的，可通过局部给药(例如，直接注射或植入)或全身给药，也可通过口服、直肠或胃肠外途径进行给药，所述肠胃外途径包括但不限于皮下注射、静脉注射、肌肉注射、腹腔注射、透皮给药、吸入给药(如气溶胶)、粘膜给药(如舌下、鼻内给药)、颅内给药等。
在一些实施方案中，本公开提供的dsRNA或药物组合物可以通过注射给予，例如，静脉内、肌内、皮内、皮下、十二指肠内或腹膜内注射。
在一些实施方案中，本公开提供的dsRNA或药物组合物可被包装在试剂盒中。
在一些实施方案中，所述的dsRNA或药物组合物的有效量或有效剂量为约0.001mg/kg体重至约200mg/kg体重、约0.01mg/kg体重至约100mg/kg体重或约0.5mg/kg体重至约50mg/kg体重。
本公开提供了一种上述的dsRNA或上述的药物组合物在制备药物中的应用。
在一些实施方案中，所述的药物可用于降低受试者中的低密度脂蛋白水平，或用于预防和/或治疗由升高的甘油三酯水平或升高的胆固醇水平介导的疾病。在一些实施方案中，所述疾病选自高甘油三酯血症、肥胖症、高脂血症、脂质和/或胆固醇代谢异常、动脉粥样硬化、心血管疾病、冠状动脉疾病、高甘油三酯血症诱导的胰腺炎、代谢综合征、II型糖尿病、家族性乳糜微粒血症综合征或家族性部分脂质营养不良。
在一些实施方案中，所述的药物可用于预防和/或治疗与APOC3基因表达相 关的疾病。在一些实施方案中，所述疾病选自高甘油三酯血症、肥胖症、高脂血症、脂质和/或胆固醇代谢异常、动脉粥样硬化、心血管疾病、冠状动脉疾病、高甘油三酯血症诱导的胰腺炎、代谢综合征、II型糖尿病、家族性乳糜微粒血症综合征或家族性部分脂质营养不良。
本公开提供了一种预防和/或治疗疾病的方法，其包括向受试者给予有效量或有效剂量的上述的dsRNA或上述的药物组合物。
在一些实施方案中，所述的疾病可为由升高的甘油三酯水平或升高的胆固醇水平介导的疾病。在一些实施方案中，所述疾病选自高甘油三酯血症、肥胖症、高脂血症、脂质和/或胆固醇代谢异常、动脉粥样硬化、心血管疾病、冠状动脉疾病、高甘油三酯血症诱导的胰腺炎、代谢综合征、II型糖尿病、家族性乳糜微粒血症综合征或家族性部分脂质营养不良。
在一些实施方案中，所述的疾病可为与APOC3基因表达相关的疾病。在一些实施方案中，所述疾病选自高甘油三酯血症、肥胖症、高脂血症、脂质和/或胆固醇代谢异常、动脉粥样硬化、心血管疾病、冠状动脉疾病、高甘油三酯血症诱导的胰腺炎、代谢综合征、II型糖尿病、家族性乳糜微粒血症综合征或家族性部分脂质营养不良。
本公开提供了一种用于在体内或在体外沉默细胞中APOC3基因或其mRNA的方法，其包括将上述的dsRNA或上述的药物组合物引入该细胞中的步骤。
本公开提供了一种抑制APOC3基因或其mRNA表达的方法，其包括向受试者给予有效量或有效剂量的上述的dsRNA或上述的药物组合物。
在一些实施方案中，所述的dsRNA或药物组合物的有效量或有效剂量为约0.001mg/kg体重至约200mg/kg体重、约0.01mg/kg体重至约100mg/kg体重或约0.5mg/kg体重至约50mg/kg体重。
本公开的dsRNA或药物组合物可以在细胞、细胞群、组织或受试者等对象中降低靶基因或其mRNA的表达水平，包括：向对象给予治疗有效量的本文所述的dsRNA或药物组合物，从而抑制靶基因或其mRNA在对象中的表达。
在一些实施方式中，所述对象已在先前被鉴定为在靶向的细胞、细胞群、组织或受试者中具有靶基因或其mRNA的病理性上调。
本公开提供了一种递送寡核苷酸至肝脏的方法，其包括向受试者给予有效量或有效剂量的上述的dsRNA或上述的药物组合物。
本公开提供了一种RNA干扰(RNAi)试剂，其包含上述的dsRNA或上述的药物组合物。
另一方面，本公开还提供了一种细胞，其包含上述的dsRNA或上述的药物组合物。
另一方面，本公开还提供了一种试剂盒，其包含上述的dsRNA或上述的药物组合物。
一些实施方案中，本公开的dsRNA选自siRNA。
本公开中，上述dsRNA或药物组合物当接触到表达靶基因的细胞时，由例如：psiCHECK活性筛选和荧光素酶报告基因检测法，其他如PCR或基于分支DNA(bDNA)的方法、或基于蛋白质的方法，如免疫荧光分析法，例如Western Blot或流式细胞术测定的，上述dsRNA或药物组合物会抑制靶基因的表达至少5％、至少10％、至少15％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％。
本公开中，上述dsRNA或药物组合物当接触到表达靶基因的细胞时，由例如：psiCHECK活性筛选和荧光素酶报告基因检测法，其他如PCR或基于分支DNA(bDNA)的方法、或基于蛋白质的方法，如免疫荧光分析法，例如Western Blot或流式细胞术测定的，上述dsRNA或药物组合物引起的靶基因mRNA剩余表达百分比为不高于99％、不高于95％、不高于90％、不高于85％、不高于80％、不高于75％、不高于70％、不高于65％、不高于60％、不高于55％、不高于50％、不高于45％、不高于40％、不高于35％、不高于30％、不高于25％、不高于20％、不高于15％、或不高于10％。
本公开中，上述dsRNA或药物组合物当接触到表达靶基因的细胞时，由例如：psiCHECK活性筛选和荧光素酶报告基因检测法，其他如PCR或基于分支DNA(bDNA)的方法、或基于蛋白质的方法，如免疫荧光分析法，例如Western Blot、或流式细胞术测定的，dsRNA在保持在靶活性的同时，将脱靶活性减少了至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％或至少75％。
本公开中，上述dsRNA或药物组合物当接触到表达靶基因的细胞时，由例如：psiCHECK活性筛选和荧光素酶报告基因检测法，其他如PCR或基于分支DNA(bDNA)的方法、或基于蛋白质的方法，如免疫荧光分析法，例如Western Blot、或流式细胞术测定的，dsRNA使在靶活性降低至多20％、至多19％、至多15％、至多10％、至多5％或超过1％的同时，将脱靶活性减少了至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％或至少75％。
本公开中，上述dsRNA或药物组合物当接触到表达靶基因的细胞时，由例如：psiCHECK活性筛选和荧光素酶报告基因检测法，其他如PCR或基于分支DNA(bDNA)的方法、或基于蛋白质的方法，如免疫荧光分析法，例如Western Blot、或流式细胞术测定的，dsRNA使在靶活性提高至少1％、至少5％、至少10％、至少15％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％或至少80％的同 时，将脱靶活性减少了至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％或至少75％。
本公开化合物可以存在特定的几何或立体异构体形式。本公开设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本公开的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本公开的范围之内。本公开的含有不对称碳原子的化合物可以以光学活性纯的形式或外消旋形式被分离出来。光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性试剂合成。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本公开某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本公开所述化合物的化学结构中，键表示未指定构型，即如果化学结构中存在手性异构体，键可以为或者同时包含两种构型。本公开所述化合物的化学结构中，键并未指定构型，即键的构型可以为E型或Z型，或者同时包含E和Z两种构型。
在本公开的化学结构式中，可以根据本文所述发明范围连接一个或多个任何基团；星号“*”表示手性中心。
在不指明构型的情况下，本公开的化合物和中间体还可以以不同的互变异构体形式存在，并且所有这样的形式包含于本公开的范围内。术语“互变异构体”或“互变异构体形式”是指可经由低能垒互变的不同能量的结构异构体。例如，质子互变异构体(也称为质子转移互变异构体)包括经由质子迁移的互变，如酮-烯醇及亚胺-烯胺、内酰胺-内酰亚胺异构化。内酰胺-内酰亚胺平衡实例是在如下所示的A和B之间。
本公开中的所有化合物可以被画成A型或B型。所有的互变异构形式在本公开的范围内。化合物的命名不排除任何互变异构体。
本公开还包括一些与本文中记载的那些相同的，但一个或多个原子被原子量或质量数不同于自然中通常发现的原子量或质量数的原子置换的同位素标记的本公开化合物。可结合到本公开化合物的同位素的实例包括氢、碳、氮、氧、磷、硫、氟、碘和氯的同位素，诸如分别为2H、3H、11C、13C、14C、13N、15N、15O、17O、18O、31P、32P、35S、18F、123I、125I和36Cl等。
除另有说明，当一个位置被特别地指定为氘(D)时，该位置应理解为具有大于氘的天然丰度(其为0.015％)至少1000倍的丰度的氘(即，至少10％的氘掺入)。示例中化合物的具有大于氘的天然丰度可以是至少1000倍的丰度的氘、至少2000倍的丰度的氘、至少3000倍的丰度的氘、至少4000倍的丰度的氘、至少5000倍的丰度的氘、至少6000倍的丰度的氘或更高丰度的氘。本公开还包括各种氘化形式的式(I)、式(I’)、式(II)化合物。与碳原子连接的各个可用的氢原子可独立地被氘原子替换。本领域技术人员能够参考相关文献合成氘化形式的式(I)、式(I’)、式(II)化合物。在制备氘代形式的式(I)、式(I’)、式(II)化合物时可使用市售的氘代起始物质，或它们可使用常规技术采用氘代试剂合成，氘代试剂包括但不限于氘代硼烷、三氘代硼烷四氢呋喃溶液、氘代氢化锂铝、氘代碘乙烷和氘代碘甲烷等。
本公开还提供了一种制备dsRNA或药物组合物的方法，其包括：合成本公开所述的dsRNA或药物组合物。
一些实施方案中，“包含”可替换为“由……组成”。
本公开引入WO2022028462A1、WO2023274395A、PCT/CN2022/139462全文。
术语解释
为了更容易理解本公开，以下具体定义了一些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
除另有说明，“任选地”、“任选”、“可选的”或“可选”是指意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如“任选地，R1和R2直接相连成环”是指R1和R2直接相连成环可以发生但不必须存在，该说明包括R1和R2直接相连成环的情形和R1和R2不成环的情形。
术语“约”、“大约”是指数值在由本领域一般技术人员所测定的具体值的可接受 误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围，例如1％至15％之间、在1％至10％之间、在1％至5％之间、在0.5％至5％之间、在0.5％至1％之间变化，本公开中，数字或数值范围之前有术语“约”的每种情况也包括给定数的实施方案。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
如无特殊说明，本公开的“化合物”、“化学修饰”、“配体”、“dsRNA”、“核酸”和“RNAi”均可独立地以盐、混合盐或非盐(例如游离酸或游离碱)的形式存在。当以盐或混合盐的形式存在时，其可为药学上可接受的盐。
“药学上可接受的盐”可选自无机盐或有机盐，也可包括药学上可接受的酸加成盐和药学上可接受的碱加成盐。
“药学上可接受的酸加成盐”是指能够保留游离碱的生物有效性而无其它副作用的，与无机酸或有机酸所形成的盐。无机酸盐包括但不限于盐酸盐、氢溴酸盐、硫酸盐、硝酸盐、磷酸盐等；有机酸盐包括但不限于甲酸盐、乙酸盐、2,2-二氯乙酸盐、三氟乙酸盐、丙酸盐、己酸盐、辛酸盐、癸酸盐、十一碳烯酸盐、乙醇酸盐、葡糖酸盐、乳酸盐、癸二酸盐、己二酸盐、戊二酸盐、丙二酸盐、草酸盐、马来酸盐、琥珀酸盐、富马酸盐、酒石酸盐、柠檬酸盐、棕榈酸盐、硬脂酸盐、油酸盐、肉桂酸盐、月桂酸盐、苹果酸盐、谷氨酸盐、焦谷氨酸盐、天冬氨酸盐、苯甲酸盐、甲磺酸盐、苯磺酸盐、对甲苯磺酸盐、海藻酸盐、抗坏血酸盐、水杨酸盐、4-氨基水杨酸盐、萘二磺酸盐等。这些盐可通过本领域已知的方法制备。
“药学上可接受的碱加成盐”是指能够保持游离酸的生物有效性而无其它副作用的、与无机碱或有机碱所形成的盐。衍生自无机碱的盐包括但不限于钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐、锌盐、铜盐、锰盐、铝盐等。优选的无机盐为铵盐、钠盐、钾盐、钙盐及镁盐，优选钠盐。衍生自有机碱的盐包括但不限于以下的盐：伯胺类、仲胺类及叔胺类，被取代的胺类，包括天然的被取代胺类、环状胺类及碱性离子交换树脂，例如氨、异丙胺、三甲胺、二乙胺、三乙胺、三丙胺、乙醇胺、二乙醇胺、三乙醇胺、二甲基乙醇胺、2-二甲氨基乙醇、2-二乙氨基乙醇、二环己胺、赖氨酸、精氨酸、组氨酸、咖啡因、普鲁卡因、胆碱、甜菜碱、乙二胺、葡萄糖胺、甲基葡萄糖胺、可可碱、嘌呤、哌嗪、哌啶、N-乙基哌啶、聚胺树脂等。优选的有机碱包括异丙胺、二乙胺、乙醇胺、三甲胺、二环己基胺、胆碱及咖啡因。这些盐可通过本领域已知的方法制备。
“烷基”指饱和的脂族烃基团，例如包括1至30个碳原子的直链和支链基团(C1-C30烷基)，又例如含有1至6个碳原子的烷基(C1-C6烷基)，又例如1至3个碳原子的烷基(C1-C3烷基)。非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙 基、2,2-二甲基丙基及其各种支链异构体等。
术语“烯基”是指含有至少一个双键的烃基。烯基的非限制性实例包括但不限于：乙烯基、1-丙烯基、2-丙烯基、1-丁烯基或2-丁烯基及其各种支链异构体。
术语“炔基”指含有至少一个三键的烃基。炔基的非限制性实例包括但不限于：乙炔基、1-丙炔基、2-丙炔基、1-丁炔基或2-丁炔基及其各种支链异构体。
术语“烷氧基”指-O-(烷基)，其中烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基。
“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至6个碳原子，更优选包含5-6个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基等；多环环烷基包括螺环、并环和桥环的环烷基。
“杂环烷基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子；更优选包含3至7个环原子。“杂环烷基”非限制性实例包括：

等等。
所述杂环烷基环可以稠合于芳基或杂芳基环上，其中与母体结构连接在一起的环为杂环烷基，其非限制性实例包括：
等。
“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享 毗邻碳原子对的环)基团，优选为6至12元，例如苯基和萘基。所述芳基环可以稠合于杂芳基、杂环烷基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性实例包括：
“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为6至12元，更优选为5元或6元。例如。其非限制性实例包括：咪唑基、呋喃基、噻吩基、噻唑基、吡唑基、噁唑基(oxazolyl)、异噁唑基(isoxazolyl)、吡咯基、四唑基、吡啶基、嘧啶基、噻二唑、吡嗪基、三唑基、吲唑基、苯并咪唑基、等。
所述杂芳基环可以稠合于芳基、杂环烷基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性实例包括：
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氰基”指-CN。
术语“氨基”指-NH2。
术语“硝基”指-NO2。
术语“氧代”指＝O取代基。
本公开中，“磷酸酯基团”可为磷酸一酯基团、磷酸二酯基团或磷酸三酯基团，优选磷酸二酯基团；“硫代磷酸酯基团”中的“磷酸酯基团”也具有同样的含义。
本公开中，硫代磷酸二酯基是指一个非桥接氧原子被硫原子替代而修饰的磷 酸二酯基，可用(M为S原子)互换使用。
“取代”指基团中的一个或多个氢原子，优选为最多5个，更优选为1～3个氢原子彼此独立地被相应数目的取代基取代。当取代基是酮或氧代(即，＝O)时，则原子上有两个(2个)氢被替代。
本公开上下文中，基团中的部分可以替换为能够与相邻核苷酸实现连接的任意基团。
术语“连接”，当表示两个分子之间的联系时，指两个分子通过共价键连接或者两个分子经由非共价键(例如，氢键或离子键)关联，包括直接连接、间接连接。
术语“直接连接”指第一化合物或基团与第二化合物或基团在没有任何间插原子或原子基团的情况下连接。
术语“间接连接”指第一化合物或基团与第二化合物或基团通过中间基团、化合物或分子(例如，连接基团)连接。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
“药学上可接受的赋形剂”包括但不限于任何已经被美国食品和药物管理局(FDA)批准对于人类或家畜动物使用可接受的任何助剂、载体、助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、增香剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂。
如本文所使用的，术语“抑制”，可以与“减少”、“沉默”、“下调”、“阻抑”和其他类似术语交替使用，并且包括任何水平的抑制。抑制可通过这些变量中的一个或多个与对照水平相比的绝对或相对水平的减少来评估。该对照水平可以是本领域中使用的任何类型的对照水平，例如给药前基线水平或从类似的未经处理或经对照(例如仅缓冲液对照或惰性剂对照)处理的受试者、细胞、或样品确定的水平。例如，可以采用mRNA剩余表达量来表征dsRNA对靶基因表达的抑制程度，如mRNA剩余表达量为不高于99％、不高于95％、不高于90％、不高于85％、不高 于80％、不高于75％、不高于70％、不高于65％、不高于60％、不高于55％、不高于50％、不高于45％、不高于40％、不高于35％、不高于30％、不高于25％、不高于20％、不高于15％、或不高于10％。靶基因表达的抑制率可以采用Luciferase Assay System检测，分别读取萤火虫(Firefly)化学发光值和海肾(Renilla)化学发光值，计算相对值Ratio＝Ren/Fir，抑制率(％)＝1-(Ratio+dsRNA/Ratioreporter only)*100％；本公开中，剩余mRNA表达量比例(或剩余活性％)＝100％-抑制率(％)。
“有效量”或“有效剂量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
如本文所使用的，“对象”、“患者”、“受试者”或“个体”可互换使用，包括人类或者非人类动物，例如哺乳动物，例如人或猴。
如本文所使用的，有义链(又称SS、SS链或正义链)是指包含与靶mRNA序列相同或基本上相同的序列的链；反义链(又称AS或AS链)是指具有与靶mRNA序列互补的序列的链。
本公开中，有义链或反义链的“5’区域”也即“5’端”、“5’末端”，可替换使用。例如反义链5’区域的第2位至第8位的核苷酸，也可替换为反义链5’端的第2位至第8位的核苷酸。同理，有义链或反义链的“3’区域”、“3’末端”和“3’端”也可替换使用。
在描述本文所述的dsRNA有义链的上下文中，术语“SEQ ID NO:1的核苷酸序列相差不超过3个核苷酸序列的至少15个连续核苷酸”旨在表示本文所述的dsRNA有义链包含如与SEQ ID NO:1所示的有义链的至少15个连续核苷酸，或与SEQ ID NO:1所示的有义链的至少15个连续核苷酸相差不超过3个核苷酸序列，任选地，相差不超过2个核苷酸序列，任选地，相差1个核苷酸序列。任选地，本文所述的dsRNA有义链包含SEQ ID NO:1所示有义链的至少16个连续核苷酸，或与SEQ ID NO:1所示的有义链的至少16个连续核苷酸相差不超过3个核苷酸序列，任选地，相差不超过2个核苷酸序列，任选地，相差1个核苷酸序列；
在描述本文所述的dsRNA反义链的上下文中，术语“与SEQ ID NO:2所示的反义链相差不超过3个核苷酸序列的至少15个连续核苷酸”旨在表示本文所述的SEQ ID NO:2所示的反义链的至少15个连续核苷酸，或与SEQ ID NO:2所示的反义链的至少15个连续核苷酸相差不超过3个核苷酸序列，任选地，相差不超过2个核苷酸序列，任选地，相差1个核苷酸序列。
如无特别说明，在本公开上下文中，“G”、“C”、“A”、“T”与“U”分别代表核苷酸，其分别包含鸟嘌呤、胞嘧啶、腺嘌呤、胸苷与尿嘧啶的碱基。小写字母m表 示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸二酯基连接。
如本公开所使用的，术语“2'-氟代(2’-F)修饰的核苷酸”指核苷酸的核糖基2'位的羟基被氟取代形成的核苷酸，“非氟代修饰的核苷酸”指核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物。
如本公开所使用的，术语“2'-甲氧基(2’-OMe)修饰的核苷酸”指核糖基的2'-羟基被甲氧基取代而形成的核苷酸。
在本公开的上下文中，一个核苷酸序列与另外一个核苷酸序列存在“核苷酸差异”，是指前者与后者相比，相同位置的核苷酸的碱基种类发生了改变，例如，在后者中一个核苷酸碱基为A时，在前者的相同位置处的对应核苷酸碱基为U、C、G或者T的情况下，认定为两个核苷酸序列之间在该位置处存在核苷酸差异。在一些实施方式中，以无碱基核苷酸或其等同物代替原位置的核苷酸时，也可认为在该位置处产生了核苷酸差异。
如本文所使用的，术语“互补”或“反向互补”一词可互相替代使用，并具有本领域技术人员周知的含义，即，在双链核酸分子中，一条链的碱基与另一条链上的碱基以互补的方式相配对。在DNA中，嘌呤碱基腺嘌呤始终与嘧啶碱基胸腺嘧啶(或者在RNA中为尿嘧啶)相配对；嘌呤碱基鸟嘌呤始终与嘧啶碱基胞嘧啶相配对。每个碱基对都包括一个嘌呤和一个嘧啶。当一条链上的腺嘌呤始终与另一条链上的胸腺嘧啶(或尿嘧啶)配对，以及鸟嘌呤始终与胞嘧啶配对时，两条链被认为是彼此相互补的，以及从其互补链的序列中可以推断出该链的序列。与此相应地，“错配”在本领域中意指在双链核酸中，对应位置上的碱基并未以互补的形式配对存在。
术语“dsRNA”是指能够进行RNA干扰的双链RNA分子，包含有义链和反义链。
术语“化学修饰”或“修饰”包括核苷酸经化学手段的所有改变，例如化学部分的添加或去除、或以一个化学部分取代另一个化学部分。
术语“碱基”包含任何已知的DNA和RNA碱基、碱基类似物，例如嘌呤或嘧啶，其还包括天然化合物腺嘌呤、胸腺嘧啶、鸟嘌呤、胞嘧啶、尿嘧啶、次黄苷和天然类似物。碱基类似物还可以是通用碱基。
术语“平端”或“平末端”可互换使用，是指在dsRNA的给定的末端没有非配对的核苷酸或核苷酸类似物，即，没有核苷酸突出。大多数情况下，两个末端都是平末端的dsRNA将在其整个长度范围内是双链的。
本公开提供的dsRNA可以通过本领域常规的制备方法(例如固相合成和液相合成的方法)得到。其中，固相合成已经有商业化订制服务。可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的dsRNA中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入dsRNA的方法也是本领域 技术人员所熟知的。
附图说明
图1为TRD002218、TRD007205在给药后第7天TTR mRNA的表达量。
图2为TRD002218、TRD007205在给药后第28天TTR mRNA的表达量。
具体实施方式
以下结合实施例进一步描述本公开，但这些实施例并非限制着本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，则该试剂可自任意分子生物学试剂的供应商以用于分子生物学应用的质量/纯度而获得。
实施例1：化学修饰的制备
1.1合成化合物1-1a和化合物1-1b
将化合物1(500mg,3.42mmol)和三乙胺(Et3N，692mg,6.84mmol,0.95mL)溶于二氯甲烷(DCM，10mL)中，冰浴下滴加4-甲苯磺酰氯(TsCl,717mg,3.76mmol)的二氯甲烷(10mL)溶液，滴加完毕后反应在室温下搅拌过夜，待反应完毕后，用水淬灭，水相用二氯甲烷(15mL)提取三次，合并的有机相先用饱和碳酸氢钠水溶液(10mL)洗涤，再用饱和食盐水(20mL)洗涤，随后减压蒸干溶剂得到粗品2(820mg,80％)，直接用于下一步反应。MS m/z：C14H21O5S，[M+H]+理论：301.10实测：301.2。
将化合物3(239mg,1.22mmol)溶解于二甲基甲酰胺(DMF，10mL)中，冰浴下加入NaH(60％溶解在矿物油中,93mg,2.33mmol)溶液，该反应下搅拌30分钟，然后滴加化合物2(350mg,1.16mmol)，滴加完毕后反应在60℃下搅拌5小时，反应完毕后，加水淬灭，水相用乙酸乙酯(15mL)提取三次，合并的有机相先用水(10mL)洗涤三次，再用饱和食盐水(10mL)洗涤，随后减压蒸干溶剂，经反相制备HPLC(C18,条件:5-50％(A:H2O,B:CH3CN),流速:70mL/min)，冻干后得到220 mg化合物4。MS m/z：C19H21N5O3Na，[M+Na]+理论：390.16，实测：390.3。
室温下将化合物4(1.50g,4.08mmol)溶解于20mL的醋酸和水(4:1)的混合溶液中，60℃下搅拌30分钟，待反应完毕后减压蒸干溶剂，经反相制备HPLC(C18,条件:5-25％(A:H2O,B:CH3CN),流速:70mL/min)，冻干后得到1.10g化合物5。MS m/z：C16H18N5O3，[M+H]+理论：328.13，实测：328.4。
将化合物5(1.00g,3.05mmol)溶于吡啶(Py，10mL)中，冰浴下滴4,4'-双甲氧基三苯甲基氯(DMTrCl,1.50g,4.58mmol)的吡啶(5mL)溶液，滴加完毕后反应在室温下搅拌过夜，待反应完毕后，用水淬灭，减压蒸干溶剂，经反相制备HPLC(C18,条件:5-80％(A:H2O,B:CH3CN),流速:70mL/min)，冻干后得到1.00g化合物6。MS m/z：C37H36N5O5，[M-H]+理论：630.26，实测：630.5。消旋体化合物6经手性柱(DaicelIE 250*4.6mm,5μm,A:正己烷,B:乙醇)拆分得410mg 6A(-)和435mg 6B(+)。
将化合物6A(-)(200mg,0.32mmol)，四氮唑(11mg,0.16mmol)，N-甲基咪唑(5mg,0.06mmol)，3A分子筛(500mg)溶于10mL的乙腈中，室温下加入化合物7(144mg,0.48mmol)，在室温下搅拌过夜。反应完毕后，将分子筛过滤掉，加入 二氯甲烷(30mL)，饱和碳酸氢钠水溶液(10mL)洗涤三次，再用饱和食盐水(20mL)洗涤，滤液旋干并经反相制备HPLC(C18,条件:5-100％(A:水,B:CH3CN),流速:70mL/min)，冻干后得到200mg化合物1-1a。MS m/z：C40H39N6O7P，[M-二异丙基+OH]+理论：747.26，实测：747.6。1H NMR(400MHz,Acetonitrile-d3)δ7.56,7.54(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)。
将化合物6B(+)(200mg,0.32mmol)，四氮唑(11mg,0.16mmol)，N-甲基咪唑(5mg,0.06mmol)，3A分子筛(500mg)溶于10mL的乙腈中，室温下加入化合物7(144mg,0.48mmol)，在室温下搅拌过夜。反应完毕后，将分子筛过滤掉，加入二氯甲烷(30mL)，饱和碳酸氢钠水溶液(10mL)洗涤三次，再用饱和食盐水(20mL)洗涤，滤液旋干并经反相制备HPLC(C18,条件:5-100％(A:水,B:CH3CN),流速:70mL/min)，冻干后得到200mg化合物1-1b。MS m/z：C40H39N6O7P，[M-二异丙基+OH]+理论：747.26，实测：747.5。
1.2合成化合物1-6a
将化合物1(10g,68.404mmol),化合物2(15g,62.186mmol)和三苯基膦(32.62g,124.371mmol)溶于无水THF(30mL),于0℃下缓慢滴加DIAD(24.656mL，124.371mmol)。该反应液在25℃下反应12h。LCMS显示反应完成。将该反应液用乙酸乙酯(200mL)和水(200mL)萃取，有机相干燥将滤液浓缩，得到的残留物用正向柱纯化(DCM/MeOH＝10/1)得目标产物3(20g)。
将化合物3(20g,28.585mmol)溶于醋酸(24mL,426.016mmol)和H2O(12mL)中，60℃搅拌1小时。之后将反应液旋干加入THF(12mL)和H2O(12mL)，80℃搅拌7小时。LCMS显示反应完成。将反应液加入乙酸乙酯(200mL)和水(100mL)萃取，水相加入碳酸钠固体直到水相有大量固体析出。将固体过滤，用水洗涤，将滤饼用油泵拉干，得到目标化合物5(9g)。
在氮气保护下,将化合物5(6.8g,18.581mmol)溶于吡啶(80mL)中，于0℃下缓慢加入TMSCl(14.250mL,111.489mmol)，搅拌2h。之后在0℃下加入Isobutyryl chloride(2.044mL,19.511mmol)，于25℃下搅拌1h。LCMS显示反应完成。用二氯甲烷(200mL)和水(200mL)萃取，有机相干燥旋干后拌样，用正向柱纯化(DCM:MeOH＝10:1)过柱，在4.8％处出峰)，得到黄色油状化合物6(12g)。
在氮气保护下,将化合物6(5.5g,12.392mmol)溶于吡啶(30mL)，加入MOLECULAR SIEVE 4A 1/16(7g,12.392mmol)，然后在0℃下分批加入DMTrCl(5.04g,14.870mmol)固体，25℃反应2h。TLC(PE:EtOAc＝1:1,Rf＝0.69)显示反应已经完成。该反应液和TJN200879-040-P1合并一起处理。将反应液用乙酸乙酯(200mL)和水(200mL)萃取，有机相干燥旋干后拌样用正向柱纯化(PE:EtOAc过柱，在84％处出峰)，得到黄色油状化合物7(12g)。
将化合物7(12g,15.389mmol)溶于EtOAc(140mL)，加入湿钯碳Pd/C(7g,15.389mmol)该反应液在25℃，氢气(15Psi)下反应2小时。TLC(PE:EtOAc＝0:1,Rf＝0.09)显示反应已经完成。将反应液过滤，滤饼用乙酸乙酯(30mL)冲洗三遍后，收集滤液。滤液旋干后加入50mL二氯甲烷和2mL三乙胺拌样用正向柱纯化(DCM:MeOH＝10:1过柱，在0.5％处出峰)，得到9g(黄色泡沫状固体)。将所得消旋化合物SFC拆分，得到产品目标化合物7A(-)(3.9g)和目标化合物7B(+)(3.8g)。
将化合物7A(-)(3.30g,5.40mmol)，四氮唑(190mg,2.70mmol)，1-甲基咪唑(90mg,1.10mmol)，3A分子筛(500mg)溶于30mL的乙腈中，室温下加入化合物8(2.50g,8.10mmol)，在室温下搅拌2h。反应完毕后，将分子筛过滤掉，加入DCM(150mL)，饱和碳酸氢钠水溶液洗涤(30mL*3)，再用饱和食盐水(30mL)洗涤，滤液旋干并经反相制备HPLC(C18,Condition:5-100％(A:water,B:CH3CN),流速:70mL/min)，冻干后得到1-6a(2.9g,66％)。MS m/z：C43H55N7O7P[M+H]+，理论：812.38，实测：812.5。1H NMR(400MHz,Acetonitrile-d3)δ7.56,7.54(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)。
1.3合成化合物1-7a
在氮气保护下，将化合物1(5g,23.1272mmol),化合物2(6.76g,46.254mmol) 和三苯基磷(7.28g,27.753mmol)溶于30mL二氧六环中，于0℃缓慢滴加入DEAD(5.502mL,27.753mmol)。滴加完成后，反应缓慢升温至25℃继续反应1h。在反应液里加入100mL H2O和100mL EtOAc萃取，有机相合并干燥过滤浓缩后拌样过柱，用正向柱纯化(PE:EtOAc＝1:1过柱得目标产物(4g)。
将化合物3(3.3g)溶于HOAc(16mL)和H2O(4mL),油浴60℃加热0.5h。将反应液旋干得到的残留物用正向柱纯化(PE:EtOAc＝0:1过柱)，得到目标产物4(3g)。
将化合物4(3g,8.873mmol)溶于5mL吡啶中，在氮气保护下于0℃缓慢滴加DMTrCl(3.91g,11.535mmol)的10mL吡啶的溶液。滴加完毕后反应升温至25℃并继续反应1h。在反应液中加入50mL水和100mL乙酸乙酯萃取。水相再用100mL乙酸乙酯萃取三次，有机相合并干燥过滤浓缩用正向柱纯化(用PE:EtOAc＝2:1)。得到目标产物5(4g)。
将化合物5(4g,5.769mmol)溶于甲醇(10mL),加入饱和的NH3甲醇溶液(40mL),0℃反应6h。将反应液旋干用正向柱纯化(用PE:EtOAc＝0:1)得消旋化合物2.4g SFC拆分，得到目标产物6A(750mg,100％纯度)和目标产物6B(400mg,99.16％纯度)。
将化合物6A(-)(700mg,1.40mmol)，四氮唑(50mg,0.70mmol)，1-甲基咪唑(23mg,0.28mmol)，3A分子筛(500mg)溶于10mL的乙腈中，室温下加入化合物7(630mg,2.10mmol)，在室温下搅拌2h。反应完毕后，将分子筛过滤掉，加入DCM(50mL)，饱和碳酸氢钠水溶液洗涤(10mL*3)，再用饱和食盐水(20mL)洗涤，滤液旋干并经反相制备HPLC(C18,Condition:5-100％(A:水B:CH3CN),流速:70mL/min)，冻干后得到1-7a(700mg,72％)。MS m/z：C38H47N4O7PNa[M+Na]+，理论：725.32，实测：725.5。
1.4合成化合物1-8a
将化合物1(8.5g,76.508mmol),化合物2(30.64g,91.809mmol)溶于DMF(150mL),加入CS2CO3(29.91g,91.809mmol)，反应于氮气保护下，90℃反应12h。LCMS检测反应完成。将反应液过滤，油泵旋干，正向柱分离纯化(80g,DCM/MeOH＝10/1～5/1)得到目标产物3(13.5g,80％纯度)。
将化合物3(10.5g,35.105mmol)溶于吡啶(65mL)和CH3CN(65mL)，向溶液中滴加BzCl(4.894mL,42.126mmol)，于25℃反应2h。LCMS检测大部分原 料反应完成，加H2O(100mL)淬灭，EtOAc(100mL X 3)萃取，干燥旋干，柱分离(合并TJN200872-101)纯化(80g,PE/EtOAc＝10/1～0/1,DCM/MeOH＝10/1)得到目标产物4(14g,90％纯度)。
将化合物4(14g,36.694mmol)溶于HOAc(56mL,314.796mmol)和H2O(14mL)，于60℃反应2h,LCMS显示反应完成。油泵浓缩，正向柱分离(40g,DCM/MeOH＝1/0～5/1)得到目标产物5(8.4g,90％纯度&2.4g,80％纯度)。
将化合物5(7.4g,21.957mmol),DMAP(0.54g,4.391mmol),MOLECULAR SIEVE 4A(11.1g,2.967mmol)溶于吡啶(60mL)，冰浴下搅拌10min，然后加入DMTrCl(8.93g,26.348mmol)，反应搅拌1.8h。LCMS检测约19％原料剩余，约60％目标MS。合并(TJN200872-105&106)一起纯化。向反应液中加入H2O(50mL)，经DCM(50mL X 3)萃取，干燥，旋干，柱分离(120g,PE/(EA:DCM:TEA＝1:1:0.05)＝1/0～0/1至DCM/MeOH＝10/1)得到目标化合物6(11g,89％纯度,TJN200872-105&106&107),回收原料(3.0g,70％纯度)。
化合物6(15g,22.041mmol)经SFC(DAICEL CHIRALPAK AD(250mm*50mm,10um)；0.1％NH3H2O EtOH，B:45％-45％；200ml/min)分离得到目标产物6A(+)(5.33g,94.29％纯度),目标产物6B(-)(6.14g,97.91％纯度),化合 物6B(-)回收1.0g。
将化合物6B(-)(5.4g,8.92mmol)，四氮唑(312mg,4.46mmol)，1-甲基咪唑(146mg,1.78mmol)，3A分子筛(500mg)溶于40mL的乙腈中，室温下加入化合物7(4g,13.4mmol)，在室温下搅拌2h。反应完毕后，将分子筛过滤掉，加入DCM(200mL)，饱和碳酸氢钠水溶液洗涤(30mL*3)，再用饱和食盐水(50mL)洗涤，滤液旋干并经反相制备HPLC(C18,Condition:5-100％(A:water,B:CH3CN),流速:70mL/min)，冻干后得到1-8a(5.8g,80％)。MS m/z：C45H51N5O7P，[M+H]+，理论：804.36，实测：804.4。
实施例2：不同化学修饰表征
其中：我们将由2-羟甲基-1,3-丙二醇为起始原料合成的核苷酸定义hmpNA；
(+)hmpNA(A)为实施例1.1节中核苷亚磷酰胺单体1-1b通过固相合成获得，绝对构型为(S)-hmpNA(A)；
(-)hmpNA(A)为实施例1.1节中核苷亚磷酰胺单体1-1a通过固相合成获得，绝对构型为(R)-hmpNA(A)；
类似的，替换hmpNA的碱基种类，通过固相合成获得以下结构并确认绝对构型：
(+)hmpNA(G)，绝对构型为(S)-hmpNA(G)；
(-)hmpNA(G)，绝对构型为(R)-hmpNA(G)；
(+)hmpNA(C)，绝对构型为(S)-hmpNA(C)；
(-)hmpNA(C)，绝对构型为(R)-hmpNA(C)；
(+)hmpNA(U)，绝对构型为(R)-hmpNA(U)；
(-)hmpNA(U)，绝对构型为(S)-hmpNA(U)。
(S)-hmpNA(G)，(R)-hmpNA(G)，(S)-hmpNA(C)，(R)-hmpNA(C)，(S)-hmpNA(U)和(R)-hmpNA(U)的绝对构型由其中间体或衍生物经X-Ray衍射而确认。
中间体或衍生物的结构为：
TJ-NA067:检测晶体为无色块状(0.30×0.10×0.04mm3)，属于单斜晶系P21空间群。晶胞参数α＝90°,β＝118.015(4)°,γ＝90°,Z＝4。计算密度Dc＝1.389g/cm3，单胞中电子数F(000)＝504.0，单胞的线性吸收系数μ(Cu Kα)＝0.840mm–1，衍射实验温度T＝150.00(11)K。
6A(+):检测晶体为无色块状(0.30×0.20×0.10mm3)，属于单斜晶系P21空间群。晶胞参数α＝90°,β＝113.876(3)°,γ＝90°,Z＝2。计算密度Dc＝0.999g/cm3，单胞中电子数F(000)＝1318.0，单胞的线性吸收系数μ(Cu Kα)＝0.570mm–1，衍射实验温度T＝100.01(18)K。
TJ-NA048:检测晶体为无色针状(0.30×0.04×0.04mm3)，属于单斜晶系P1空间群。晶胞参数α＝85.007(4)°,β＝88.052(4)°,γ＝70.532(4)°,Z＝2。计算密度Dc＝1.366g/cm3，单胞中电子数F(000)＝620.0，单胞的线性吸收系数μ(Cu Kα)＝0.856mm–1，衍射实验温度T＝150.00(13)K。
TJ-NA092:检测晶体为无色棱柱状(0.30×0.10×0.10mm3)，属于三斜晶系P1空间群。晶胞参数α＝93.146(2)°,β＝101.266(2)°,γ＝96.134(2)°,Z＝2。计算密度Dc＝1.412g/cm3，单胞中电子数F(000)＝228.0，单胞的线性吸收系数μ(Cu Kα)＝0.945mm–1，衍射实验温度T＝100.00(10)K。
实施例3：制备NAG0052、L96
化合物NAG0024、NAG0026购买自天津药明康德新药开发有限公司。除非特别说明，以下实施例中所用的试剂均为市售商品。
化合物NAG0052的合成
起始原料化合物1采购自江苏倍达医药科技有限公司。化合物NAG0052的合成路线如下所示：
上述路线中涉及的具体中间产物以及终产物的合成以及鉴定如下所述：
化合物2
在0℃以及氮气保护下，往化合物1(12.3mL,101mmol)的THF(300mL)溶 液中分批加入NaH(12.2g,304mmol，纯度60％)。该混合物在20℃下搅拌1小时之后再次冷却到0℃，接着往体系中逐滴加入苄溴(36.3mL,304mmol)，并且在20℃搅拌12小时。将该反应液用H2O(100mL)淬灭后，用EtOAc(200mL x 2)萃取。合并后的有机相用饱和食盐水(100mL)洗涤,Na2SO4干燥，过滤，浓缩得到的残留物经过硅胶柱层析分离后得到目标化合物2(20.0g,51.8mmol,产率51％)。
LCMS:tR＝2.615and 2.820min in 30-90AB_7min_220&254_Shimadzu.lcm(Xtimate C18,3um,2.1*30mm),MS(ESI)m/z＝351.2[M+Na]+。
1H NMR:(400MHz,CDCl3)δppm 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)。
化合物3和4
在20℃以及氮气保护下，往化合物2(13.0g,33.6mmol)的DCM(300mL)溶液中一次性加入TMSCN(13.5mL,101mmol)，接着逐滴加入TMSOTf(9.14mL,50.5mmol)的DCM(30mL)溶液。该反应液在20℃下搅拌15小时。反应结束之后用饱和NaHCO3水溶液(80mL)淬灭该体系，并且用DCM(150mL x 2)萃取，合并后的有机相用饱和食盐水(80mL)洗涤，Na2SO4干燥，过滤以及浓缩后通过硅胶柱层析分离后得到目标化合物3(3.30g,9.18mmol,产率27％)以及淡黄色油状液体化合物4(8.50g,9.18mmol,产率70％)。
化合物3
1H NMR:(400MHz,CDCl3)δppm 7.42-7.29(m,10H),4.81(t,J＝7.8Hz,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)。
化合物4
1H NMR:(400MHz,CDCl3)δppm 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＝4.0Hz,1H),2.45-2.37(m,2H)。
化合物5
在0℃及氮气保护下将化合物4(3.00g,9.28mmol)的THF(15mL)溶液，滴加到LiAlH4(0.79g,20.9mmol)的THF(15mL)溶液中，滴加完后体系在0℃反应1小时。TLC(PE:EtOAc＝3:1)监测到原料完全消失。向反应液中缓慢加入十水硫酸钠，加至不冒泡为止。之后将反应液过滤，滤饼用二氯甲烷(60mL)洗涤三次后，收集滤液旋干，得目标化合物5(3.00g,产率90％)。
1H NMR:(400MHz,DMSO-d6)δppm 7.40-7.14(m,10H),4.54-4.38(m,4H),4.06-3.99(m,2H),3.91(q,J＝6.4Hz,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)。
化合物6
在氮气保护下，将化合物5(3.00g,8.25mmol)溶于DCM(30mL)，加入TEA (3.44mL,24.7mmol)和CbzCl(1.76mL,12.4mmol)，20℃反应2小时。LCMS显示反应完成。将反应液加入二氯甲烷(30mL)和水(60mL)萃取。有机相用水(60mL x 3)洗涤三次，无水硫酸钠干燥，浓缩用正向柱纯化(PE:EtOAc＝1:1)，得到目标化合物6(2.5g,产率90％)。
LCMS:tR＝0.810min in 5-95AB_1min,MS(ESI)m/z＝462.2[M+H]+。
1H NMR:(400MHz,CDCl3)δppm 7.39-7.29(m,15H),5.35(s,1H),5.15-5.01(m,2H),4.72(d,J＝6.0Hz,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)。
化合物7
在氮气保护下，将化合物6(2.00g,3.90mmol)溶于DCM(5mL)，在-78℃下加入BCl3的THF溶液(1M,27.3mL)，反应1小时。TLC(DCM:MeOH＝10:1)监测到原料完全消失。将反应液在-78℃下加入甲醇(20mL)淬灭，浓缩，用正向柱纯化(DCM:MeOH＝10:1)，得到目标化合物7(2.00g,产率60％)。
1H NMR:(400MHz,CD3OD)δppm 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)。
化合物8
在氮气保护下，将化合物7(0.50g,1.78mmol)溶于吡啶(5mL)中，在0℃下加入4A分子筛(500mg)和DMTrCl(0.66mL,2.13mmol)，之后升温至20℃反应1.5小时。TLC(PE:EtOAc＝2:1)监测到原料完全消失。将反应液加入乙酸乙酯(60mL)和水(60mL)萃取，有机相用水(60mL x 3)洗涤三次后用无水硫酸钠干燥，浓缩，用正向柱纯化(PE:EtOAc＝1:1)，得到目标化合物8(800mg,产率90％)。
1H NMR:(400MHz,CDCl3)δppm 7.44(d,J＝7.6Hz,2H),7.37-7.23(m,11H),7.22-7.15(m,1H),6.84(d,J＝8.8Hz,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)。
化合物9
将化合物8(800mg,1.234mmol)溶于EtOAc(5mL)，加入Pd/C 10％(800mg,7.517mmol)，反应在H2条件(15Psi)，20℃下反应1小时。LCMS显示反应已经完成。反应液过滤，滤饼用二氯甲烷(100mL)和甲醇(100mL)洗涤三次，浓缩，经过反相柱分离得到化合物9(300mg,54％)。
LCMS:tR＝2.586min in 10-80CD_3min MS(ESI)m/z＝450.2[M+H]+。
化合物11
将化合物10(435mg,1.780mmol)溶于DCM(10mL)，加入DIEA(0.441mL,2.67mmol)和HATU(677mg,1.78mmol)后,再加入化合物9(400mg,0.890mmol)，20℃反应1小时。TLC(DCM:MeOH＝10:1)监测反应完成。将反应液加入二氯甲烷 (60mL)和水(60mL)萃取，有机相用水(60mL x 3)洗涤三次,无水硫酸钠干燥，浓缩用正向柱纯化(PE:EtOAc＝0:1过柱，在100％处出产品峰),得到目标化合物11(600mg,产率90％)。
LCMS:tR＝2.745min in 30-90CD_3min,MS(ESI)m/z＝698.4[M+Na]+。
1H NMR:(400MHz,CD3OD)δppm 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)。
化合物12
将化合物11(600mg,0.799mmol)溶于THF(3mL)和H2O(1mL)，加入LiOH.H2O(134mg,3.20mmol)，20℃反应12小时。TLC(DCM:MeOH＝10:1)显示反应完成。将反应液旋干，用水(5mL)和甲醇(5mL)溶解，用反向柱纯化(H2O:CH3CN＝1:1,在35％左右出峰)，得到目标化合物12(460mg,产率100％，锂盐)。
LCMS:tR＝1.346min in 10-80CD_3min,MS(ESI)m/z＝684.3[M+Na]+。
HPLC:tR＝1.879min in 10-80CD_6min。
1H NMR:(400MHz,CD3OD)δppm 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)。
化合物13
室温环境，氮气保护下，将化合物NAG0024(271mg,0.151mmol)溶解于无水THF(2mL)和无水DMF(4mL)，加入3A分子筛，再依次加入化合物12(100mg,0.151mmol),HOBt(25mg,0.181mmol),DCC(38mg,0.181mmol)和DIEA(39mg,0.302mmol)。反应液45℃反应16h。LC-MS显示反应完全后，加水淬灭，过滤。滤液浓缩后，经C18反相柱纯化(H2O/MeCN)，得到化合物13(210mg，产率57％)。
化合物NAG0052
室温环境下，化合物13(230mg，0.094mmol)溶于吡啶(5mL)，加入分子筛，加入DMAP(12mg，0.283mmol)，丁二酸酐(28mg，0.283mmol)。氮气保护，50℃搅拌16小时。LCMS检测反应完全，过滤旋干。过C18反相柱纯化后，由制备HPLC二次纯化，得到目标化合物NAG0052(123mg，0.048mmol，产率51％)。
MS(ESI)m/z＝2535.3[M-1]-.理论：2536.2。
1H NMR(400MHz,Acetonitrile-d3)δ7.48-7.43(m,2H),7.37-7.12(m,11H),7.00-6.85(m,10H),6.66(s,1H),5.31(dd,J＝3.4,1.1Hz,3H),5.20-5.13(m,1H),5.05(dd,J＝11.3,3.4Hz,3H),4.56(d,J＝8.5Hz,3H),4.30(dd,J＝7.7,5.3Hz,1H),4.18-3.93(m,14H),3.79(s,10H),3.65(q,J＝4.7,3.6Hz,13H),3.56-3.07(m,24H), 2.56(s,6H),2.37(t,J＝5.8Hz,10H),2.17(t,J＝7.5Hz,9H),2.02-1.96(m,20H),1.88(s,8H),1.82-1.73(m,2H),1.60(dt,J＝15.0,7.3Hz,16H),1.27(s,13H)。
L96的合成
按照专利申请WO2014025805A1记载的方法制备获得如上结构式所示的L96。
实施例4：dsRNA的合成
1.自制带有载体的树脂
将含有羧酸基团的化合物NAG0052(157mg,0.062mmol)溶于无水DMF(3mL)，待底物完全溶解后，依次加入无水乙腈(4mL),DIEA(0.03mL,0.154mmol，2.5eq)和HBTU(35mg,0.093mmol，1.5eq)。反应液混合均匀后，再加入大孔胺甲基树脂(476mg,空白载量为0.41mmol/g,目标载量为0.1mmol/g)。将反应液放入摇床上(温度：25℃，转速：200rpm)振摇过夜。反应液过滤，滤饼依次分别用DCM，无水乙腈洗涤，收集固体，真空干燥过夜。
将上步固体分散于无水乙腈(5mL)，依次加入吡啶(0.18mL)，DMAP(3mg)，NMI(0.12mL)和CapB1(2.68mL)。将反应液放入摇床上(温度：25℃，转速：200rpm)振摇2h。反应液过滤，滤饼用无水乙腈洗涤，收集固体，真空干燥过夜，得到带有载体的树脂。载量经过测定为0.1mmol/g。
2.对于已经连接在树脂上的NAG0052，使用该树脂作为起始，按照核苷酸排布顺序自3’-5’方向逐一连接核苷单体。每连接一个核苷单体都包括脱保护、偶联、盖帽、氧化或硫化四步反应。操作为本领域常规。
化合物NAG0052经过固相合成连接到序列上，再经过胺解后，NAG0052结构脱去一部分官能团成为NAG0052’。制得的dsRNA具有表1和表2中所示的有义链和反义链。
表1.dsRNA列表
表2.有义链和反义链的核酸序列
在上表中，大写字母G、A、C、U分别表示包含鸟嘌呤、腺嘌呤、胞嘧啶和尿嘧啶的核苷酸，小写字母m表示2'-甲氧基修饰，小写字母f表示2'-氟代修饰，小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸二酯基连接；以下同。
实施例5：dsRNA在体内对靶基因mRNA表达量的抑制
本实验考察本公开的缀合不同的dsRNA在体内对靶基因mRNA表达量的抑制效率
将雄性6-8周龄C57BL/6小鼠随机分组，每组共6只，每个时间点各3只，分别向每组小鼠给予TRD007205、参比阳性TRD002218以及PBS。
所有动物依据体总计算给药量，采用皮下注射方式单次给药，dsRNA给药剂量(以无配体核苷酸的量计)为1mg/kg，给药体积为5mL/kg。给药7天、28天后处死小鼠，收集肝脏，用RNA later(Sigma Aldrich公司)保存；随后用组织匀浆仪匀浆肝组织，再用组织RNA提取试剂盒(凡知医疗科技，FG0412)根据操作说明书标注的操作步骤提取得到肝组织总RNA。将总RNA反转录成cDNA并采用实时荧光定量PCR方法检测肝组织中的TTR mRNA(转甲状腺蛋白mRNA)的表达量。在该荧光定量PCR法中，以甘油醛3-磷酸脫氫酶(GAPDH)基因作为内参基因，使用针对TTR和GAPDH的Taqman探针引物分别检测TTR和GAPDH的mRNA表达量。
表3.小鼠体内实验分组信息：
表4.检测引物的序列参见如下：
TTR mRNA表达量按照如下等式计算：
TTR mRNA表达量＝【(测试组TTR mRNA表达量/测试组GAPDH mRNA表达量)/(对照组TTR mRNA表达量/对照组GAPDH mRNA表达量)】x 100％。
给药7天、28天后，本公开的缀合不同结构的dsRNA的在体内对靶基因mRNA表达量的抑制效率分别见图1和图2。由图1的结果可知，TRD007205在给药后7天对于TTR mRNA的表达抑制均具有良好的效果。由图2可知，给药28天后，TRD007205对靶基因mRNA表达量的抑制作用均优于TRD002218。
实施例6：合成dsRNA
1.自制带有载体的树脂。
2.使用带有NAG0052的树脂作为起始，按照核苷酸排布顺序自3’-5’方向逐一连接核苷单体。每连接一个核苷单体都包括脱保护、偶联、盖帽、氧化或硫化四步反应。
制得的dsRNA具有表5和表6中所示的有义链和反义链。
表5.dsRNA列表

表6.dsRNA的有义链和反义链

其中，
(-)hmpNA(A)表示(-)hmpNA(G)表示
NAG0052’的结构为：
A(GNA)的结构为：
L10的结构为：
L96结构为
表7.dsRNA的有义链和反义链对应的裸序列
实施例7：人原代肝细胞(PHH)中人APOC3的抑制-7个浓度点抑制活性
在人原代肝细胞(PHH)中采用7个浓度梯度对dsRNA进行人原代肝细胞(PHH)活性筛选。各个dsRNA样品转染起始终浓度为10nM，5倍梯度稀释和7 个浓度点。
PHH冻存于液氮中，转染前24h，将人原代肝细胞(PHH)复苏后接种于96孔板，接种密度为每孔3×104个细胞，每孔80μL培养基。参照产品说明手册，使用Lipofectamine RNAi MAX(ThermoFisher，13778150)转染dsRNA，dsRNA转染的梯度终浓度为10nM，2nM，0.4nM，0.08nM，0.016nM，0.0032nM和0.00064nM。在处理24小时后，使用高通量细胞RNA提取试剂盒进行细胞总RNA提取、RNA逆转录实验和定量实时PCR检测，测定人APOC3的mRNA水平，根据GAPDH内参基因水平对人APOC3的mRNA水平进行校正。
其中，在实时定量PCR检测时，采用的是探针Q-PCR检测实验，其引物信息如表8所示。
表8.Taqman引物信息表
结果分析方法
Q-PCR检测实验完毕后，按照系统自动设定的阈值获取相应的Ct值，可以通过Ct值比较，相对定量某个基因的表达：比较Ct指的是通过与内参基因Ct值之间的差值来计算基因表达差异,也称之是2-△△Ct，△△Ct＝[(Ct实验组目的基因-Ct实验组内参)-(Ct对照组目的基因-Ct对照组内参)]。抑制率(％)＝(1-目的基因表达剩余量)*100％。
结果以相对于经过对照dsRNA处理的细胞的人APOC3mRNA表达剩余百分比来表示。抑制率的IC50结果见表9。表9实验结果表明，TRD008043优于其余组别。
表9.siRNA在PHH细胞中多剂量抑制活性

以相同的方法考察TRD007972-1与TJR100385、TJR100386的活性差异，抑制率的IC50结果见表10。
表10结果表明，TRD007972-1在PHH细胞中针对APOC3基因具有高水平的在靶抑制活性，优于TJR100385、TJR100386。
表10.dsRNA在PHH细胞中多剂量抑制活性
实施例8：dsRNA的psiCHECK反义链(AS链)脱靶水平验证
在HEK-293A细胞中采用9个浓度梯度对dsRNA进行体外分子水平模拟在靶及脱靶水平筛选。结果表明，TRD008043具有高活性的同时，还具有低脱靶性。
构建dsRNA序列对应的脱靶序列，插入到psiCHECK-2质粒中。该质粒包含海肾荧光素酶基因及萤火虫荧光素酶基因。作为双报告基因系统，dsRNA的靶序列插入到海肾荧光素酶基因的3’UTR区域，dsRNA对于靶标序列的活性可以通过经萤火虫荧光素酶校准后的海肾荧光素酶表达情况的检测来反映，检测使用Dual-Luciferase Reporter Assay System(Promega，E2940)。
dsRNA序列对应的GSSM靶标质粒构建规则如下：
针对dsRNA的反义链，构建与反义链5’端1-8位完全互补，而其它位置的碱基完全不匹配的脱靶质粒(GSSM)，碱基错配对应规则为A与C互配、G与T互配。
HEK293A细胞培养于含10％胎牛血清的DMEM高糖培养基中，在37℃，5％CO2条件下培养。转染前24h，将HEK293A细胞接种于96孔板，接种密度为每孔8×103个细胞，每孔100μL培养基。
按照说明书，使用Lipofectamine2000(ThermoFisher，11668019)对细胞共转染dsRNA及对应质粒，Lipofectamine2000每孔使用0.2μL，质粒转染量为20ng每孔。对于脱靶序列质粒，dsRNA共设置9个浓度点，最高浓度点终浓度为20nM， 3倍梯度稀释，20nM，6.6667nM，2.2222nM，0.7407nM，0.2469nM，0.0823nM，0.0274nM，0.0091nM，0.0030nM。转染后24h，采用Dual-Luciferase Reporter Assay System(Promega，E2940)检测脱靶水平。
结果如表11所示，结果表明，与TJR100383和TJR100384相比，TRD008043无脱靶风险。
表11.AS链种子区psiCHECK脱靶活性筛选结果
实施例9：Tg小鼠的药效学研究
小鼠为B6；CBA-Tg(APOC3)3707Bres/J，品系：#006907(Jackson实验室，11-12w)，以下简称APOC3Tg小鼠，雌雄各9只。动物到达后分笼饲养N＝2/笼，适应10天。每周测量一次体重。
给药前14天收集至少40μL血浆样本(～90μL血液)，用于适应期进行血浆中HDL、LDL、TG、TC和ApoC3第一次基线测量。第二次基线在给药前7天进行，收集至少40μL血浆样本(～90μL血液)进行第二次血浆中TG、TC、HDL-C和ApoC3检测；选取17只动物，根据TG和ApoC3(载脂蛋白C3)水平，分成3组。第三次基线在给药当天上午进行，收集至少40μL血浆样本(～90μL血液)进行TG、TC、HDL-C和ApoC3检测，这组数据将作为0点(第0天)基线。
供试品预配制时，用生理盐水(PH＝7.4)溶解，每种溶液取5μL稀释10倍，再用NanoDrop检测浓度3次/种。给药配方的最终浓度将根据NanoDrop的结果进行调整。单次给予生理盐水组和受试样品，均为皮下注射，注射体积根据给药前的体重计算而得。
给药后每周测量一次体重，第7、14、21、28、35和42天收集血浆样本(采集血液前，过夜禁食)进行血浆中ApoC3、TG、TC和HDL-C检测。血样采集后半小时内离心(离心条件：4℃,3000g,5min)。将血浆收集到2支聚丙烯管中，体积分别为30μL(准确体积)和10～15μL(其余)，干冰速冻，样品分析前保存在-80℃冰箱中。结果如表13-表15所示。
表12.实验分组及给药方案
表13.APOC3Tg小鼠单次给予受试物后血浆中APOC3与0天基线的百分比

***p<0.001,**p<0.01,*p<0.05，p均为与生理盐水组相比。One-way ANOVA 
Dunnett's test。
表13结果表明：APOC3Tg小鼠单次给予受试样品后，TRD008043组小鼠血浆中APOC3与生理盐水组比较，第7天、第14天、第21天、第28天、第35天、第42天与0天的基线的百分比有显著的降低。而TRD007972-1组小鼠血浆中APOC3与生理盐水组比较，第35天、第42天与0天的基线的百分比无明显差异。同时，从数值上看，TRD008043组在单次给药后血浆中APOC3与0天基线百分比在D14-D35约比TRD007972-1低3倍。
结果表明，TRD008043与TRD007972-1相比，TRD008043降低人APOC3的效果优于TRD007972-1。
表14.APOC3Tg小鼠单次给予受试物后血浆中TG与0天基线的百分比


***p<0.001,**p<0.01,*p<0.05，p均为与生理盐水组相比,One-way ANOVA 
Dunnett's test
表14结果表明，APOC3Tg小鼠单次给予受试样品后，至第28天，与生理盐水组比较，TRD008043仍保持显著降低血浆中TG的作用，但TRD007972-1与生理盐水组比较已无显著性区别。同时，从数值上看，TRD008043组在单次给药后血浆中TG与0天的基线百分比在D14-D35约比TRD007972-1组低2-4倍。
结果表明，TRD008043与TRD007972-1相比，TRD008043降低TG的效果优于TRD007972-1。
表15.APOC3Tg小鼠单次给予受试物后血浆中TC与0天基线的百分比

*p<0.05，p为与生理盐水组相比,One-way ANOVA Dunnett's test
表15结果表明，APOC3Tg小鼠单次给予受试样品后，至第28天，与生理盐水组比较，TRD008043仍保持显著降低血浆中TC的作用，但TRD007972-1与生理盐水组比较均无显著性区别。同时，从数值上看，TRD008043组在单次给药后血浆中TC与0天的基线百分比在D14-D35约比TRD007972-1组低2倍。
结果表明，TRD008043与TRD007972-1相比，TRD008043降低TC的效果优于TRD007972-1。

Claims (21)

  1. 一种双链核糖核酸(dsRNA)，其包含有义链和反义链，所述有义链和反义链按照5'端到3'端的方向包含连续的核苷酸，其中
    所述有义链第7、8和9位的核苷酸为2'-氟代修饰的核苷酸，第5位的核苷酸独立地为2'-甲氧基修饰的核苷酸或2'-氟代修饰的核苷酸，其余位置的核苷酸为2'-甲氧基修饰的核苷酸；
    所述反义链第2和14位的核苷酸为2'-氟代修饰的核苷酸，第4、6、8、9、10、12、16和18位的核苷酸独立地为2'-甲氧基或2'-氟代修饰的核苷酸，其余位置的核苷酸为2'-甲氧基修饰的核苷酸；
    所述反义链中2'-氟代修饰的核苷酸的个数为2-7个；
    所述反义链第7位的核苷酸位置处包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐：
    所述式(I)所示的化学修饰选自以下任一结构：
    B是所述反义链在其5’区域的第7位
    核苷酸未被修饰时的碱基；
    并且
    所述dsRNA抑制载脂蛋白C3(APOC3)的表达。
  2. 如权利要求1所述的dsRNA，其中，
    所述有义链含有如下式所示的核苷酸：
    5’-NaNaNaNaNaNaNbNbNbNaNaNaNaNaNaNaNaNaNa-3’；
    其中，Na为2'-甲氧基修饰的核苷酸，Nb为2'-氟代修饰的核苷酸。
  3. 如权利要求1或2所述的dsRNA，其中，
    所述反义链包含如下式所示的核苷酸序列：
    5’-Na’Nb’Na’X’Na’X’W’X’X’X’Na’X’Na’Nb’Na’X’Na’X’Na’Na’Na’-3’；
    优选地，所述反义链包含如下式所示的核苷酸序列：
    5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’；
    5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’；
    5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’；
    5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’；
    5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Nb’Na’Na’Na’-3’；
    5’-Na’Nb’Na’Nb’Na’Nb’W’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’；
    5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Nb’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’；
    5’-Na’Nb’Na’Na’Na’Na’W’Nb’Nb’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’；
    5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’；
    5’-Na’Nb’Na’Na’Na’Nb’W’Na’Na’Na’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’；
    5’-Na’Nb’Na’Na’Na’Na’W’Na’Na’Na’Na’Na’Na’Nb’Na’Nb’Na’Na’Na’Na’Na’-3’；或，
    5’-Na’Nb’Na’Na’Na’Na’W’Na’Na’Na’Na’Na’Na’Nb’Na’Na’Na’Na’Na’Na’Na’-3’；
    其中，每个X’独立地为Na’或Nb’；Na’为2'-甲氧基修饰的核苷酸，Nb’为2'-氟代修饰的核苷酸；
    W’表示包含式(I)所示的化学修饰、其互变异构体或其药学上可接受的盐的核苷酸。
  4. 如权利要求1-3中任一项所述的dsRNA，其中，所述有义链和/或反义链中至少一个磷酸酯基为具有修饰基团的磷酸酯基，优选为硫代磷酸酯基，更优选为硫代磷酸二酯基。
  5. 如权利要求4所述的dsRNA，其中，所述硫代磷酸二酯基存在于以下位置中的至少一处：
    所述有义链的5'端第1个核苷酸和第2个核苷酸之间；
    所述有义链的5'端第2个核苷酸和第3个核苷酸之间；
    所述反义链的5'端第1个核苷酸和第2个核苷酸之间；
    所述反义链的5'端第2个核苷酸和第3个核苷酸之间；
    所述反义链的3'端第1个核苷酸和第2个核苷酸之间；以及
    所述反义链的3'端第2个核苷酸和第3个核苷酸之间；
    优选地，所述有义链和/或反义链中包括多个硫代磷酸二酯基，所述硫代磷酸二酯基存在于：
    所述有义链的5'端第1个核苷酸和第2个核苷酸之间；和，
    所述有义链的5'端第2个核苷酸和第3个核苷酸之间；和，
    所述反义链的5'端第1个核苷酸和第2个核苷酸之间；和，
    所述反义链的5'端第2个核苷酸和第3个核苷酸之间；和，
    所述反义链的3'端第1个核苷酸和第2个核苷酸之间；和，
    所述反义链的3'端第2个核苷酸和第3个核苷酸之间。
  6. 如权利要求1-5中任一项所述的dsRNA，其中，
    所述有义链包含与SEQ ID NO:1的核苷酸序列相差不超过3个核苷酸的至少15个连续核苷酸；并且
    所述反义链包含与SEQ ID NO:2的核苷酸序列相差不超过3个核苷酸的至少19个连续核苷酸；
    优选地，
    所述有义链包含SEQ ID NO:1所示的核苷酸序列，并且反义链包含SEQ ID NO:2所示的核苷酸序列。
  7. 如权利要求1-6中任一项所述的dsRNA，其中，
    所述反义链包含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和SEQ ID NO:14中任一项所示的核苷酸序列；
    优选地，所述有义链包含SEQ ID NO:3所示的核苷酸序列。
  8. 如权利要求1-7中任一项所述的dsRNA，其中，所述dsRNA还包括与其缀合的配体，所述配体包含N-乙酰基-半乳糖胺。
  9. 如权利要求8所述的dsRNA，其中，
    所述配体具有如下结构或其药学上可接受的盐，
  10. 如权利要求8或9中任一项所述的dsRNA，其中，所述dsRNA有义链的3’端与所述配体缀合。
  11. 如权利要求8-10中任一项所述的dsRNA，其中，所述配体通过磷酸酯基团或硫代磷酸酯基团与所述dsRNA有义链的3’端连接；优选通过磷酸二酯基团或硫代磷酸二酯基团连接，更优选通过磷酸二酯基团连接。
  12. 如权利要求1-11中任一项所述的dsRNA，其中，
    所述反义链包含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、SEQ ID NO:14任一项所示的核苷酸序列；
    优选地，所述有义链包含SEQ ID NO:4所示的核苷酸序列。
  13. 如权利要求1-12中任一项所述的dsRNA，其中，所述dsRNA具有如下结构或其药学上可接受的盐：
    其中，Af＝腺嘌呤2'-F核糖核苷；Cf＝胞嘧啶2'-F核糖核苷；Uf＝尿嘧啶2'-F核糖核苷；Gf＝鸟嘌呤2'-F核糖核苷；Am＝腺嘌呤2'-OMe核糖核苷；Cm＝胞嘧啶2'-OMe核糖核苷；Gm＝鸟嘌呤2'-OMe核糖核苷；Um＝尿嘧啶2'-OMe核糖核苷；
    表示硫代磷酸二酯基，表示磷酸二酯基；
    NAG0052’表示(-)hmpNA(A)表示
  14. 一种药物组合物，其包含如权利要求1-13中任一项所述的dsRNA；任选地，所述的药物组合物还包含一种或多种药学上可接受的赋形剂。
  15. 一种载体，其包含如权利要求1-13中任一项所述的dsRNA。
  16. 一种细胞，其包含如权利要求1-13中任一项所述的dsRNA，或如权利要求15所述的载体。
  17. 一种如权利要求1-13中任一项所述的dsRNA、如权利要求14所述的药物组合物、如权利要求15所述的载体、或如权利要求16所述的细胞在制备药物中的应用；
    所述的药物用于降低受试者中的甘油三酯水平，或用于预防和/或治疗由升高的甘油三酯水平或升高的胆固醇水平介导的疾病；优选地，所述的由升高的甘油三酯水平或升高的胆固醇水平介导的疾病选自高甘油三酯血症、肥胖症、高脂血症、脂质和/或胆固醇代谢异常、动脉粥样硬化、心血管疾病、冠状动脉疾病、高甘油三酯血症诱导的胰腺炎、代谢综合征、II型糖尿病、家族性乳糜微粒血症综合征或家族性部分脂质营养不良。
  18. 一种抑制APOC3基因或其mRNA表达的方法，其包括向受试者给予有效量或有效剂量的如权利要求1-13中任一项所述的dsRNA、如权利要求14所述的药物组合物、如权利要求15所述的载体、或如权利要求16所述的细胞。
  19. 一种递送寡核苷酸至肝脏的方法，其包括向受试者给予有效量或有效剂量的如权利要求8-13中任一项所述的dsRNA、如权利要求14所述的药物组合物、如权利要求15所述的载体、或如权利要求16所述的细胞。
  20. 一种试剂盒，其包含如权利要求1-13中任一项所述的dsRNA、如权利要求14中任一项所述的药物组合物、如权利要求15所述的载体、或如权利要求16所述的细胞。
  21. 一种制备dsRNA或药物组合物的方法，其包括：合成如权利要求1-13中任一项所述dsRNA。
PCT/CN2023/073166 2022-01-20 2023-01-19 一种dsRNA、其应用及制备方法 Ceased WO2023138659A1 (zh)

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