Sirna used for inhibiting HSD17b13 expression, conjugate thereof, pharmaceutical composition and use thereof

Abstract

Provided are a siRNA capable of inhibiting HSD17B13 gene expression, a siRNA conjugate, a prodrug, a pharmaceutical composition comprising same, and the use thereof. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, and the siRNA contains a sense strand and an antisense strand. The siRNA, the conjugate thereof and the pharmaceutical composition can effectively treat and / or prevent diseases related to the overexpression of the HSD17B13 gene.

Description

siRNA for inhibiting HSD17B13 expression, its conjugate and pharmaceutical composition and use thereof Technical Field

[0001] The present application relates to siRNA, siRNA conjugates, prodrugs, pharmaceutical compositions containing the same, and preparation methods and uses thereof that can inhibit HSD17B13 gene expression. Background Art

[0002] The 17β-hydroxysteroid dehydrogenase (17β-HSD) family consists of 15 enzymes, most of which are involved in activation or inactivation by sex hormones (e.g., HSD17B1, HSD17B2, HSD17B3, HSD17B5, and HSD17B6). Other members are involved in fatty acid metabolism, cholesterol biosynthesis, and bile acid production. Members of the HSD17B family vary in tissue distribution, subcellular localization, catalytic preference, and substrate specificity (Marchais Oberwinkler, et al. (2011) J Steroid Biochem Mol Biol 125(1-2):66–82).

[0003] HSD17B13, a member of the 17β-hydroxysteroid dehydrogenase family, is primarily localized in hepatocytes. Its highest expression levels are found in liver hepatocytes, while only lower levels can be detected in the ovary, bone marrow, kidney, brain, lung, skeletal muscle, bladder, and testis. It is a hepatocyte-specific lipid droplet (LD)-associated protein, and increasing evidence suggests that it plays a key role in hepatic lipid metabolism. The function of HSD17B13 is not fully understood; however, several 17β-HSD family members, including 17β-HSD-4, -7, -10, and -12, have been shown to be involved in carbohydrate and fatty acid metabolism. This suggests that HSD17B13 may also play a role in lipid metabolism pathways. Hepatic upregulation of HSD17B13 has been reported in patients with fatty liver disease, supporting a role for this enzyme in the pathogenesis of non-alcoholic fatty liver disease (NAFLD).

[0004] Non-alcoholic fatty liver disease (NAFLD), also known as metabolic (dysfunction)-associated fatty liver disease (MAFLD), is the accumulation of excess fat in the liver in the absence of other clear causes (such as alcohol consumption). NAFLD is the most common liver disease in the world, affecting approximately 25% of the world's population. The prevalence of NAFLD is currently on the rise, a trend that will undoubtedly increase the economic burden and lead to a sharp increase in the number of patients with end-stage liver disease requiring liver transplantation and the number of people suffering from hepatocellular carcinoma. There is currently no specific treatment for NAFLD, which mainly involves weight loss through dietary changes and exercise. Preliminary studies have shown that pioglitazone and vitamin E have therapeutic potential.

[0005] Wen Su et al. have previously identified HSD17B13 as a lipid droplet (LD)-associated protein in NAFLD patients and reported that HSD17B13 is one of the most abundantly expressed LD proteins that is specifically localized on the surface of LDs (Wen Su, et al., Comparative proteomic study reveals 17β-HSD13 as a pathogenic protein in nonalcoholic fatty live disease, 111 PNAS 11437-11442 (2014)). Furthermore, it was found that the level of HSD17B13 is upregulated in the livers of patients and mice with NAFLD. Overexpression leads to an increase in the number and size of LDs, while gene silencing of HSD17B13 attenuates oleic acid-induced LD formation in cultured hepatocytes. It has also been shown that hepatic overexpression of the HSD17B13 protein in C57BL / 6 mice significantly increases lipogenesis and triglyceride (TG) content in the liver, leading to a fatty liver phenotype. NSAbul-Husn et al. provide additional evidence implicating HSD17B13 gene expression in the pathogenesis of NAFLD and non-alcoholic steatohepatitis (NASH) (NSAbul-Husn et al., A Protein-Truncating HSD17B13 Variant and Protection from Chronic Liver Disease, 378 N.Eng.J.Med.1096-1106 (2018)). The team conducted a genome-wide association study, which revealed that an HSD17B13 splice variant (rs72613567:TA) associated with reduced levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indicated less liver damage and inflammation in patients with fatty liver disease. The splice variant produces a truncated loss of functional protein, suggesting that HSD17B13 normally produces products that can promote liver cell damage. ARO-HSD, an RNAi therapy targeting HSD17B13, co-developed by GlaxoSmithKline and Arrowhead, is entering Phase I clinical trials for the treatment of non-alcoholic steatohepatitis. Currently, there are no marketed nucleic acid drugs targeting HSD17B13.

[0006] The present invention aims to provide siRNA, siRNA conjugates, prodrugs, and pharmaceutical compositions thereof, which can affect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the HSD17B13 gene, thereby inhibiting the expression of the HSD17B13 gene in the liver and achieving the purpose of disease treatment. Summary of the Invention

[0007] The present invention provides an siRNA capable of inhibiting HSD17B13 gene expression, wherein the siRNA comprises a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I and the nucleotide sequence II are selected from the following sequences:

[0008] (1) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 25, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 26;

[0009] (2) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 40, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 41;

[0010] (3) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 53, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 54;

[0011] (4) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 140, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 141:

[0012] 5'-GUAUGCAGAAUAUU-3'(SEQ ID NO:140)

[0013] 5'-AAUAUUCUGCAUAC-3' (SEQ ID NO: 141);

[0014] (5) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 142, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 143:

[0015] 5'-GAAUAUUCAAUUUGAA-3'(SEQ ID NO:142)

[0016] 5'-UUCAAAUUGAAUAUUC-3' (SEQ ID NO: 143);

[0017] (6) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 1, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 2;

[0018] (7) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 28.

[0019] In one embodiment, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary, or completely reverse complementary; the substantially reverse complementary refers to that there are no more than 3 base mismatches between the two nucleotide sequences; the substantially reverse complementary refers to that there is no more than 1 base mismatch between the two nucleotide sequences; and the completely reverse complementary refers to that there is no mismatch between the two nucleotide sequences.

[0020] In one embodiment, the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV, and the length of the nucleotide sequence III and the nucleotide sequence IV are each independently 0-7 nucleotides, wherein the nucleotide sequence III is linked to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is linked to the 3' end of the nucleotide sequence II, and the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the substantially reverse complementary means that there is no more than one base mismatch between the two nucleotide sequences; and completely reverse complementary means that there is no mismatch between the two nucleotide sequences; and / or,

[0021] The nucleotide sequence III is connected to the 3' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 5' end of the nucleotide sequence II. The nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the substantially reverse complementary means that there is no more than one base mismatch between the two nucleotide sequences; and the completely reverse complementary means that there is no mismatch between the two nucleotide sequences.

[0022] In one embodiment, the siRNA comprises a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains nucleotide sequences I and III, and the antisense strand contains nucleotide sequences II and IV, and the nucleotide sequences I and III are at least partially reverse complementary to the nucleotide sequences II and IV to form a double-stranded region, wherein the nucleotide sequences I and III, the nucleotide sequences II and IV are selected from the following sequences:

[0023] (1) the nucleotide sequences I and III comprise or consist of the nucleotide sequence shown in SEQ ID NO: 112, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequence shown in SEQ ID NO: 113;

[0024] (2) The nucleotide sequences I and III comprise or consist of the nucleotide sequence shown in SEQ ID NO: 208, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequence shown in SEQ ID NO: 209.

[0025] In one embodiment, the sense strand further contains a nucleotide sequence V and / or the antisense strand further contains a nucleotide sequence VI, the length of the nucleotide sequences V and VI is 0 to 3 nucleotides, the nucleotide sequence V is connected to the 3' end of the sense strand to form the 3' overhang of the sense strand, and / or the nucleotide sequence VI is connected to the 3' end of the antisense strand to form the 3' overhang of the antisense strand. In a preferred embodiment, the length of the nucleotide sequence V or VI is 2 nucleotides. In a preferred embodiment, the nucleotide sequence V or VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides. In a preferred embodiment, the nucleotide sequence V is identical to or different from the nucleotide at the corresponding position of the target mRNA, or the nucleotide sequence VI is mismatched or complementary to the nucleotide at the corresponding position of the target mRNA.

[0026] In one embodiment, the length of the double-stranded region is 15-30 nucleotide pairs; preferably, the length of the double-stranded region is 17-23 nucleotide pairs; more preferably, the length of the double-stranded region is 19-21 nucleotide pairs.

[0027] In another embodiment, the sense strand or antisense strand has 15-30 nucleotides; preferably, the sense strand or antisense strand has 19-25 nucleotides; more preferably, the sense strand or antisense strand has 19-23 nucleotides.

[0028] In one embodiment, at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide, and / or at least one phosphate group is a phosphate group having a modified group; preferably, the phosphate group having a modified group is a thiophosphate group formed by replacing one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom.

[0029] In one embodiment, the siRNA comprises a sense strand that does not include 3' overhanging nucleotides.

[0030] In one embodiment, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate-derivative group, or the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derivative group.

[0031] In one embodiment, the 3' terminal nucleotide of the sense strand is linked to an inverted abasic deoxyribose residue, and / or the 5' terminal nucleotide of the sense strand is linked to an inverted abasic deoxyribose residue.

[0032] In one embodiment, the inverted abasic deoxyribose residue is linked to the 3' terminal nucleotide and / or the 5' terminal nucleotide of the sense strand via a phosphodiester bond, a phosphorothioate group or other internucleoside linkage.

[0033] In one embodiment, the modified nucleotide is selected from 2'-fluoro-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-deoxyribonucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, nucleotide analogs or a combination of any two or more thereof.

[0034] In one embodiment, the modified nucleotide is selected from 2'-fluoro modified nucleotides, 2'-methoxy modified nucleotides, 2'-O-CH2-CH2-O-CH3 modified nucleotides, 2'-O-CH2-CH=CH2 modified nucleotides, 2'-CH2-CH2-CH=CH2 modified nucleotides, 2'-deoxyribonucleotides, nucleotide analogs or a combination of any two or more thereof.

[0035] In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

[0036] In a preferred embodiment, in the sense strand, from 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11, and the remaining positions are non-fluoro-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 9, 10 and 11, and the remaining positions are non-fluoro-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 8, 9 and 10, and the remaining positions are non-fluoro-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 8, 9, 10 and 11, and the remaining positions are non-fluoro-modified nucleotides; or Alternatively, the 2'-fluoro-modified nucleotides are located at positions 7 and 9, and the remaining positions are non-fluorinated modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 7, 9 and 11, and the remaining positions are non-fluorinated modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 11, 13 and 15, and the remaining positions are non-fluorinated modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 11 and 13, and the remaining positions are non-fluorinated modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 8 and 9, and the remaining positions are Non-fluorinated modified nucleotides; or, 2'-fluorinated modified nucleotides are located at positions 3, 9, 11 and 13, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluorinated modified nucleotides are located at positions 3, 7, 9 and 11, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluorinated modified nucleotides are located at positions 7, 11 and 13, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluorinated modified nucleotides are located at positions 7, 11, 12 and 13, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluorinated modified nucleotides are located at positions 3, 7, 11 and 16, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 3, 7, 11, 16 and 17, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 3, 7 and 9, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 7, 9 and 14, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 5, 7, 8 and 9, and the remaining positions are non-fluorinated modified nucleotides; and / or

[0037] In the antisense strand, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16, and the remaining positions are non-fluoro-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16, and the remaining positions are non-fluoro-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 2, 14 and 16, and the remaining positions are non-fluoro-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 2, 6 and 14, and the remaining positions are non-fluoro-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9 and 14, and the remaining positions are non-fluoro-modified nucleotides. The remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 5, 8, 10, 14, 16 and 18, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro-modified nucleotides are located at positions 2, 4, 6, 12, 14, 16 and 18, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro-modified nucleotides are located at positions 2 and 14, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro-modified nucleotides are located at positions 2 and 14, and the remaining positions are non-fluorinated modified nucleotides; Nucleotides are located at positions 2, 4, 5, 6, 8, 10, 12, 14, 16 and 18, and the remaining positions are non-fluorinated modified nucleotides; alternatively, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10 and 14, and the remaining positions are non-fluorinated modified nucleotides; alternatively, 2'-fluoro-modified nucleotides are located at positions 2, 5, 7 and 14, and the remaining positions are non-fluorinated modified nucleotides; alternatively, 2'-fluoro-modified nucleotides are located at positions 2, 7, 12 and 14, and the remaining positions are non-fluorinated modified nucleotides; alternatively, 2'-fluoro-modified nucleotides are located at positions 2, 6, 12 and 14, and the remaining positions are non-fluorinated modified nucleosides Acid; or, the 2'-fluoro modified nucleotides are located at positions 2, 3, 6 and 14, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro modified nucleotides are located at positions 2, 6, 8 and 14, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro modified nucleotides are located at positions 2, 10 and 14, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro modified nucleotides are located at positions 2, 7 and 14, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro modified nucleotides are located at positions 2, 3, 5, 7, 10, 12 and 14, and the remaining positions are non-fluorinated modified nucleotides.

[0038] In one embodiment, each non-fluorinated modified nucleotide is independently selected from a nucleotide or nucleotide analogue formed by replacing the hydroxyl group at the 2' position of the ribose group of the nucleotide with a non-fluorinated group, and the nucleotide analogue is selected from one of pseudouracil, isonucleotide, LNA, ENA, cET BNA, UNA and GNA.

[0039] In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a 2'-deoxyribonucleotide, a GNA-modified nucleotide, or a combination of any two or more thereof.

[0040] In a preferred embodiment, in the sense strand, from 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 9, 10 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 8, 9 and 10, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 8, 9, 10 and 1 ... The modified nucleotides are located at positions 7 and 9, the 2'-deoxyribonucleotide is located at position 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 7, 9, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 11, 13, and 15, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 11, and 13, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 8, and 9 2'-methoxy modified nucleotides at positions 3, 9, 11, and 13, and 2'-methoxy modified nucleotides at other positions; or, 2'-fluoro modified nucleotides at positions 3, 7, 9, and 11, and 2'-methoxy modified nucleotides at other positions; or, 2'-fluoro modified nucleotides at positions 7, 11, and 13, and 2'-methoxy modified nucleotides at other positions; or, 2'-fluoro modified nucleotides at positions 7, 11, 12, and 13, and 2'-methoxy modified nucleotides at other positions; or, 2'-fluoro modified nucleotides at positions 7, 11, 12, and 13, and 2'-methoxy modified nucleotides at other positions; or The nucleotides are located at positions 3, 7, 11 and 16, and the remaining positions are 2'-methoxy modified nucleotides; or, the 2'-fluoro modified nucleotides are located at positions 3, 7, 11, 16 and 17, and the remaining positions are 2'-methoxy modified nucleotides; or, the 2'-fluoro modified nucleotides are located at positions 3, 7 and 9, and the remaining positions are 2'-methoxy modified nucleotides; or, the 2'-fluoro modified nucleotides are located at positions 7, 9 and 14, and the remaining positions are 2'-methoxy modified nucleotides; or, the 2'-fluoro modified nucleotides are located at positions 5, 7, 8 and 9, and the remaining positions are 2'-methoxy modified nucleotides; and / or

[0041] In the antisense strand, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 2, 14 and 16, the GNA-modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16, the GNA-modified nucleotide is located at position 7 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 2, 6 and 14, and the remaining positions are 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 2, 6, 8, 9 and 14, and the remaining positions are 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 2, 3, 5, 8, 10, 14, 16 and 18, and the remaining positions are 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14, and the remaining positions are 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 2, 4, 6, 12, 14, 16 and 18, with 2'-methoxy-modified nucleotides at the remaining positions; alternatively, 2'-fluoro-modified nucleotides are located at positions 2 and 14, with 2'-methoxy-modified nucleotides at the remaining positions; alternatively, 2'-fluoro-modified nucleotides are located at positions 2, 4, 5, 6, 8, 10, 12, 14, 16 and 18, with 2'-methoxy-modified nucleotides at the remaining positions; alternatively, 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14, a GNA-modified nucleotide is located at position 6 of the antisense strand, with 2'-methoxy-modified nucleotides at the remaining positions; alternatively, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10 and 14, with 2'-methoxy-modified nucleotides at the remaining positions 2'-fluoro-modified nucleotides at positions 2, 5, 7 and 14, with 2'-methoxy-modified nucleotides at the remaining positions; or, 2'-fluoro-modified nucleotides at positions 2, 7, 12 and 14, with 2'-methoxy-modified nucleotides at the remaining positions; or, 2'-fluoro-modified nucleotides at positions 2, 6, 12 and 14, with 2'-methoxy-modified nucleotides at the remaining positions; or, 2'-fluoro-modified nucleotides at positions 2, 3, 6 and 14, with 2'-methoxy-modified nucleotides at the remaining positions; or, 2'-fluoro-modified nucleotides at positions 2, 6, 8 and 14, with 2'-methoxy-modified nucleotides at the remaining positions;Alternatively, 2'-fluoro modified nucleotides are located at positions 2, 10, and 14, the nucleotide at position 7 is replaced by a deoxyribonucleotide, a 2'-deoxyribonucleotide is located at position 5 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, 2'-fluoro modified nucleotides are located at positions 2, 7, and 14, the nucleotide at position 12 is replaced by a deoxyribonucleotide, a 2'-deoxyribonucleotide is located at position 5 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, 2'-fluoro modified nucleotides are located at positions 2 and 14, the nucleotides at positions 7 and 12 are replaced by deoxyribonucleotides, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, 2'-fluoro modified nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14, a GNA modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides.

[0042] In some embodiments, the siRNA is directed from the 5' end to the 3' end.

[0043] (1) The sense strand contains phosphorothioate groups located at the following positions:

[0044] Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand; and

[0045] Between the second and third nucleotides starting from the 5' end of the sense strand; and

[0046] Between the first nucleotide and the second nucleotide starting from the 3' end of the sense strand; and

[0047] Between the second and third nucleotides starting from the 3' end of the sense strand;

[0048] or,

[0049] (2) The sense strand comprises a phosphorothioate group located at the following position:

[0050] Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand; and

[0051] Between the second and third nucleotides starting from the 5' end of the sense strand;

[0052] or,

[0053] (3) The sense strand comprises a phosphorothioate group located at the following position:

[0054] Between the first and second nucleotides starting from the 5' end of the sense strand;

[0055] or,

[0056] (4) The sense strand comprises a phosphorothioate group located at the following position:

[0057] Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand; and

[0058] Between the second and third nucleotides starting from the 5' end of the sense strand; and

[0059] between the reverse abasic deoxyribose residue starting at the 3' end of the sense strand and the first nucleotide;

[0060] or

[0061] (5) The sense strand comprises a phosphorothioate group located at the following position:

[0062] between the inverted abasic deoxyribose residue starting at the 5' end of the sense strand and the first nucleotide; and

[0063] Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand; and

[0064] The positive strand is located between the inverted abasic deoxyribose residue starting at the 3' end and the first nucleotide.

[0065] In some embodiments, the antisense strand of the siRNA comprises phosphorothioate groups located at the following positions along the 5' to 3' direction:

[0066] Between the first nucleotide and the second nucleotide starting from the 5' end of the antisense strand; and

[0067] Between the second and third nucleotides starting from the 5' end of the antisense strand; and

[0068] Between the first nucleotide and the second nucleotide starting from the 3' end of the antisense strand; and

[0069] between the second and third nucleotides starting from the 3' end of the antisense strand.

[0070] In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a 2'-deoxyribonucleotide, a GNA-modified nucleotide, or a combination of any two or more thereof.

[0071] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group.

[0072] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group.

[0073] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, the GNA-modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group.

[0074] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, the GNA-modified nucleotide is located at position 7 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group.

[0075] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is not connected to a 5' phosphate group or a 5' phosphate derivative group.

[0076] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is not connected to a 5' phosphate group or a 5' phosphate-derived group.

[0077] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0078] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0079] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is not connected to a 5' phosphate group or a 5' phosphate-derived group.

[0080] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is not connected to a 5' phosphate group or a 5' phosphate-derived group.

[0081] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0082] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 8, 9 and 10 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0083] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 5, 8, 10, 14, 16 and 18 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0084] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0085] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 8, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0086] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 4, 6, 12, 14, 16 and 18 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0087] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0088] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7 and 9 of the sense strand, 2'-deoxyribonucleotides are located at position 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 4, 5, 6, 8, 10, 12, 14, 16 and 18 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0089] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0090] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 11, 13 and 15 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0091] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 11 and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0092] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 8 and 9 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0093] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 9, 11 and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0094] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 9 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0095] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 11 and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0096] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 11, 12 and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0097] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 11 and 16 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0098] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 11, 16 and 17 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0099] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14 of the antisense strand, the 6th position is a GNA-modified nucleotide, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0100] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0101] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 5, 7 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0102] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 8 and 9 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0103] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 9 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0104] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 9 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 5, 7 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0105] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, and 9 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 5, 7, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0106] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, and 9 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0107] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 9 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7, 12 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0108] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 9 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 12 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0109] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 9 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 6 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0110] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 9 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0111] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 3, 7, 9 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 10 and 14 of the antisense strand, the 7th nucleotide is replaced by a thymine nucleotide, a 2'-deoxyribonucleotide is located at position 5 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0112] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 3, 7, 9 and 11 of the sense strand, and 2'-methoxy-modified nucleotides are located at the remaining positions; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7 and 14 of the antisense strand, the 12th nucleotide is replaced by a thymine nucleotide, a 2'-deoxyribonucleotide is located at position 5 of the antisense strand, and 2'-methoxy-modified nucleotides are located at the remaining positions, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0113] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 3, 7, 9 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2 and 14 of the antisense strand, the 7th and 12th nucleotides are replaced with thymine nucleotides, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0114] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9 and 14 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0115] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 8 and 9 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 5, 7, 10, 12 and 14 of the antisense strand, the 6th position is a GNA-modified nucleotide, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0116] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 9 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 5, 7, 10, 12 and 14 of the antisense strand, the 6th position is a GNA-modified nucleotide, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

[0117] In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 5, 7, 8 and 9 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides.

[0118] In a specific embodiment, the present invention provides an siRNA selected from Table 1, preferably, the siRNA is selected from N-ER-FY007180, N-ER-FY007180M8, N-ER-FY007180M44, N-ER-FY007180M56, N-ER-FY007180M58, N-ER-FY007180M62, N-ER-FY007180M2, N-ER-FY007180M3, N-ER-FY007180M4, N-ER-FY007180M5, N-ER-FY007180M6, N-ER-FY007180M7 M7、N-ER-FY007180M9、N-ER-FY007180M10、N-ER-FY007180M11、N-ER-FY007180M12、N-ER-FY007180M13、N-ER-FY007180M14、N-ER-FY00718 0M15、N-ER-FY007180M16、N-ER-FY007180M17、N-ER-FY007180M18、N-ER-FY007180M19、N-ER-FY007180M21、N-ER-FY007180M24、N-ER-FY007 180M26, N-ER-FY007180M27, N-ER-FY007180M28, N-ER-FY007180M29, N-ER-FY007180M30, N-ER-FY007180M31, N-ER-FY007180M32, N-ER-FY 007180M34, N-ER-FY007180M35, N-ER-FY007180M37, N-ER-FY007180M40, N-ER-FY007180M45, N-ER-FY007180M46, N-ER-FY007180M47, N-ER- FY007180M48, N-ER-FY007180M49, N-ER-FY007180M50, N-ER-FY007180M51, N-ER-FY007180M52, N-ER-FY007180M53, N-ER-FY007180M54, N- ER-FY007180M55, N-ER-FY007180M57, N-ER-FY007180M59, N-ER-FY007180M60, N-ER-FY007180M61, N-ER-FY007180M63, N-ER-FY007180M64.

[0119] The present invention also provides an siRNA conjugate or a prodrug thereof, wherein the siRNA conjugate contains the siRNA of the present invention and a conjugated group conjugated to the siRNA (as shown in the following formula, a double helix structure represents the siRNA, and the conjugated group is connected to the 3' end of the sense strand of the siRNA):

[0120] In the above conjugate structure, X can be selected from O or S. In one embodiment, X is O.

[0121] In one embodiment, the conjugate group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked.

[0122] Preferably, in the siRNA conjugate, the sense strand and the antisense strand of the siRNA are complementary to each other to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region;

[0123] or,

[0124] In the siRNA conjugate, the sense strand and the antisense strand of the siRNA are complementary to each other to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, while the 3' end of the antisense strand forms a blunt end.

[0125] In one embodiment, the conjugated group is selected from:

[0126] In a specific embodiment, the siRNA conjugate is an siRNA conjugate selected from Table 2; preferably, the siRNA conjugate is selected from N-ER-FY007180M8L96, N-ER-FY007180M44L96, N-ER-FY007180M56L96, N-ER-FY007180M58L96, N-ER-FY007180M62L96, N-ER-FY007180M2L96, N-ER-FY007180M3L96, N-ER-FY007180M6L96, N-ER-FY007180 M7L96, N-ER-FY007180M9L96, N-ER-FY007180M10L96, N-ER-FY007180M11L96, N-ER-FY007180M13L96, N-ER-FY007180M15L96, N-ER-F Y007180M21L96, N-ER-FY007180M24L96, N-ER-FY007180M26L96, N-ER-FY007180M27L96, N-ER-FY007180M29L96, N-ER-FY007180M31L 96. N-ER-FY007180M32L96, N-ER-FY007180M34L96, N-ER-FY007180M35L96, N-ER-FY007180M37L96, N-ER-FY007180M40L96, N-ER-FY 007180M45L96, N-ER-FY007180M46L96, N-ER-FY007180M47L96, N-ER-FY007180M48L96, N-ER-FY007180M49L96, N-ER-FY007180M50L9 6. N-ER-FY007180M51L96, N-ER-FY007180M52L96, N-ER-FY007180M53L96, N-ER-FY007180M54L96, N-ER-FY007180M55L96, N-ER-FY00 7180M57L96, N-ER-FY007180M59L96, N-ER-FY007180M60L96, N-ER-FY007180M61L96, N-ER-FY007180M63L96, N-ER-FY007180M64L96.

[0127] The present invention also provides a pharmaceutical composition comprising the siRNA of the present invention, or the siRNA conjugate of the present invention or a prodrug thereof, and a pharmaceutically acceptable carrier.

[0128] The present invention also provides a kit comprising the siRNA of the present invention, or the siRNA conjugate of the present invention or a prodrug thereof, or the pharmaceutical composition of the present invention.

[0129] The present invention also provides use of the siRNA of the present invention, or the siRNA conjugate of the present invention or a prodrug thereof, or the pharmaceutical composition of the present invention for preparing a medicament for inhibiting HSD17B13 gene expression.

[0130] The present invention also provides use of the siRNA of the present invention, or the siRNA conjugate of the present invention or a prodrug thereof, or the pharmaceutical composition of the present invention for preparing a medicament for preventing and / or treating diseases associated with HSD17B13 gene overexpression.

[0131] In one embodiment, the disease is selected from nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, cirrhosis, alcoholic hepatitis, liver fibrosis, liver cancer.

[0132] The present invention also provides a method for inhibiting HSD17B13 gene expression, comprising contacting a therapeutically effective amount of the siRNA of the present invention, or the siRNA conjugate of the present invention or a prodrug thereof, or the pharmaceutical composition of the present invention with cells expressing HSD17B13 or administering the method to a subject in need thereof.

[0133] The present invention also provides a method for treating and / or preventing diseases associated with HSD17B13 gene overexpression, comprising administering a therapeutically effective amount of the siRNA of the present invention, or the siRNA conjugate or prodrug thereof of the present invention, or the pharmaceutical composition of the present invention to a subject in need.

[0134] The present invention also provides the siRNA of the present invention, or the siRNA conjugate of the present invention or a prodrug thereof, or the pharmaceutical composition of the present invention for use as a medicament. Beneficial effects

[0135] The siRNAs, pharmaceutical compositions, and siRNA conjugates provided herein demonstrate excellent inhibitory activity against HSD17B13 gene expression in in vitro cell experiments and have promising potential for treating diseases associated with HSD17B13 overexpression. For example, the siRNAs and their conjugates disclosed herein can reduce HSD17B13 mRNA expression in the liver, exhibit low toxicity and side effects, exhibit good plasma stability, and exhibit long-lasting efficacy, suggesting promising clinical application prospects.

[0136] The siRNA and siRNA conjugates provided in the present application show a good inhibitory effect on the HSD17B13 gene in human liver cancer cell Huh7 cells.

[0137] The siRNA conjugates provided herein demonstrate a potent inhibitory effect on the HSD17B13 gene in primary human hepatocytes (PHHs). In some embodiments, the siRNA conjugates provided herein exhibit an inhibition rate of greater than about 70% at 100 nM and about 65% at 10 nM when freely taken up by PHHs.

[0138] In some specific embodiments, the siRNA conjugates of the present application have high inhibitory activity on the hHSD17B13 gene in vivo and can reduce the hHSD17B13 protein level for a long time.

[0139] In some specific embodiments, the siRNA conjugate of the present application can significantly reduce the expression level of HSD17B13 mRNA in the liver of cynomolgus monkeys, which is better than the clinical drug ARO-HSD. DETAILED DESCRIPTION

[0140] definition

[0141] Throughout the specification, unless otherwise specified, in the art, "G", "C", "A", "T" and "U" generally represent the bases guanine, cytosine, adenine, thymine and uracil, respectively. However, it is also generally known in the art that "G", "C", "A", "T" and "U" each generally represent a nucleotide containing guanine, cytosine, adenine, thymine and uracil as a base, respectively, which is a common way to represent deoxyribonucleic acid sequences and / or ribonucleic acid sequences. Therefore, in the context of the present disclosure, the meanings represented by "G", "C", "A", "T" and "U" include the above-mentioned various possible situations. In this application, "nucleotide" and "ribonucleotide" are used interchangeably, and in this application, "deoxyribonucleotide" and "2'-deoxyribonucleotide" are used interchangeably. Lowercase letters a, u, c, g: indicate 2'-methoxy modified nucleotides; Af, Gf, Cf, Uf: indicate 2'-fluoro modified nucleotides; "dG", "dC", "dA": indicate 2'-deoxyribonucleotides; when "T" is at the overhang position, it indicates a thymine ribonucleotide (T), and when "T" is in the double-stranded region of the motif, it indicates that the uracil ribonucleotide (U) at the corresponding position in the motif sequence is replaced by a thymine ribonucleotide (T); (invAb) is an inverted abasic deoxyribose residue; lowercase letter s indicates that the two nucleotides adjacent to the letter s are connected by a thiophosphate group; P1: indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide; EVP: indicates that the nucleotide adjacent to the right of EVP is a 5'-trans vinylphosphonate nucleotide; (Underline + Bold + Italic): indicates GNA-modified nucleotides; Base indicates a base, such as A, U, G, C or T.

[0142] In the above and below, the "2'-fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine. "Non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluoro group. In the present disclosure, "non-fluoro modification" also includes the replacement of nucleotides, such as replacing uracil (U) with thymine (T); accordingly, "non-fluoro-modified nucleotide" also includes nucleotides after the above-mentioned base replacement. In some embodiments, each non-fluoro-modified nucleotide is independently selected from one of the nucleotides or nucleotide analogues in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluoro group. The nucleotides formed by replacing the hydroxyl group at the 2' position of these ribose groups with non-fluorinated groups are well known to those skilled in the art. These nucleotides can be selected from 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, and 2'-deoxyribonucleotides.

[0143] As used herein, "nucleotide substitution with a deoxyribonucleotide" refers to the replacement of ribonucleotides A, U, C, and G with corresponding deoxyribonucleotides A, T, C, and G during the modification process. For example, in the 5' to 3' direction, the 7th nucleotide of the antisense strand of N-ER-FY007180 is replaced with a deoxyribonucleotide, which means that the 7th ribonucleotide "U" is replaced with "T"; similarly, ribonucleotide A is replaced with the corresponding deoxyribonucleotide A; ribonucleotide C is replaced with the corresponding deoxyribonucleotide C; and ribonucleotide G is replaced with the corresponding deoxyribonucleotide G.

[0144] "Alkyl" includes straight chain, branched chain or cyclic saturated alkyl. For example, alkyl includes but is not limited to methyl, ethyl, propyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclohexyl and the like. For example, "C 1-6 "C" in "alkyl" 1-6 ” refers to a group containing 1, 2, 3, 4, 5 or 6 carbon atoms in a straight chain, branched or cyclic form.

[0145] "Alkoxy" as used herein refers to an alkyl group attached to the rest of the molecule through an oxygen atom (-O-alkyl), wherein the alkyl group is as defined herein. Non-limiting examples of alkoxy include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, and the like.

[0146] "Nucleotide analogs" refer to groups that can replace nucleotides in nucleic acids but have structures different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides, such as pseudouracil (Ψ), isonucleotides, bridged nucleic acids (BNA), or acyclic nucleotides.

[0147] Pseudouracil (Ψ) refers to a natural structural analog of uridine nucleoside, in which the ribose is not attached to the N1 of uracil, but to the C5 of the pyrimidine ring. The structural formula of pseudouracil is shown below:

[0148] BNA refers to a constrained or inaccessible nucleotide. BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endo sugar condensed bridge structure. The bridge is usually incorporated into the 2'- and 4'-positions of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET BNA, etc., where LNA is shown in formula (1), ENA is shown in formula (2), and cET BNA is shown in formula (3):

[0149] Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of a nucleotide, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), where UNA is shown in formula (4) and GNA is shown in formula (5):

[0150] In the above formulae (4) and (5), R is selected from H, OH or alkoxy (O-alkyl).

[0151] Isonucleotides are compounds formed by a change in the position of the base on the ribose ring of a nucleotide, for example, a compound formed by the base moving from the 1'-position to the 2'-position or the 3'-position of the ribose ring, as shown in formula (6) or (7):

[0152] In the compounds of formula (6)-formula (7) above, Base represents a base, such as A, U, G, C or T; and R is selected from H, OH, F or the non-fluorinated groups described above.

[0153] In some embodiments, the nucleotide analog is selected from the group consisting of pseudouracil, isonucleotide, LNA, ENA, cET BNA, UNA, and GNA. In some embodiments, each non-fluorinated modified nucleotide is a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, or a combination of any two or more thereof. In some preferred embodiments, each non-fluorinated modified nucleotide is a 2'-methoxy-modified nucleotide. As used herein and hereinbelow, the term "2'-methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0154] The "2'-methoxy modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group. The "phosphorothioate group" refers to a phosphorothioate group in which one of the oxygen atoms in the phosphodiester bond of the phosphate group is replaced by a sulfur atom.

[0155] The "phosphorothioate group" refers to the following formula:

[0156] The "5'-phosphate nucleotide" refers to the structure of the following formula:

[0157] In the context of this specification, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one chain are each paired with bases on the other chain in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (G) is always paired with the pyrimidine base cytosine (C). Each base pair includes a purine and a pyrimidine. When adenine on one chain is always paired with thymine (or uracil) on the other chain, and guanine is always paired with cytosine, the two chains are considered to be complementary to each other, and the sequence of the chain can be inferred from the sequence of its complementary chain. Accordingly, "mismatch" means in the art that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary form.

[0158] In the above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there is no more than 1 base mismatch between the two nucleotide sequences; and "completely reverse complementary" means that there is no base mismatch between the two nucleotide sequences.

[0159] As used above and below, a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence refers to a change in the base type of the nucleotide at the same position in the former compared to the latter. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, the two nucleotide sequences are considered to have a nucleotide difference at that position. In some embodiments, when a nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, a nucleotide difference at that position can also be considered.

[0160] In this context, "double-stranded region" refers to the double-stranded region of the siRNA where the sense and antisense strands complement each other to form the siRNA; "overhang" refers to one or more unpaired nucleotides that protrude from the duplex structure of the siRNA when one 3' end of one strand of the siRNA extends beyond the 5' end of the other strand, or vice versa. "Blunt end" or "blunt end" means that there are no unpaired nucleotides at that end of the siRNA, i.e., no nucleotide overhang. A "blunt-ended" siRNA is one that is double-stranded throughout its length, i.e., has no nucleotide overhangs at either end of the molecule.

[0161] Throughout this specification, particularly when describing the methods for preparing the siRNA, pharmaceutical compositions, or siRNA conjugates of the present application, unless otherwise specified, the nucleoside monomers referred to herein are modified or unmodified nucleoside phosphoramidite monomers used in solid-phase phosphoramidite synthesis, depending on the type and sequence of nucleotides in the siRNA or siRNA conjugate to be prepared. Solid-phase phosphoramidite synthesis is a method used in RNA synthesis well known to those skilled in the art. The nucleoside monomers used herein are all commercially available.

[0162] In the context of this application, unless otherwise specified, "conjugation" refers to the covalent bonding of two or more chemical moieties, each with a specific function, to each other; accordingly, "conjugate" refers to a compound formed by covalent bonding of the chemical moieties. Furthermore, "siRNA conjugate" refers to a compound formed by covalent bonding of one or more chemical moieties with specific functions to siRNA. siRNA conjugates should be understood as a general term for multiple siRNA conjugates or an siRNA conjugate represented by a certain chemical formula, depending on the context. In the context of this application specification, "conjugated molecule" should be understood as a specific compound that can be conjugated to siRNA through a reaction to ultimately form the siRNA conjugate of the present application.

[0163] Various hydroxy protecting groups can be used in the present application. Generally speaking, protecting groups make chemical functional groups insensitive to specific reaction conditions and can be attached to and removed from the functional group in the molecule without substantially damaging the rest of the molecule. In some embodiments, the protecting group is stable under alkaline conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxy protecting groups that can be used in the present application include monomethoxytrityl, 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxy protecting groups that can be used in the present application include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl) and TMTr (4,4',4"-trimethoxytrityl).

[0164] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0165] The term "subject," as used herein, refers to any animal, such as a mammal or marsupial. Subjects of the present application include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, rabbits, or any type of poultry.

[0166] As used herein, "treatment" refers to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Furthermore, a therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, even though the subject may still be afflicted with the underlying disorder.

[0167] As used herein, "prevention" refers to an approach for obtaining beneficial or desired results, including but not limited to prophylactic benefit. To obtain a "prophylactic benefit," siRNA, siRNA conjugate, or pharmaceutical composition can be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0168] As used herein, "prodrug" refers to a compound that can be converted into an active compound by certain chemical or physiological processes (e.g., enzymatic processes and metabolic hydrolysis). Therefore, the term "prodrug" also refers to a precursor of a pharmaceutically acceptable biologically active compound.

[0169] In this article, considering the situation where the 5' terminal nucleotide of the antisense chain is connected to the 5' hydroxyl group (i.e., there is no phosphate group), such an antisense chain will first be phosphorylated in the body to convert the 5' terminal nucleotide into a nucleotide carrying a 5' phosphate group, and then play a role in the body. Therefore, in the present invention, such siRNA, siRNA modifications, and siRNA conjugates are also referred to as prodrugs. For example, in this application, an siRNA modification or conjugate with an M6 pattern modification is a prodrug of an siRNA modification or conjugate with an M2 pattern modification, because the difference between the M2 pattern modification and the M6 ​​pattern modification is whether the 5' end of the antisense chain has P1. Similarly, the relationship between the M7 pattern modification and the M3 pattern modification is the same. Therefore, siRNA herein includes its corresponding prodrug.

[0170] siRNA

[0171] This application relates to an siRNA capable of inhibiting HSD17B13 gene expression. The siRNA herein comprises a nucleotide group as a basic structural unit, which, as is well known to those skilled in the art, comprises a phosphate group, a ribose group, and a base. Typically, an active, i.e., functional, siRNA is approximately 12-40 nucleotides in length, and in some embodiments, approximately 15-30 nucleotides in length.

[0172] The siRNA of the present application contains a sense strand and an antisense strand, and each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complemented to form a double-stranded region. In some embodiments, the length of the double-stranded region is 15-30 nucleotide pairs. In other embodiments, the length of the double-stranded region is 17-23 nucleotide pairs. In other embodiments, the length of the double-stranded region is 19-21 nucleotide pairs. In yet other embodiments, the length of the double-stranded region is 19 or 21 nucleotide pairs.

[0173] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV are each independently 0-7 nucleotides in length, nucleotide sequence III is linked to the 5' end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3' end of nucleotide sequence II, and nucleotide sequence III and nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; substantially reverse complementary means that there is no more than one base mismatch between the two nucleotide sequences; completely reverse complementary means that there is no mismatch between the two nucleotide sequences. In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV are each independently 0-7 nucleotides in length, nucleotide sequence III is linked to the 3' end of nucleotide sequence I, and nucleotide sequence IV is linked to the 5' end of nucleotide sequence II, and nucleotide sequence III and nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; substantially reverse complementary means that there is no more than one base mismatch between the two nucleotide sequences; completely reverse complementary means that there is no mismatch between the two nucleotide sequences. In some embodiments, the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV, the length of the nucleotide sequence III and the nucleotide sequence IV are each independently 0-7 nucleotides, the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; and the nucleotide sequence III is connected to the 3' end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 5' end of the nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the substantially reverse complementary means that there is no more than one base mismatch between the two nucleotide sequences; and the completely reverse complementary means that there is no mismatch between the two nucleotide sequences.

[0174] In some embodiments, the sense strand further contains a nucleotide sequence V and / or the antisense strand further contains a nucleotide sequence VI, the length of the nucleotide sequences V and VI is 0 to 3 nucleotides, the nucleotide sequence V is connected to the 3' end of the sense strand to form a 3' overhang of the sense strand and / or the nucleotide sequence VI is connected to the 3' end of the antisense strand to form a 3' overhang of the antisense strand. In some embodiments, the length of the nucleotide sequence V or VI is 2 nucleotides. In other embodiments, the nucleotide sequence V or VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides. In other embodiments, the nucleotide sequence V is identical to or different from the nucleotide at the corresponding position of the target mRNA, or the nucleotide sequence VI is mismatched or complementary to the nucleotide at the corresponding position of the target mRNA.

[0175] The lengths of the sense strands and antisense strands provided herein are the same or different. In some embodiments, the sense strand or antisense strand has 15-30 nucleotides. In other embodiments, the sense strand or antisense strand has 19-25 nucleotides. In other embodiments, the sense strand or antisense strand has 19-23 nucleotides. The length ratios of the sense strands and antisense strands of the siRNA provided herein can be 15 / 15, 16 / 16, 17 / 17, 18 / 18, 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 20 / 19, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 21 / 19, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 22 / 19, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 23 / 19, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 24 / 24, 25 / 25, 26 / 26, 27 / 27, 28 / 28, 29 / 29, 30 / 30, 22 / 24, 22 / 25, 22 / 26, 23 / 24, 23 / 25 or 23 / 26, etc. In some embodiments, the length ratio of the siRNA sense strand to the antisense strand is 19 / 19, 21 / 21, 19 / 21, 21 / 23 or 23 / 23. In this case, the siRNA disclosed herein has better cellular mRNA silencing activity.

[0176] Studies have found that different modification strategies can have distinct effects on siRNA stability, bioactivity, and cytotoxicity. For example, CN102140458B investigated various chemical modification strategies for siRNA and identified seven effective modification methods. Compared to unmodified siRNA, one of these modification methods resulted in siRNA with improved blood stability while maintaining comparable inhibitory activity.

[0177] The nucleotides in the siRNA of the present invention are each independently modified or unmodified. In some embodiments, each nucleotide in the siRNA of the present invention is an unmodified nucleotide; in some embodiments, some or all of the nucleotides in the siRNA of the present invention are modified nucleotides, and these modifications to the nucleotide groups do not significantly weaken or abolish the function of the siRNA of the present invention to inhibit HSD17B13 gene expression.

[0178] In some embodiments, the siRNA of the present application contains at least one modified nucleotide. In the context of the present application, the term "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribose group of the nucleotide with other groups, or a nucleotide having a modified base. The modified nucleotide does not significantly weaken or lose the function of the siRNA to inhibit gene expression. For example, the modified nucleotides disclosed in JK Watts, GF Deleavey, and MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13 (19-20): 842-55 can be selected.

[0179] In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided herein is a modified nucleotide, and / or at least one phosphate group is a phosphate group having a modified group; in other words, at least a portion of the phosphate groups and / or ribose groups in the phosphate-sugar backbone of at least one single strand of the sense strand or the antisense strand is a phosphate group having a modified group and / or a ribose group having a modified group. In some embodiments, the phosphate group having a modified group is a phosphorothioate group formed by replacing one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom.

[0180] In some embodiments, the siRNA includes a sense strand that does not include a 3' overhanging nucleotide; that is, the sense strand of the siRNA may have a 3' overhanging nucleotide, and the 3' overhanging nucleotide of the sense strand is excluded to form a blunt end.

[0181] In some embodiments, when the nucleotide sequences of the sense and antisense strands complement each other to form a double-stranded region, and no overhanging nucleotides are present at the 3' end of the sense strand, a nucleotide sequence V is added to the 3' end of the sense strand to serve as an overhanging nucleotide. Subsequently, after chemical modification, the nucleotide sequence formed by nucleotide sequence V ligating the 3' end of the sense strand is removed, and accordingly, the sense strand of the siRNA forms a blunt end.

[0182] In some embodiments, when the nucleotide sequences of the sense strand and the antisense strand complement each other to form a double-stranded region, and the 3' end of the sense strand has protruding nucleotides extending out of the double-stranded region, the protruding nucleotides at the 3' end of the sense strand are excluded and used as the nucleotide sequence of the sense strand. Accordingly, the sense strand of the siRNA forms a blunt end.

[0183] In some embodiments, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate-derived group.

[0184] An exemplary structure of a 5' phosphate group is: The structures of the 5' phosphate derivative group include but are not limited to: wait.

[0185] The 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group to form the following structure:

[0186] Wherein, Base represents a base, such as A, U, G, C or T. R' is a hydroxyl group or is substituted by various groups known to those skilled in the art. For example, the substituted modified nucleotide can be a 2'-fluoro (2'-F) modified nucleotide, a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, or a 2'-deoxyribonucleotide.

[0187] In some embodiments, the 5' terminal nucleotide of the sense strand or antisense strand is not connected to a 5' phosphate group or a 5' phosphate derivative group (i.e., the ribose group of the 5' terminal nucleotide of the sense strand or antisense strand is a 5' hydroxyl group), and its structure is shown below:

[0188] Wherein, Base represents a base, such as A, U, G, C or T. R is hydroxyl or hydrogen or substituted by various groups known to those skilled in the art. For example, R can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, or 2'-substituted amino.

[0189] Exemplary modified nucleotides have the following structures:

[0190] Wherein, Base represents a base, such as A, U, G, C or T. The hydroxyl group at the 2' position of the ribose group is substituted by R. The hydroxyl group at the 2' position of these ribose groups can be substituted by various groups known to those skilled in the art. For example, the modified nucleotides after substitution can be 2'-fluoro (2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxyribonucleotides.

[0191] In some embodiments, the sense strand may include one or more blocking residues or moieties, sometimes referred to in the art as "caps," "end caps," or "blocking residues." As used herein, a "blocking residue" is a non-nucleotide compound or other moiety that can be incorporated into one or more ends of the nucleotide sequence of the siRNA disclosed herein. In some cases, blocking residues can provide certain beneficial properties for the siRNA, such as protection from exonuclease degradation. In some embodiments, an inverted abasic deoxyribose residue (invAb) is added as a blocking residue. In some embodiments, the blocking residue appears at the 5' end, the 3' end, or both the 5' and 3' ends of the sense strand.

[0192] In some embodiments, one or more reverse abasic deoxyribose residues (invAb) are added to the 3' end of the sense strand. In some embodiments, one or more reverse abasic deoxyribose residues (invAb) are added to the 5' end of the sense strand. In some embodiments, one or more reverse abasic deoxyribose residues (invAb) are added to the 5' end of the sense strand and the 3' end of the sense strand. The reverse abasic deoxyribose residues and the terminal nucleotides can be connected via a phosphodiester bond, a thiophosphate group, or other internucleoside bond. The chemical structure of the reverse abasic deoxyribose residue is as follows:

[0193] When (invAb) is located inside the siRNA, it is Formula A; when (invAb) is located at the 3' end of the siRNA, it is Formula B; when (invAb) is located at the 5' end of the siRNA, it is Formula C:

[0194] The phosphodiester bond in Formula A is directed toward the 3' end of the siRNA.

[0195] siRNA conjugates

[0196] The present application relates to an siRNA conjugate, which contains the above-mentioned siRNA and a conjugation group conjugated to the siRNA.

[0197] In the present application, the sense strand and the antisense strand of the siRNA conjugate form a double-stranded region of the siRNA conjugate, and a blunt end is formed at the 3' end of the sense strand of the siRNA conjugate. In some embodiments, the 3' end of the sense strand of the siRNA conjugate forms a blunt end, and the 3' end of the antisense strand of the siRNA conjugate has 1-3 protruding nucleotides extending out of the double-stranded region. In other embodiments, the 3' end of the sense strand of the siRNA conjugate forms a blunt end, and the 3' end of the antisense strand of the siRNA conjugate forms a blunt end.

[0198] In some preferred embodiments, the siRNA conjugate is obtained by conjugating siRNA with a conjugating group, wherein the sense strand and antisense strand of the siRNA are complementary to form a double-stranded region of the siRNA, and the 3' end of the sense strand of the siRNA forms a blunt end, and the conjugating group is conjugated to the 3' end of the sense strand having the blunt end to form the siRNA conjugate.

[0199] In some preferred embodiments, the 3' end of the sense strand of the siRNA has a protruding nucleotide extending out of the double-stranded region, and the sequence with a 3' blunt end formed after excluding the protruding nucleotide at the 3' end of the sense strand is used as the nucleotide sequence for connecting the conjugated group, and the conjugated group is connected to the 3' blunt end of the sense strand to form an siRNA conjugate.

[0200] In some more preferred embodiments, when the nucleotide sequences of the sense and antisense strands complement each other to form a double-stranded region, if no overhanging nucleotides are present at the 3' end of the sense strand, a nucleotide sequence V is added to the 3' end of the sense strand as an overhanging nucleotide. The sequence with a 3' blunt end formed after removing the overhanging nucleotides at the 3' end of the sense strand is used as the nucleotide sequence for attaching a conjugated group, and the conjugated group is attached to the 3' blunt end of the sense strand to form an siRNA conjugate.

[0201] In some more preferred embodiments, when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region, and the 3' end of the sense strand has a protruding nucleotide extending out of the double-stranded region, the sequence with a 3' blunt end formed after excluding the protruding nucleotide at the 3' end of the sense strand is used as the nucleotide sequence for connecting the conjugated group, and the conjugated group is connected to the 3' blunt end of the sense strand to form an siRNA conjugate.

[0202] Exemplarily, the siRNA sequence is as shown in N-ER-FY007166M2, and the 3' end of the sense chain of the siRNA has a protruding nucleotide extending out of the double-stranded region. The gsusaagaAfgUfCfUfgauagaua blunt-end sequence formed after excluding the protruding -sTsT nucleotides at the 3' end of the sense chain is used as the nucleotide sequence for connecting the L96 conjugation group. Therefore, the sequence of the siRNA conjugate formed is: the sense chain is gsusaagaAfgUfCfUfgauagauaL96, and the antisense chain is P1usAfsucuAfucagacuUfcUfuacsTsT.

[0203] In general, the conjugated group includes at least one pharmaceutically acceptable targeting group, or further includes a linker, and the siRNA, the linker and the targeting group are connected in sequence. In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The siRNA molecule can be non-covalently or covalently conjugated to the conjugated group, for example, it can be covalently conjugated to the conjugated group. The conjugation site of siRNA and conjugated group can be at the 3' end or 5' end of the siRNA sense strand, or at the 5' end of the antisense strand, or in the internal sequence of siRNA. In some embodiments, the conjugation site of siRNA and conjugated group is at the 3' end of the siRNA sense strand.

[0204] In some embodiments, the conjugate group can be connected to the phosphate group, 2'-hydroxyl group or base of the nucleotide. In some embodiments, the conjugate group can also be connected to the 3'-hydroxyl group, in which case the nucleotides are connected by a 2'-5' phosphodiester bond. When the conjugate group is connected to the end of the siRNA chain, the conjugate group is usually connected to the phosphate group of the nucleotide; when the conjugate group is connected to the internal sequence of the siRNA, the conjugate group is usually connected to the ribose sugar ring or the base. Various connection methods can be referred to in the literature: Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.ACS Chemical biology, 2015, 10(5): 1181-7.

[0205] In some embodiments, the siRNA and conjugated group can be linked via acid-labile or reducible chemical bonds. These bonds can degrade in the acidic environment of endosomes, freeing the siRNA. For non-degradable conjugations, the conjugated group can be attached to the sense strand of the siRNA to minimize the effect of conjugation on siRNA activity.

[0206] In some embodiments, the pharmaceutically acceptable targeting group may be a ligand commonly used in the field of siRNA administration, such as the various ligands described in WO2009082607A2, which is incorporated herein by reference in its entirety.

[0207] In some embodiments, the pharmaceutically acceptable targeting group can be selected from one or more of the ligands formed by the following targeting molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acid, vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-permeable peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); receptor ligands expressed by hepatocytes, such as asialoglycoproteins, asialosugar residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin, etc.

[0208] In some embodiments, each ligand is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to an asialoglycoprotein receptor (ASGPR) on the liver surface. The types of these ligands are well known to those skilled in the art, and their function is generally to bind to specific receptors on the surface of target cells, mediating the delivery of siRNA connected to the ligand to the target cells.

[0209] In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In some embodiments, each ligand is independently an asialoglycoprotein, such as asialo serum mucin (ASOR) or asialo fetuin (ASF). In some embodiments, the ligand is a sugar or a sugar derivative.

[0210] In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, a disaccharide, or a trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is independently selected from a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, or a monosaccharide derivative. In some embodiments, each or at least one ligand is selected from the group consisting of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives, and sialic acid.

[0211] In some embodiments, each of the ligands can be independently selected from D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucose, β-D-glucose, Sugar, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine , 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2, 3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside heptoside ethyl ester, 2,5-anhydro-D-allose nitrile, ribose, D-ribose, D-4-thioribose, L-ribose or L-4-thioribose. Other options of the ligand can be found in, for example, the description of CN105378082A, which is incorporated herein by reference in its entirety.

[0212] In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the monovalent, divalent, trivalent, and tetravalent herein refer to the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate after the siRNA molecule forms the siRNA conjugate with the conjugated group containing the galactose or N-acetylgalactosamine molecule as the targeting group. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA described herein is conjugated to a conjugated group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described herein is conjugated to a conjugation group comprising N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0213] The targeting group can be connected to the siRNA molecule via a suitable linker. Those skilled in the art can select a suitable linker based on the specific type of the targeting group. For information on these linkers, the type of targeting group, and the method of connection to the siRNA, please refer to the disclosure of WO2015006740A2, which is incorporated herein by reference in its entirety.

[0214] siRNA synthesis method

[0215] By the conventional solid-phase phosphoramidite method in the art, nucleoside monomers are connected one by one in the 3'-5' direction according to the order of nucleotide arrangement. Each connection of a nucleoside monomer includes a four-step reaction of deprotection, coupling, oxidation or sulfurization, and capping. Among them, when a phosphate is used to connect two nucleotides, the four-step reaction of deprotection, coupling, oxidation, and capping is included when the next nucleoside monomer is connected. When a thiophosphate is used to connect two nucleotides, the four-step reaction of deprotection, coupling, sulfurization, and capping is included when the next nucleoside monomer is connected. The present invention selects nucleotide monomers according to the target sequence for synthesis. The selected nucleotide monomers are nucleotide monomers commonly used by those skilled in the art. For example, the nucleotide monomer for synthesizing A can be, but is not limited to, adenosine-3-phosphate. It should be understood that these monomers, when present in an oligonucleotide, can be interconnected by a 5'-3' phosphodiester bond or a 5'-3' phosphorothioate group. When, for example, the 3' position of the last nucleotide in the 5' to 3' direction is a hydroxyl group, this is achieved according to conventional means in the art.

[0216] For example, the synthesis conditions of the siRNA of the present application can be as follows:

[0217] The deprotection conditions include: reaction temperature of 25° C., reaction time of 70 seconds, deprotection reagent selected from dichloroacetic acid in dichloromethane solution (3% V / V), and a molar ratio of the deprotection reagent to the 4,4'-dimethoxytrityl protecting group on the solid support of 5:1.

[0218] The coupling reaction conditions include: a reaction temperature of 25° C., a reaction time of 600 seconds, a coupling reagent selected from a 0.25 M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT), a molar ratio of the nucleic acid sequence connected to the solid phase support to the nucleoside monomer of 1:10, and a molar ratio of the nucleic acid sequence connected to the solid phase support to the coupling reagent of 1:65.

[0219] The oxidation reaction conditions included a reaction temperature of 25°C, a reaction time of 15 seconds, an oxidizing agent selected from a 0.05 M iodine tetrahydrofuran solution, and a molar ratio of the oxidizing agent to the nucleic acid sequence attached to the solid support during the coupling step of 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1.

[0220] The sulfurization reaction conditions include a reaction temperature of 25°C, a reaction time of 300 seconds, a sulfurization reagent selected from hydrogenated xanthan gum, and a molar ratio of the sulfurization reagent to the nucleic acid sequence attached to the solid support in the coupling step of 120:1. The reaction is carried out in a mixed solvent of acetonitrile:pyridine = 1:1.

[0221] The capping reaction conditions include: a reaction temperature of 25° C., a reaction time of 15 seconds, a capping reagent selected from a mixed solution of CapA (10% acetic anhydride acetonitrile solution) and CapB (10% N-methylimidazole pyridine / acetonitrile solution) with a molar ratio of 1:1, and a molar ratio of the capping reagent to the nucleic acid sequence connected to the solid phase support of acetic anhydride: N-methylimidazole: nucleic acid sequence connected to the solid phase support of 1:1:1.

[0222] After all nucleoside monomers are connected, the nucleic acid sequence connected to the solid phase carrier is aminolyzed, purified, and desalted in sequence to obtain the siRNA sense chain and antisense chain. Finally, the two chains are heated and annealed to obtain the product.

[0223] Methods for aminolysis, purification, desalting, and annealing are well known in the art. For example, aminolysis can be performed by contacting the nucleotide sequence attached to a solid support with concentrated aqueous ammonia; purification can be performed by chromatography; desalting can be performed by reverse-phase chromatography; and mixing the sense and antisense strands in equal molar ratios under varying stringency conditions followed by gradual cooling.

[0224] The synthesized siRNAs are shown in Table 1 and Table 1-1.

[0225] siRNA conjugate synthesis method

[0226] Take the synthesis of L96 as an example:

[0227] In the first step, DMTr-L96 and succinic anhydride are reacted to obtain compound L96-A:

[0228] Preparation process: DMTr-L96, succinic anhydride, 4-dimethylaminopyridine and diisopropylethylamine are added to dichloromethane and stirred at 25°C for 24 hours. The reaction solution is then washed with 0.5M triethylamine phosphate, and the aqueous phase is washed three times with dichloromethane. The combined organic phases are evaporated to dryness under reduced pressure to obtain a crude product; then purified by column chromatography to obtain pure L96-A.

[0229] In the second step, L96-A is reacted with NH2-SPS to obtain L96-B:

[0230] Preparation process: L96-A, O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), and diisopropylethylamine (DIPEA) were mixed and dissolved in acetonitrile. Stir at room temperature for 5 minutes to obtain a homogeneous solution. Aminomethyl resin (NH2-SPS, 100-200 mesh) was added to the reaction liquid. The reaction was shaken at 25°C for 18 hours. After filtration, the filter cake was washed with dichloromethane and acetonitrile. The resulting filter cake was capped with a CapA / CapB mixture to obtain L96-B, which is the solid support containing the conjugated molecule.

[0231] Step 3: Synthesis of siRNA conjugates:

[0232] L96-B was used as a solid phase carrier to synthesize the sense strand of the siRNA conjugate according to the siRNA synthesis method described above, and the antisense strand of the siRNA conjugate was synthesized using the siRNA synthesis method described above, and annealing was performed to generate the siRNA conjugate of this application.

[0233] The synthesized siRNA conjugates are shown in Table 2.

[0234] Pharmaceutical composition

[0235] The present application provides a pharmaceutical composition comprising the siRNA described above as an active ingredient and a pharmaceutically acceptable carrier.

[0236] The pharmaceutically acceptable carrier may be a carrier conventionally used in the field of siRNA administration, such as, but not limited to, lipid nanoparticles (LNP), magnetic nanoparticles (e.g., nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine, PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly (D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate), ... phosphate), PPEEA) and poly (methacrylate-N, N-dimethylaminoethyl ester) (poly (2-dimethylaminoethyl methacrylate), PDMAEMA) and one or more of their derivatives.

[0237] There is no special requirement for the content of siRNA and pharmaceutically acceptable carrier in the pharmaceutical composition, and the conventional content of each component can be used.

[0238] In some embodiments, the pharmaceutical composition may further include other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds conventionally used in the art. For example, the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0239] The pH buffer may be a tris hydrochloride buffer with a pH value of 7.5-8.5 and / or a phosphate buffer with a pH value of 5.5-8.5, for example, a phosphate buffer with a pH value of 5.5-8.5.

[0240] The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. The content of the protective agent may be 0.01-30% by weight based on the total weight of the pharmaceutical composition.

[0241] The osmotic pressure regulator can be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmoles / kilogram (mOsm / kg). According to the desired osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.

[0242] In some embodiments, the pharmaceutical composition can be a liquid preparation, such as an injection; or a lyophilized powder injection, which is mixed with a liquid excipient during administration to form a liquid preparation. The liquid preparation can be, but is not limited to, administered subcutaneously, intramuscularly, or intravenously, and can also be, but is not limited to, administered via spray to the lungs, or administered via spray through the lungs to other organs and tissues (such as the liver). In some embodiments, the pharmaceutical composition is administered intravenously.

[0243] In some embodiments, the pharmaceutical composition can be in the form of a liposome formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid and / or a pegylated lipid.

[0244] The following examples are intended to further illustrate the present invention, but are not intended to limit the present invention in any way.

[0245] Example

[0246] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples (although indicating specific embodiments of the present disclosure) are given for illustrative purposes only, as various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art after reading the detailed description.

[0247] The experimental techniques and methods used in this example are conventional unless otherwise specified. For example, in the following examples, where specific conditions are not specified, conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer are generally followed. Materials and reagents used in the examples were obtained through commercial channels unless otherwise specified.

[0248] Example 1 Preparation of siRNA

[0249] The siRNA molecules with the following sequences were synthesized by Tianlin Biotechnology (Shanghai) Co., Ltd.

[0250] Table 1 siRNA and its sequence

[0251] Table 1-1

[0252] Among them, the capital letters "G", "C", "A", "T" and "U" each generally represent a nucleotide containing guanine, cytosine, adenine, thymine and uracil as a base, respectively; the lowercase letters a, u, c, g represent: 2'-methoxy modified nucleotides; Af, Gf, Cf, Uf represent: 2'-fluoro modified nucleotides; "dG", "dC", "dA": represent 2'-deoxyribonucleotides; when "T" is at the overhang position, it represents a thymine ribonucleotide (T) , and when "T" is in the double-stranded region of the motif, it indicates that the uracil ribonucleotide (U) at the corresponding position in the motif sequence is replaced by a thymine ribonucleotide (T); (invAb) is an inverted abasic deoxyribose residue; the lowercase letter s indicates that the two nucleotides adjacent to the letter s are connected by a thiophosphate group; P1: indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide; EVP: indicates that the nucleotide adjacent to the right of EVP is a 5'-trans vinylphosphonate nucleotide; (Underlined + Bold + Italic): Indicates GNA-modified nucleotides.

[0253] siRNA conjugates with the following sequences were synthesized by Tianlin Biotechnology (Shanghai) Co., Ltd.

[0254] Table 2 siRNA conjugates and their sequences:

[0255] Wherein, L96 is connected to the 3' end of the sense strand in Table 1 or the blunt end formed by the 3' end of the sense strand through a phosphodiester bond, and L96 is:

[0256] In Tables 1 and 2, if the left side of the 5'-terminal nucleotide of the sense strand, modified sense strand, and modified sense strand connected to a conjugated group is not labeled with P1 or EVP, it means that the 5'-terminal nucleotide is not connected to a 5'-phosphate group or a 5'-phosphate derivative group (i.e., the ribose group of the 5'-terminal nucleotide is a 5'-hydroxyl group), and its structure is shown in Formula X:

[0257] Wherein, Base represents a base, such as A, U, G, C or T; R is hydroxyl or hydrogen or is substituted by various groups known to those skilled in the art, for example, R can be fluorine, alkoxy, substituted alkoxy, alkyl, substituted alkyl, amino, or substituted amino.

[0258] In Tables 1 and 2, if the left side of the 5' terminal nucleotide of the antisense strand and the modified antisense strand is not labeled with P1 or EVP, it means that the 5' terminal nucleotide is not linked to a 5' phosphate group or a 5' phosphate derivative group, and its structure is also shown in Formula X.

[0259] In Table 1 and Table 2, the 3'-terminal nucleotide of the sense strand and the modified sense strand, and the 3'-terminal nucleotide of the antisense strand and the modified antisense strand is at the 3' position a hydroxyl group.

[0260] Example 2 siRNA and siRNA conjugates inhibit HSD17B13 gene expression

[0261] The inhibitory activity of the siRNA of the present invention on HSD17B13 gene expression was evaluated using a dual-luciferase reporter gene vector.

[0262] Main experimental reagents:

[0263] Experimental steps:

[0264] Day 0: Transform psiCHECK2-HSD17B13 plasmid into Huh7 cells

[0265] The psiCHECK2-HSD17B13 plasmid was diluted to 10 ng / μL with Opti-MEM. Huh7 cells were washed with DPBS and then digested with trypsin. The cell density was adjusted to 1×10 5cells / mL. Mix Fugene-HD transfection reagent and 10 ng / μL psiCHECK2-HSD17B13 diluent at a ratio of 3:100 (volume ratio). Incubate at room temperature for 10 minutes. After incubation, add the mixture to Huh7 cells at a ratio of 1:9 (volume ratio). Plate 10,000 cells per well in a 96-well plate with 100 μL of culture medium per well. Culture Huh7 cells overnight in a 5% CO2, 37°C incubator.

[0266] Day 1: Treatment of siRNA compounds (siRNA and siRNA conjugates)

[0267] Will RNAiMAX transfection reagent and Opti-MEM were mixed at a ratio of 1.5:48.5 (volume ratio) to prepare Mixture A. The mixture was incubated at room temperature for 15 minutes. The test siRNA compounds (final concentrations of 1 nM, 0.1 nM, and 0.05 nM, respectively) were mixed with Mixture A at a 1:1 ratio and incubated at room temperature for 15 minutes. After incubation, 20 μL of the resulting mixture was added to 100 μL of fresh Opti-MEM medium at a ratio of 1:5 and mixed thoroughly to prepare Mixture B. The Huh7 cell supernatant was discarded, and 120 μL / well of Mixture B was added to a 96-well plate. The 96-well plate was then placed in a CO2 cell incubator and incubated for 48 hours.

[0268] Day 2: Detection of reporter genes

[0269] Observe the cell status under a microscope, discard the cell supernatant, and add 75 μL of & Luciferase reagent and 75 μL fresh 10% FBS-DMEM medium were shaken for 10 minutes in a shaker in the dark. 100 μL of the above sample was transferred to a 96-well all-white detection plate and the luminescence value of firefly luciferase was measured on a multifunctional microplate reader. Then 50 μL of Stop& Reagent, shake the plate in the dark for 10 minutes, and detect the luminescence value of Renilla luciferase (Renilla lum).

[0270] The experiment was set up as follows: a control group, in which the siRNA compound was replaced by RNA-free H2O, and the other conditions were the same as those of the experimental group; a blank group, in which Huh7 cells were not transfected with the psiCHECK2-HSD17B13 plasmid and no siRNA compound was added.

[0271] Data processing:

[0272] The ratio of the fluorescence value of Renilla luciferase to that of Firefly luciferase is denoted as α, and the formula is:

[0273] α = (average value of Renilla lum in test wells - average value of Renilla lum in blank group) / (average value of Firefly lum in test wells - average value of Firefly lum in blank group);

[0274] The experimental group ratio calculated according to the above ratio formula is recorded as: α(experimental group), and the control group ratio is recorded as: α(control group).

[0275] The inhibition rate of siRNA inhibiting the expression of the target gene HSD17B13 was calculated according to the following formula:

[0276] Inhibition rate (%) = [1-α (average value of experimental group) / α (average value of control group)] × 100%

[0277] Table 3 Inhibition rate of siRNA of the present invention

[0278] Table 3-1

[0279] Table 3-2

[0280] The sequence of siRNA 2 is shown in Table 3-3:

[0281] Table 3-3

[0282] It can be seen from Table 3, Table 3-1 and Table 3-2 that the siRNA and siRNA conjugate of the present invention can significantly inhibit the expression of the HSD17B13 gene at 1 nM, 0.1 nM and 0.05 nM.

[0283] Example 3 Determination of the inhibition rate of siRNA conjugates in inhibiting HSD17B13 gene expression

[0284] Material:

[0285] Primary human hepatocytes PHH cells were provided by Shanghai WuXi AppTec Pharmaceutical Development Co., Ltd.;

[0286] PHH medium: invitroGRO CP Medium serum free BIOVIT, catalog number: S03316

[0287] RNAiMAX transfection reagent, purchased from Invitrogen, catalog number: 13778-150;

[0288] RNA extraction kit 96Kit, catalog number: QIAGEN-74182;

[0289] Reverse transcription kit FastKing RT Kit (with gDNase), catalog number: Tiangen-KR116-02;

[0290] FastStart Universal Probe Mast(Roche-04914058001);

[0291] TaqMan Gene Expression Assay(GAPDH,Thermo,Assay ID-Hs02786624_g1);

[0292] TaqMan Gene Expression Assay (HSD17B13, Thermo, Assay ID-Hs01068199_m1).

[0293] (1) siRNA conjugates (final concentrations of siRNA conjugates were 100 nM and 10 nM, respectively, in duplicate) were freely taken up by PHH cells. The process was as follows: frozen PHH cells were taken, revived, counted, and the cell density was adjusted to 6 × 10 5 Cells / mL were added, and siRNA conjugates were added at the same time. Cells were seeded into 96-well plates at a density of 54,000 cells per well, with 100 μL of culture medium per well. Cells were cultured in a 5% CO2, 37°C incubator. After 48 hours, the culture medium was removed and the cells were collected for total RNA extraction. Use according to the kit product instructions. Total RNA was extracted using 96Kit.

[0294] (2) Reverse transcribe the extracted total RNA into cDNA using the Fastking RT Kit (With gDNase) by following the steps below:

[0295] a) Remove gDNA using gDNAase according to the table below;

[0296] Table 4

[0297] Run the program at 42 °C for 3 minutes and then place the plate at 4 °C.

[0298] b) adding the following reagents to the system obtained in step a) and performing reverse transcription:

[0299] Table 5

[0300] 42℃, 15min; 95℃, 3min.

[0301] c) The reverse transcription product obtained in step b) was stored at -20°C for real-time PCR analysis.

[0302] (3) Perform real-time qPCR analysis

[0303] a) Prepare the qPCR reaction mixture as shown in the table below. Keep all reagents on ice throughout the entire procedure.

[0304] Table 6

[0305] Table 7

[0306] b) Perform qPCR procedure as follows

[0307] 95°C, 10 minutes;

[0308] 95°C, 15 seconds, 60°C, 1 minute (40 cycles of this operation).

[0309] (4) Result analysis:

[0310] a) Quant Studio 7 software was used with default settings to automatically calculate Ct values;

[0311] b) Calculate the relative gene expression using the following formula:

[0312] ΔCt = Ct (HSD17B13 gene) – Ct (GAPDH)

[0313] ΔΔCt=ΔCt(test sample group)–ΔCt(Mock group), where the Mock group represents the group without the addition of siRNA conjugates compared with the test sample group;

[0314] mRNA expression relative to the Mock group = 2 -ΔΔCt

[0315] Inhibition rate (%) = (relative expression of mRNA in the Mock group – relative expression of mRNA in the sample group) / relative expression of mRNA in the Mock group × 100%

[0316] Table 8 Inhibition rate of siRNA conjugates in inhibiting HSD17B13 gene expression

[0317] As can be seen from Table 8, the siRNA conjugate of the present application has a very high HSD17B13 gene inhibitory activity. When the siRNA conjugate enters PHH through free uptake, the inhibition rate is about 70% or more at 100 nM and about 65% or more at 10 nM.

[0318] Example 4 Silencing Effect of siRNA Conjugates in Mice Expressing the Human HSD17B13 (hHSD17B13) Gene

[0319] (1) Construction of hHSD17B13 gene overexpressing mouse model using AAV

[0320] 6-8 week old C57BL / 6 mice (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were introduced into the facility. After 3-5 days of adaptive feeding, a single injection of adeno-associated virus AAV (pAAV[Exp]-CBh>{HSD17B13CDS(ns)}:T2A:SEAP:WPRE, provided by Yunzhou Biotechnology (Guangzhou) Co., Ltd.) carrying the hHSD17B13 gene was performed into the tail vein for target gene overexpression modeling. The administration volume was 100 μL (2*10 13 vg) / head, and then fed with normal feed.

[0321] (2) Efficacy of silencing siRNA conjugates in hHSD17B13 mouse model

[0322] Fourteen days after AAV virus injection, mice were divided into groups (6 per group) and subcutaneously administered a single 3 mg / kg dose of the siRNA conjugate of the present application. SEAP protein expression (i.e., hHSD17B13 protein expression) was measured on days 7, 14, 21, 28, 35, and 42 after administration to determine the inhibition rate of hHSD17B13 by the siRNA conjugate.

[0323] Table 9 Inhibition rate of hHSD17B13 by siRNA conjugates

[0324] Note: ARO-HSD (WO2023196941) is an arrowhead clinical drug, as Yangshen, and its sequence is shown in Table 10.

[0325] Table 10

[0326] (NAG37)s structural formula:

[0327] As can be seen from Table 9, the siRNA conjugate of the present application has a high inhibitory activity on the hHSD17B13 gene in vivo and can reduce the hHSD17B13 protein level for a long time.

[0328] Example 5 Repeated-dose toxicity test of N-ER-FY007180M8L96 in SD rats after subcutaneous injection

[0329] This study consisted of four groups. The main experimental group consisted of 15 male and female SD rats (from Guokesaifu Hebei Pharmaceutical Technology Co., Ltd.) in each group. Each group was subcutaneously injected with a vehicle control (0.9% sodium chloride injection) or 30, 100, or 300 mg / kg of N-ER-FY007180M8L96, once every two weeks for a total of three times, with a dosing volume of 5 mL / kg. Ten male and female animals in each group underwent a planned autopsy at the end of the dosing period on D30, and the remaining recovery animals underwent a planned autopsy on D84. A satellite group (dosage and administration method were the same as those of the main experimental group) was set up, with 5 male and female rats in each group for blood collection for cytokine studies. In addition, a TK group (dosage method and dose were the same as those of the main experimental group) was set up, with 4 male and female rats in the vehicle control group and 8 male and female rats in the test article group for blood collection for TK studies.

[0330] Toxicity parameters for this study included death / moribundity, general observations, body weight, food intake, ophthalmological examination, clinical pathology (hematology and coagulation parameters, serum biochemical parameters, immune function parameters, urine parameters, and bone marrow smear), gross necropsy, organ weights, histopathology, and tissue distribution. Blood samples were collected from animals in the satellite group during the acclimation period, and 4 and 24 hours after dosing on Day 1 and Day 29 for cytokine analysis. Blood samples were collected from animals in the TK group before dosing and at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after dosing on Day 1 and Day 29 for toxicokinetic analysis.

[0331] During the trial, all animals survived until planned autopsy. General observations, body weight, food intake, ophthalmological examinations, cytokines, clinical pathology (hematology, coagulation, serum biochemistry, immune function indicators, urine indicators, bone marrow smears), and gross autopsy showed no significant abnormalities related to the test article. Therefore, the no-observed-adverse-effect level (NOAEL) for subcutaneous administration of N-ER-FY007180M8L96 to SD rats was 300 mg / kg.

[0332] Example 6 Pharmacological efficacy study of siRNA conjugates in cynomolgus monkeys with NASH model

[0333] 1. Twelve male cynomolgus macaques (weight > 8 kg) aged 10 years and older with NASH model (liver tissue pathology scores must meet the requirements of steatosis ≥ 2, inflammatory response ≥ 1, ballooning ≥ 1, and liver fibrosis score ≥ 1) (provided by Kunming Keling Biotechnology Co., Ltd.) were divided into 4 groups and fed in the breeding facility. MRI scans of liver fat were performed 1 week before administration (D-7).

[0334] 2. Observation period of medication

[0335] Animals will undergo a 12-week observation period. The day of dosing is defined as Day 0 (D0). The specific dosing schedule is outlined in Table 11.

[0336] Table 11

[0337] The injection site was the loose skin area around the scapula / lateral neck. Hair around the injection site was shaved, and the skin was disinfected with iodine and alcohol before subcutaneous administration. Liver biopsies were performed on days 56 and 112 after administration for mRNA expression analysis. The change rate (%) of liver mRNA was calculated according to the following formula:

[0338] ΔCt = Ct (HSD17B13 gene) – Ct (GAPDH)

[0339] ΔΔCt = ΔCt (after administration) – ΔCt (before administration);

[0340] mRNA expression relative to pre-dose = 2 -ΔΔCt

[0341] Change rate (%) = (relative mRNA expression level after administration – relative mRNA expression level before administration) / relative mRNA expression level before administration × 100%

[0342] The results are shown in Table 12.

[0343] Table 12

[0344] The siRNA conjugates N-ER-FY007104M8L96 and N-ER-FY007180M8L96 of the present application can significantly reduce the expression level of HSD17B13 mRNA in the liver of crab-eating monkeys, which is better than the clinical drug ARO-HSD.

[0345] Example 7 In vitro stability test of rat liver homogenate

[0346] 1. Experimental reagents and consumables

[0347] 2. Experimental Procedure

[0348] 2.1 Preparation of liver homogenate

[0349] 2.1.1 Preparation of grinding fluid

[0350] Table 13

[0351] 2.1.2 Tissue homogenization

[0352] Rat liver tissue and grinding solution were prepared at a ratio of 100 mg:5 mL (concentration of 20 mg / mL), and grinding beads were added to the homogenizer. The grinding parameters were set as follows.

[0353] Table 14

[0354] 2.2 Sample preparation

[0355] The siRNA conjugate sample was prepared with enzyme-free water to a 1 mg / mL solution for later use. The internal standard sample was prepared with enzyme-free water to a concentration of 0.125 mg / mL.

[0356] 2.3 Sample incubation

[0357] (1) Add 250 μL of prepared liver homogenate to a 2 mL enzyme-free tube;

[0358] (2) Add 50 μL of nucleic acid sample based on step 1;

[0359] (3) The system is 300 μL of biological sample, vortexed, and allowed to stand for 5 min;

[0360] (4) Divide into 2 tubes, 100 μL each;

[0361] (5) System incubation time: 48h.

[0362] 2.4 Biological sample processing

[0363] Vortex and mix every 100 μL of biological sample system, add 300 μL of clarity OTX lysis buffer and vortex, let it stand for 30 minutes, add 100 μL of internal standard solution, vortex, let it stand for 5 minutes, centrifuge for 1 minute, and set aside (the total sample volume is about 500 μL).

[0364] 2.5 Solid Phase Extraction:

[0365] (1) Preparation of solid phase extraction reagents

[0366] Activator: Add 200 mL of methanol to the mobile phase bottle and mark it as activator;

[0367] Equilibration solution: Prepare 1 M phosphate buffer solution [877 mL sodium dihydrogen phosphate (1.56 g / L) + 123 mL sodium dihydrogen phosphate (3.58 g / L)], dilute 100-fold, adjust the pH to 5.5 with phosphoric acid, and mark it as equilibrium solution;

[0368] Flushing solution: Take 500 mL of the equilibrium solution and transfer it to a 1 L mobile phase bottle. Add 500 mL of acetonitrile and adjust the pH to 5.5 with phosphoric acid. Mix well and mark as flushing solution.

[0369] Eluent: Weigh 7.9 g of ammonium bicarbonate into a 1 L mobile phase bottle, add 1 L of water, take 500 mL of ammonium bicarbonate solution into a 1 L mobile phase bottle, add 500 mL of acetonitrile, adjust the pH to 9 with sodium hydroxide, mix well, and mark as eluent;

[0370] (2) The extraction steps are as follows:

[0371] Table 15

[0372] 2.6 Post-processing

[0373] The eluate (taken twice, 600 mL each time, for a total of 1200 mL) was placed in a 2 mL EP tube and concentrated under vacuum for 6 hours at 1800 rpm. The concentrated sample was reconstituted with 100 μL of mobile phase (initial ratio) and centrifuged at low speed for 2 minutes. 10 μL of the supernatant was injected into a high-resolution mass spectrometer. LC-MS / MS was used to semi-quantitatively determine the antisense strand ratio of the siRNA conjugates of this application. The metabolic results after 48 hours of in vitro incubation in rat liver homogenate are shown in Table 16 below.

[0374] Table 16 Remaining percentage of antisense strand of siRNA conjugates

[0375] Among them, the larger the remaining amount of AS, the better the stability of the drug and the better the long-term effect of the drug. The siRNA conjugate of the present application has good stability.

Claims

1. An siRNA capable of inhibiting HSD17B13 gene expression, the siRNA comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I and the nucleotide sequence II are selected from the following sequences: (1) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 25, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 26; (2) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 40, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 41; (3) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 53, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 54; (4) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 140, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 141: 5'-GUAUGCAGAAUAUU-3'(SEQ ID NO:140) 5'-AAUAUUCUGCAUAC-3' (SEQ ID NO: 141); (5) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 142, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 143: 5'-GAAUAUUCAAUUUGAA-3'(SEQ ID NO:142) 5'-UUCAAAUUGAAUAUUC-3' (SEQ ID NO: 143); (6) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 1, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 2; (7) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:

28.

2. The siRNA according to claim 1, wherein the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary, or completely reverse complementary; the substantially reverse complementary refers to that there are no more than 3 base mismatches between the two nucleotide sequences; the substantially reverse complementary refers to that there are no more than 1 base mismatch between the two nucleotide sequences; and the completely reverse complementary refers to that there are no mismatches between the two nucleotide sequences.

3. The siRNA according to claim 1 or 2, wherein the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV, and the lengths of the nucleotide sequence III and the nucleotide sequence IV are each independently 0-7 nucleotides, wherein the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, and the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the substantially reverse complementary means that there is no more than one base mismatch between the two nucleotide sequences; and completely reverse complementary means that there is no mismatch between the two nucleotide sequences; and / or, the nucleotide sequence III is connected to the 3' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 5' end of the nucleotide sequence II, and the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the substantially reverse complementary means that there is no more than one base mismatch between the two nucleotide sequences; and completely reverse complementary means that there is no mismatch between the two nucleotide sequences.

4. The siRNA according to any one of claims 1 to 3, comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises nucleotide sequences I and III, and the antisense strand comprises nucleotide sequences II and IV, wherein the nucleotide sequences I and III are at least partially reverse complementary to the nucleotide sequences II and IV to form a double-stranded region, wherein the nucleotide sequences I and III, and the nucleotide sequences II and IV are selected from the following sequences: (1) the nucleotide sequences I and III comprise or consist of the nucleotide sequence shown in SEQ ID NO: 112, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequence shown in SEQ ID NO: 113; (2) The nucleotide sequences I and III comprise or consist of the nucleotide sequence shown in SEQ ID NO: 208, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequence shown in SEQ ID NO:

209.

5. The siRNA according to any one of claims 1 to 4, wherein the sense strand further comprises a nucleotide sequence V and / or the antisense strand further comprises a nucleotide sequence VI, wherein the length of the nucleotide sequences V and VI is 0 to 3 nucleotides, the nucleotide sequence V is linked to the 3' end of the sense strand to form a 3' overhang of the sense strand and / or the nucleotide sequence VI is linked to the 3' end of the antisense strand to form a 3' overhang of the antisense strand; preferably, the length of the nucleotide sequence V or VI is 2 nucleotides; more preferably, the nucleotide sequence V or VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides; Alternatively, the nucleotide sequence V is identical to or different from the nucleotide at the corresponding position of the target mRNA, or the nucleotide sequence VI is mismatched or complementary to the nucleotide at the corresponding position of the target mRNA.

6. The siRNA according to any one of claims 1-5, wherein the length of the double-stranded region is 15-30 nucleotide pairs; preferably, the length of the double-stranded region is 17-23 nucleotide pairs; more preferably, the length of the double-stranded region is 19-21 nucleotide pairs.

7. The siRNA according to any one of claims 1 to 6, wherein the sense strand or antisense strand has 15-30 nucleotides; preferably, the sense strand or antisense strand has 19-25 nucleotides; more preferably, the sense strand or antisense strand has 19-23 nucleotides.

8. The siRNA according to any one of claims 1 to 7, wherein at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide, and / or at least one phosphate group is a phosphate group having a modified group; preferably, the phosphate group having a modified group is a phosphorothioate group formed by replacing one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom; and / or, the siRNA comprises a sense strand that does not include a 3' overhanging nucleotide.

9. The siRNA according to any one of claims 1 to 8, wherein The 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate-derived group, or the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group.

10. The siRNA according to any one of claims 1 to 9, wherein The 3' terminal nucleotide of the sense strand is linked to an inverted abasic deoxyribose residue, and / or the 5' terminal nucleotide of the sense strand is linked to an inverted abasic deoxyribose residue.

11. The siRNA according to any one of claims 1 to 10, wherein the modified nucleotide is selected from 2'-fluoro-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-deoxyribonucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, nucleotide analogs, or a combination of any two or more thereof; preferably, the modified nucleotide is selected from 2'-fluoro-modified nucleotides, 2'-methoxy-modified nucleotides, 2'-O-CH2-CH2-O-CH3-modified nucleotides, 2'-O-CH2-CH=CH2-modified nucleotides, 2'-CH2-CH2-CH=CH2-modified nucleotides, 2'-deoxyribonucleotides, nucleotide analogs, or a combination of any two or more thereof.

12. The siRNA according to any one of claims 1 to 11, wherein each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide or a non-fluoro-modified nucleotide; Preferably, in the sense strand, from 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro-modified nucleotides are located at positions 9, 10 and 11, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro-modified nucleotides are located at positions 8, 9 and 10, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro-modified nucleotides are located at positions 8, 9, 10 and 11, and the remaining positions are non-fluorinated modified nucleotides; or, fluorinated modified nucleotides are located at positions 7 and 9, and the remaining positions are non-fluorinated modified nucleotides; alternatively, 2'-fluorinated modified nucleotides are located at positions 7, 9 and 11, and the remaining positions are non-fluorinated modified nucleotides; alternatively, 2'-fluorinated modified nucleotides are located at positions 7, 9, 11, 13 and 15, and the remaining positions are non-fluorinated modified nucleotides; alternatively, 2'-fluorinated modified nucleotides are located at positions 7, 9, 11 and 13, and the remaining positions are non-fluorinated modified nucleotides; alternatively, 2'-fluorinated modified nucleotides are located at positions 3, 7, 8 and 9, and the remaining positions are non-fluorinated modified nucleotides. fluorinated nucleotides at positions 3, 9, 11, and 13, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluorinated nucleotides at positions 3, 7, 9, and 11, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluorinated nucleotides at positions 7, 11, and 13, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluorinated nucleotides at positions 7, 11, 12, and 13, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluorinated nucleotides at positions 3, 9, and 11, and the remaining positions are non-fluorinated modified nucleotides , 7, 11 and 16, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 3, 7, 11, 16 and 17, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 3, 7 and 9, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 7, 9 and 14, and the remaining positions are non-fluorinated modified nucleotides; or, 2'-fluoro-modified nucleotides are located at positions 5, 7, 8 and 9, and the remaining positions are non-fluorinated modified nucleotides; and / or In the antisense strand, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16, and the remaining positions are non-fluoro-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16, and the remaining positions are non-fluoro-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 2, 14 and 16, and the remaining positions are non-fluoro-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 2, 6 and 14, and the remaining positions are non-fluoro-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9 and 14, and the remaining positions are non-fluoro-modified nucleotides. The remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 5, 8, 10, 14, 16 and 18, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro-modified nucleotides are located at positions 2, 4, 6, 12, 14, 16 and 18, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro-modified nucleotides are located at positions 2 and 14, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro-modified nucleotides are located at positions 2 and 14, and the remaining positions are non-fluorinated modified nucleotides; Nucleotides are located at positions 2, 4, 5, 6, 8, 10, 12, 14, 16 and 18, and the remaining positions are non-fluorinated modified nucleotides; alternatively, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10 and 14, and the remaining positions are non-fluorinated modified nucleotides; alternatively, 2'-fluoro-modified nucleotides are located at positions 2, 5, 7 and 14, and the remaining positions are non-fluorinated modified nucleotides; alternatively, 2'-fluoro-modified nucleotides are located at positions 2, 7, 12 and 14, and the remaining positions are non-fluorinated modified nucleotides; alternatively, 2'-fluoro-modified nucleotides are located at positions 2, 6, 12 and 14, and the remaining positions are non-fluorinated modified nucleosides Acid; or, the 2'-fluoro modified nucleotides are located at positions 2, 3, 6 and 14, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro modified nucleotides are located at positions 2, 6, 8 and 14, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro modified nucleotides are located at positions 2, 10 and 14, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro modified nucleotides are located at positions 2, 7 and 14, and the remaining positions are non-fluorinated modified nucleotides; or, the 2'-fluoro modified nucleotides are located at positions 2, 3, 5, 7, 10, 12 and 14, and the remaining positions are non-fluorinated modified nucleotides.

13. The siRNA according to claim 12, wherein each non-fluorinated modified nucleotide is independently selected from a nucleotide or nucleotide analogue in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group; The nucleotide analog is selected from one of pseudouracil, isonucleotide, LNA, ENA, cET BNA, UNA and GNA.

14. The siRNA according to any one of claims 1 to 13, wherein each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a 2'-deoxyribonucleotide, a GNA-modified nucleotide, or a combination of any two or more thereof; Preferably, in the sense strand, from 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 9, 10 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 8, 9 and 10, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 8, 9, 10 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 8, 9, 10 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 8, 9, 10 and 11, and the remaining positions are 2'-methoxy-modified nucleotides at positions 7 and 9, 2'-deoxyribonucleotides are located at position 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, 2'-fluoro-modified nucleotides are located at positions 7, 9 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, 2'-fluoro-modified nucleotides are located at positions 7, 9, 11, 13 and 15, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, 2'-fluoro-modified nucleotides are located at positions 7, 9, 11 and 13, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, 2'-fluoro-modified nucleotides are located at positions 3, 7, 8 and 9, and the remaining positions are 2'-methoxy-modified nucleotides. 2'-methoxy modified nucleotides at positions 3, 9, 11, and 13, and 2'-methoxy modified nucleotides at the remaining positions; or, 2'-fluoro modified nucleotides at positions 3, 7, 9, and 11, and 2'-methoxy modified nucleotides at the remaining positions; or, 2'-fluoro modified nucleotides at positions 7, 11, and 13, and 2'-methoxy modified nucleotides at the remaining positions; or, 2'-fluoro modified nucleotides at positions 7, 11, 12, and 13, and 2'-methoxy modified nucleotides at the remaining positions; or, 2'-fluoro modified nucleotides nucleotides at positions 3, 7, 11 and 16, with 2'-methoxy modified nucleotides at the remaining positions; or, 2'-fluoro modified nucleotides at positions 3, 7, 11, 16 and 17, with 2'-methoxy modified nucleotides at the remaining positions; or, 2'-fluoro modified nucleotides at positions 3, 7 and 9, with 2'-methoxy modified nucleotides at the remaining positions; or, 2'-fluoro modified nucleotides at positions 7, 9 and 14, with 2'-methoxy modified nucleotides at the remaining positions; or, 2'-fluoro modified nucleotides at positions 5, 7, 8 and 9, with 2'-methoxy modified nucleotides at the remaining positions; and / or In the antisense strand, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 2, 14 and 16, the GNA-modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides; alternatively, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16, the GNA-modified nucleotide is located at position 7 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 2, 6 and 14, and the remaining positions are 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 2, 6, 8, 9 and 14, and the remaining positions are 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 2, 3, 5, 8, 10, 14, 16 and 18, and the remaining positions are 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14, and the remaining positions are 2'-methoxy modified nucleotides; or, 2'-fluoro modified nucleotides are located at positions 2, 4, 6, 12, 14, 16 and 18, with 2'-methoxy-modified nucleotides at the remaining positions; alternatively, 2'-fluoro-modified nucleotides are located at positions 2 and 14, with 2'-methoxy-modified nucleotides at the remaining positions; alternatively, 2'-fluoro-modified nucleotides are located at positions 2, 4, 5, 6, 8, 10, 12, 14, 16 and 18, with 2'-methoxy-modified nucleotides at the remaining positions; alternatively, 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14, a GNA-modified nucleotide is located at position 6 of the antisense strand, with 2'-methoxy-modified nucleotides at the remaining positions; alternatively, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10 and 14, with 2'-methoxy-modified nucleotides at the remaining positions 2'-fluoro-modified nucleotides at positions 2, 5, 7 and 14, with 2'-methoxy-modified nucleotides at the remaining positions; or, 2'-fluoro-modified nucleotides at positions 2, 7, 12 and 14, with 2'-methoxy-modified nucleotides at the remaining positions; or, 2'-fluoro-modified nucleotides at positions 2, 6, 12 and 14, with 2'-methoxy-modified nucleotides at the remaining positions; or, 2'-fluoro-modified nucleotides at positions 2, 3, 6 and 14, with 2'-methoxy-modified nucleotides at the remaining positions; or, 2'-fluoro-modified nucleotides at positions 2, 6, 8 and 14, with 2'-methoxy-modified nucleotides at the remaining positions;Alternatively, 2'-fluoro modified nucleotides are located at positions 2, 10, and 14, the nucleotide at position 7 is replaced by a deoxyribonucleotide, a 2'-deoxyribonucleotide is located at position 5 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, 2'-fluoro modified nucleotides are located at positions 2, 7, and 14, the nucleotide at position 12 is replaced by a deoxyribonucleotide, a 2'-deoxyribonucleotide is located at position 5 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, 2'-fluoro modified nucleotides are located at positions 2 and 14, the nucleotides at positions 7 and 12 are replaced by deoxyribonucleotides, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, 2'-fluoro modified nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14, a GNA modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides.

15. The siRNA according to any one of claims 1 to 14, wherein, in the direction from the 5' end to the 3' end, (1) The sense strand contains phosphorothioate groups located at the following positions: Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand; and Between the second and third nucleotides starting from the 5' end of the sense strand; and Between the first nucleotide and the second nucleotide starting from the 3' end of the sense strand; and Between the second and third nucleotides starting from the 3' end of the sense strand; or, (2) The sense strand comprises a phosphorothioate group located at the following position: Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand; and Between the second and third nucleotides starting from the 5' end of the sense strand; or, (3) The sense strand comprises a phosphorothioate group located at the following position: Between the first and second nucleotides starting from the 5' end of the sense strand; or, (4) The sense strand comprises a phosphorothioate group located at the following position: Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand; and Between the second and third nucleotides starting from the 5' end of the sense strand; and between the reverse abasic deoxyribose residue starting at the 3' end of the sense strand and the first nucleotide; or (5) The sense strand comprises a phosphorothioate group located at the following position: between the inverted abasic deoxyribose residue starting at the 5' end of the sense strand and the first nucleotide; and Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand; and The positive strand is located between the inverted abasic deoxyribose residue starting at the 3' end and the first nucleotide.

16. The siRNA according to any one of claims 1 to 15, wherein the antisense strand comprises a phosphorothioate group at the following positions, from the 5' end to the 3' end: Between the first nucleotide and the second nucleotide starting from the 5' end of the antisense strand; and Between the second and third nucleotides starting from the 5' end of the antisense strand; and Between the first nucleotide and the second nucleotide starting from the 3' end of the antisense strand; and between the second and third nucleotides starting from the 3' end of the antisense strand.

17. The siRNA according to any one of claims 1 to 16, wherein each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a 2'-deoxyribonucleotide, a GNA-modified nucleotide, or a combination of any two or more thereof; Preferably, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5' phosphate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5' phosphate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, the GNA modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, the GNA-modified nucleotide is located at position 7 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5' phosphate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 8, 9, and 10 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, from the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; from the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 5, 8, 10, 14, 16, and 18 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, from the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 8, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; from the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 4, 6, 12, 14, 16, and 18 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2 and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7 and 9 of the sense strand, a 2'-deoxyribonucleotide is located at position 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 18 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 11, 13, and 15 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 11, and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 8, and 9 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 3, 9, 11, and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 11, and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 11, 12, and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 3, 7, 11, and 16 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 3, 7, 11, 16, and 17 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, the 6th position is a GNA-modified nucleotide, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 5, 7, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 8, and 9 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 5, 7, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 3, 7, and 9 of the sense strand, and 2'-methoxy-modified nucleotides are located at the remaining positions; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 5, 7, and 14 of the antisense strand, and 2'-methoxy-modified nucleotides are located at the remaining positions, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, and 9 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7, 12, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 12, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 3, 6, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, and 2'-methoxy-modified nucleotides are located at the remaining positions; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 10, and 14 of the antisense strand, the 7th nucleotide is replaced by a thymine nucleotide, a 2'-deoxyribonucleotide is located at position 5 of the antisense strand, and 2'-methoxy-modified nucleotides are located at the remaining positions, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, and 2'-methoxy-modified nucleotides are located at the remaining positions; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, and 14 of the antisense strand, the 12th nucleotide is replaced with a thymine nucleotide, a 2'-deoxyribonucleotide is located at position 5 of the antisense strand, and 2'-methoxy-modified nucleotides are located at the remaining positions, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2 and 14 of the antisense strand, the 7th and 12th nucleotides are replaced with thymine nucleotides, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, and 14 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 8, and 9 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand, the 6th position is a GNA-modified nucleotide, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand, the 6th position is a GNA-modified nucleotide, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 5, 7, 8 and 9 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides.

18. The siRNA according to any one of claims 1 to 17, which is selected from the siRNA in Table 1, preferably, the siRNA is selected from N-ER-FY007180, N-ER-FY007180M8, N-ER-FY007180M44, N-ER-FY007180M56, N-ER-FY007180M58, N-ER-FY007180M62, N-ER-FY007180M2, N-ER-FY007180M3, N-ER-FY007180M4, N-ER-FY007180M5, N-ER-FY007180M6, N-ER-FY007180M7 007180M7, N-ER-FY007180M9, N-ER-FY007180M10, N-ER-FY007180M11, N-ER-FY007180M12, N-ER-FY007180M13, N-ER-FY007180M14, N-ER-FY 007180M15, N-ER-FY007180M16, N-ER-FY007180M17, N-ER-FY007180M18, N-ER-FY007180M19, N-ER-FY007180M21, N-ER-FY007180M24, N-ER-F Y007180M26, N-ER-FY007180M27, N-ER-FY007180M28, N-ER-FY007180M29, N-ER-FY007180M30, N-ER-FY007180M31, N-ER-FY007180M32, N-ER -FY007180M34, N-ER-FY007180M35, N-ER-FY007180M37, N-ER-FY007180M40, N-ER-FY007180M45, N-ER-FY007180M46, N-ER-FY007180M47, N-E N -ER-FY007180M55, N-ER-FY007180M57, N-ER-FY007180M59, N-ER-FY007180M60, N-ER-FY007180M61, N-ER-FY007180M63, N-ER-FY007180M64.

19. An siRNA conjugate or a prodrug thereof, wherein the siRNA conjugate comprises the siRNA according to any one of claims 1 to 18 and a conjugation group conjugated to the siRNA.

20. The siRNA conjugate or a prodrug thereof according to claim 19, wherein the conjugated group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked; Preferably, in the siRNA conjugate, the sense strand and the antisense strand of the siRNA are complementary to each other to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region; or, In the siRNA conjugate, the sense strand and the antisense strand of the siRNA are complementary to each other to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, while the 3' end of the antisense strand forms a blunt end.

21. The siRNA conjugate or prodrug thereof according to claim 19 or 20, wherein the conjugated group is selected from:

22. The siRNA conjugate or prodrug thereof according to any one of claims 19 to 21, wherein the siRNA conjugate is an siRNA conjugate selected from Table 2; preferably, the siRNA conjugate is selected from N-ER-FY007180M8L96, N-ER-FY007180M44L96, N-ER-FY007180M56L96, N-ER-FY007180M58L96, N-ER-FY007180M62L96, N-ER-FY007180M2L96, N-ER-FY007180M3L96, N-ER-FY007180 M6L96, N-ER-FY007180M7L96, N-ER-FY007180M9L96, N-ER-FY007180M10L96, N-ER-FY007180M11L96, N-ER-FY007180M13L96, N-ER-FY007 180M15L96, N-ER-FY007180M21L96, N-ER-FY007180M24L96, N-ER-FY007180M26L96, N-ER-FY007180M27L96, N-ER-FY007180M29L96, N-ER -FY007180M31L96, N-ER-FY007180M32L96, N-ER-FY007180M34L96, N-ER-FY007180M35L96, N-ER-FY007180M37L96, N-ER-FY007180M40L9 6. N-ER-FY007180M45L96, N-ER-FY007180M46L96, N-ER-FY007180M47L96, N-ER-FY007180M48L96, N-ER-FY007180M49L96, N-ER-FY00718 0M50L96, N-ER-FY007180M51L96, N-ER-FY007180M52L96, N-ER-FY007180M53L96, N-ER-FY007180M54L96, N-ER-FY007180M55L96, N-ER-F Y007180M57L96, N-ER-FY007180M59L96, N-ER-FY007180M60L96, N-ER-FY007180M61L96, N-ER-FY007180M63L96, N-ER-FY007180M64L96.

23. A pharmaceutical composition comprising the siRNA according to any one of claims 1 to 18, or the siRNA conjugate according to any one of claims 19 to 22 or a prodrug thereof, and a pharmaceutically acceptable carrier. 24 . A kit comprising the siRNA according to claim 1 , or the siRNA conjugate or a prodrug thereof according to claim 19 , or the pharmaceutical composition according to claim 21 .

25. Use of the siRNA according to any one of claims 1 to 18, or the siRNA conjugate or prodrug thereof according to any one of claims 19 to 22, or the pharmaceutical composition according to claim 23 for preparing a medicament for inhibiting HSD17B13 gene expression.

26. Use of the siRNA according to any one of claims 1 to 18, or the siRNA conjugate or prodrug thereof according to any one of claims 19 to 22, or the pharmaceutical composition according to claim 23 for preparing a medicament for preventing and / or treating diseases associated with HSD17B13 gene overexpression.

27. The use according to claim 26, wherein the disease is selected from non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, alcoholic hepatitis, liver fibrosis, and liver cancer.

28. A method for inhibiting HSD17B13 gene expression, comprising contacting a therapeutically effective amount of the siRNA of any one of claims 1-18, or the siRNA conjugate or prodrug thereof of any one of claims 19-22, or the pharmaceutical composition of claim 23 with a cell expressing HSD17B13 or administering it to a subject in need thereof.

29. A method for treating and / or preventing diseases related to HSD17B13 gene overexpression, comprising administering a therapeutically effective amount of the siRNA according to any one of claims 1 to 18, or the siRNA conjugate or prodrug thereof according to any one of claims 19 to 22, or the pharmaceutical composition according to claim 23 to a subject in need thereof.

30. The siRNA of any one of claims 1-18, or the siRNA conjugate or prodrug thereof of any one of claims 19-22, or the pharmaceutical composition of claim 23, for use as a medicament.

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