Sirna for inhibiting MARC1 gene expression and conjugate thereof, and use thereof

Abstract

The present invention relates to a siRNA for inhibiting MARC1 gene expression and a siRNA conjugate thereof, a pharmaceutical composition comprising same, and a use thereof. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, and the siRNA comprises a sense strand and an antisense strand. The siRNA and the conjugate thereof and the pharmaceutical composition can effectively treat and / or prevent diseases associated with overexpression of MARC1 gene.

Description

siRNA for inhibiting expression of MARC1 gene, conjugates and uses thereof TECHNICAL FIELD

[0001] The present invention relates to siRNA for inhibiting expression of MARC1 gene, siRNA conjugates thereof, pharmaceutical compositions comprising the same and uses thereof. BACKGROUND

[0002] Liver disease is a leading cause of death and disability worldwide, with chronic disease progressively destroying liver cells and even destroying regeneration, leading to liver fibrosis and cirrhosis. Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the world, with prevalence doubling in the past 20 years and now estimated to affect approximately 20-30% of the world’s population. The pathology that leads to nonalcoholic fatty liver disease is comprised of a series of factors. In some individuals, accumulation of ectopic fat in the liver, known as steatosis, triggers inflammation and liver cell damage, leading to further disease, known as nonalcoholic steatohepatitis (NASH). NASH is defined as steatosis with evidence of cell damage, inflammation, and varying degrees of scarring or fibrosis.

[0003] Mitochondrial amidoxime reducing component 1 (MARC1) is a molybdenum-containing enzyme that can reduce N-hydroxylated compounds and is associated with the outer membrane of mitochondria. A common missense variant in the MARC1 gene was recently shown to protect the liver from the invasion of fatty liver and cirrhosis caused by various causes. The report (Luukkonen P.K., Juuti A., Sammalkorpi H., et al. MARC1 variant rs2642438 increases hepatic phosphatidylcholines and decreases severity of non-alcoholic fatty liver disease in humans. Journal of Hepatology, 2020, 73: 696-739.) said that MARC1 variant carriers (n = 53) had higher liver concentrations of polyunsaturated phosphatidylcholine than non-carriers (n = 65), which may be related to the incidence of NASH. In addition, studies have shown that by knocking out the expression of the MARC1 gene in patients, hyperglycemia, diabetes, metabolic syndrome, insulin resistance (insulin insensitivity), glucose intolerance, high blood sugar levels, pulmonary hypertension, etc. in patients can be treated.

[0004] In summary, by inhibiting the expression of the MARC1 gene in patients, obesity, non-alcoholic fatty liver disease, alcohol-related fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, liver fibrosis, elevated liver enzyme levels (ALT, AST, ALP), hepatocellular carcinoma, hypercholesterolemia and related cardiovascular diseases, insulin resistance, glucose intolerance, hyperglycemia, type II diabetes and metabolic syndrome can be prevented and treated. There is no related drug on the market targeting only such gene expression, so the development of drugs targeting the MARC1 target has important value. SUMMARY

[0005] The present application aims to provide siRNA, siRNA conjugates and pharmaceutical compositions thereof, which are effective in RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the MARC1 gene, thereby selectively and effectively inhibiting the expression of the MARC1 gene and achieving the purpose of disease treatment.

[0006] In particular, the present application provides an siRNA capable of inhibiting MARC1 gene expression, the siRNA comprising a sense strand and an antisense strand, wherein each of the nucleotides in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, 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:

[0007] (1) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 2;

[0008] (2) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 3, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 4;

[0009] (3) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 5, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 6;

[0010] (4) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 7.

[0011] In some embodiments, the sense strand further contains a nucleotide sequence V and / or the antisense strand further contains a nucleotide sequence VI, the nucleotide sequence V and VI each has a length of 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 preferred embodiments, the nucleotide sequence V or VI each has a length of 2 nucleotides. In some preferred embodiments, the nucleotide sequence V is identical to or has nucleotide difference with the nucleotide at the corresponding position of the target mRNA, or VI is mismatched or complementary to the nucleotide at the corresponding position of the target mRNA. In some preferred embodiments, the nucleotide sequence V or VI each is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides.

[0012] In some embodiments, at least one of the nucleotides in the sense strand or the antisense strand is a modified nucleotide, and / or at least one phosphate group is a phosphate group with a modification group; preferably, the phosphate group with a modification group is a phosphorothioate group formed by replacing one of the oxygen atoms in the phosphodiester bond of the phosphate group with a sulfur atom.

[0013] In some embodiments, 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.

[0014] In some embodiments, neither the 5' terminal nor the 3' terminal of the sense strand is linked to an inverted abasic deoxyribose residue. In other embodiments, only the 5' terminal of the sense strand is linked to one inverted abasic deoxyribose residue. In other embodiments, only the 3' terminal of the sense strand is linked to one inverted abasic deoxyribose residue. In other embodiments, both the 5' terminal and the 3' terminal of the sense strand are linked to one inverted abasic deoxyribose residue, respectively.

[0015] In some preferred embodiments, 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.

[0016] In some embodiments, the modified nucleotide is selected from a 2'-fluoro-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'-deoxy-modified nucleotide, a 2'-amino-modified nucleotide, a 2'-substituted amino-modified nucleotide, a nucleotide analog, or a combination of any two or more thereof.

[0017] In some embodiments, the modified nucleotide is selected from a 2'-F-modified nucleotide, a 2'-0-CH3-modified nucleotide, a 2'-0-CH2-CH2-0-CH3-modified nucleotide, a 2'-0-CH2-CH=CH2-modified nucleotide, a 2'-CH2-CH2-CH=CH2-modified nucleotide, a 2'-deoxy-modified nucleotide, a nucleotide analog, or a combination of any two or more thereof.

[0018] In some embodiments, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

[0019] In some preferred embodiments, in the sense strand, 2'-fluoro-modified nucleotides are at positions 7, 9, 10, and 11, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 5, 7, 8, and 9, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 9, 10, and 11, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 8, 9, and 10, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 8, 9, 10, and 11, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7 and 9, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, and 11, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 11, 13, and 15, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 11, and 13, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 8, and 9, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 9, 11, and 13, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 9, and 11, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 11, and 13, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 11, 12, and 13, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11, and 16, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11, and 17, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11, 16, and 17, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, and 14, and the remaining positions are non-fluoro-modified nucleotides; and / or

[0020] In the antisense strand, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 2, 6, 14, and 16, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 3, 5, 8, 10, 14, 16, and 18, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 3, 4, 5, 7, 10, and 14, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 4, 6, 12, 14, 16, 18, and 20, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2 and 14, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, 18, and 20, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 7, 10, and 14, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 5, 7, and 14; or, 2'-fluoro-modified nucleotides are at positions 2, 3, 5, 7, 10, 12, and 14, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 7, and 14, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 4, 6, 12, 14, 16, and 18, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 18, and the remaining positions are non-fluoro-modified nucleotides.

[0021] In some embodiments, each non-fluoro-modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by a non-fluoro group, the nucleotide analog being selected from one of pseudouracil, an iso-nucleotide, LNA, ENA, cET BNA, UNA, and GNA.

[0022] In some embodiments, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a 2'-deoxy-modified nucleotide, a GNA-modified nucleotide, or a combination of any two or more thereof.

[0023] In some preferred embodiments, in the sense strand, 2'-fluoro-modified nucleotides are at positions 7, 9, 10, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 5, 7, 8, and 9, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 9, 10, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 8, 9, and 10, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 8, 9, 10, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7 and 9, and the nucleotide at position 11 of the sense strand is a 2'-deoxy-modified nucleotide, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 11, 13, and 15, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 11, and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 8, and 9, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 9, 11, and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 9, and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 11, and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 11, 12, and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11, and 16, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11, and 17, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11, 16, and 17, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, and 14, and the remaining positions are 2'-methoxy-modified nucleotides; and / or

[0024] In some embodiments, the siRNA comprises a sense strand having 2'-fluoro-modified nucleotides at positions 2, 6, 14, and 16, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 3, 5, 8, 10, 14, 16, and 18, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 3, 4, 5, 7, 10, and 14, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 4, 6, 12, 14, 16, 18, and 20, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2 and 14, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 3, 4, 5, 7, 10, and 14, and a GNA-modified nucleotide at position 6, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, 18, and 20, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 7, 10, and 14, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 5, 7, and 14; or 2'-fluoro-modified nucleotides at positions 2, 3, 5, 7, 10, 12, and 14, and a GNA-modified nucleotide at position 6, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 7, and 14, 2'-deoxy-modified nucleotides at positions 5 and 12, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 4, 6, 12, 14, 16, and 18, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 18, and 2'-methoxy-modified nucleotides at the remaining positions.

[0025] In some embodiments, the siRNA comprises a sense strand having 2'-fluoro-modified nucleotides at positions 2, 6, 14, and 16, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 3, 5, 8, 10, 14, 16, and 18, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 3, 4, 5, 7, 10, and 14, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 4, 6, 12, 14, 16, 18, and 20, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2 and 14, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 3, 4, 5, 7, 10, and 14, and a GNA-modified nucleotide at position 6, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, 18, and 20, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 7, 10, and 14, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 5, 7, and 14; or 2'-fluoro-modified nucleotides at positions 2, 3, 5, 7, 10, 12, and 14, and a GNA-modified nucleotide at position 6, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 7, and 14, 2'-deoxy-modified nucleotides at positions 5 and 12, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 4, 6, 12, 14, 16, and 18, and 2'-methoxy-modified nucleotides at the remaining positions; or 2'-fluoro-modified nucleotides at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 18, and 2'-methoxy-modified nucleotides at the remaining positions.

[0026] (1) between the 1st and 2nd nucleotides from the 5' terminus of the sense strand; and

[0027] between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand;

[0028] or,

[0029] (2) between the 1st and 2nd nucleotides from the 5' terminus of the sense strand; and

[0030] the 2nd nucleotide to the 3rd nucleotide from the 5' terminus of the sense strand; and

[0031] the inverted abasic deoxyribose residue to the 1st nucleotide from the 3' terminus of the sense strand;

[0032] or,

[0033] (3) the 1st nucleotide to the 2nd nucleotide from the 5' terminus of the sense strand.

[0034] In some embodiments, the siRNA comprises, in the 5' to 3' direction, a phosphorothioate group at each of the following positions:

[0035] the 1st nucleotide to the 2nd nucleotide from the 5' terminus of the antisense strand; and

[0036] the 2nd nucleotide to the 3rd nucleotide from the 5' terminus of the antisense strand; and

[0037] the 1st nucleotide to the 2nd nucleotide from the 3' terminus of the antisense strand; and

[0038] the 2nd nucleotide to the 3rd nucleotide from the 3' terminus of the antisense strand.

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

[0040] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 7, 9, 10, and 11 of the sense strand, and 2'-methoxy-modified nucleotides are at the remaining positions; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy-modified nucleotides are at the remaining positions, the 5' terminal nucleotide of the antisense strand being linked to a 5'-trans- vinylphosphonate group.

[0041] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 5, 7, 8, and 9 of the sense strand, and 2'-methoxy-modified nucleotides are at the remaining positions; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy-modified nucleotides are at the remaining positions, the 5' terminal nucleotide of the antisense strand being linked to a 5'-trans- vinylphosphonate group.

[0042] In a preferred embodiment, 2'-fluoro modified nucleotides are at positions 9, 10, and 11 of the sense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides are at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand being linked to a 5 '-trans vinyl phosphonate ester group.

[0043] In a preferred embodiment, 2'-fluoro modified nucleotides are at positions 8, 9, and 10 of the sense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides are at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand being linked to a 5 '-trans vinyl phosphonate ester group.

[0044] In a preferred embodiment, 2'-fluoro modified nucleotides are at positions 7, 9, 10, and 11 of the sense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides are at positions 2, 3, 5, 8, 10, 14, 16, and 18 of the antisense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand being linked to a 5 '-trans vinyl phosphonate ester group.

[0045] In a preferred embodiment, 2'-fluoro modified nucleotides are at positions 7, 9, 10, and 11 of the sense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides are at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand being linked to a 5 '-trans vinyl phosphonate ester group.

[0046] In a preferred embodiment, 2'-fluoro modified nucleotides are at positions 8, 9, 10, and 11 of the sense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides are at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand being linked to a 5 '-trans vinyl phosphonate ester group.

[0047] In a preferred embodiment, 2'-fluoro modified nucleotides are at positions 7, 9, 10, and 11 of the sense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides are at positions 2, 4, 6, 12, 14, 16, 18, and 20 of the antisense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction, of the antisense strand, the 5' terminal nucleotide of which is linked to a 5 '-trans vinyl phosphonate ester group.

[0048] In a preferred embodiment, 2'-fluoro modified nucleotides are at positions 7, 9, 10, and 11 of the sense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides are at positions 2 and 14 of the antisense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction, of the antisense strand, the 5' terminal nucleotide of which is linked to a 5 '-trans vinyl phosphonate ester group.

[0049] In a preferred embodiment, 2'-fluoro modified nucleotides are at positions 7 and 9 of the sense strand, the nucleotide at position 11 of the sense strand is a 2'-deoxy modified nucleotide, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides are at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, 18, and 20 of the antisense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction, of the antisense strand, the 5' terminal nucleotide of which is linked to a 5 '-trans vinyl phosphonate ester group.

[0050] In a preferred embodiment, 2'-fluoro modified nucleotides are at positions 7, 9, and 11 of the sense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides are at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction, of the antisense strand, the 5' terminal nucleotide of which is linked to a 5 '-trans vinyl phosphonate ester group.

[0051] In a preferred embodiment, 2'-fluoro modified nucleotides are at positions 7, 9, 11, 13, and 15 of the sense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides are at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides are at the remaining positions, in the 5' to 3' direction, of the antisense strand, the 5' terminal nucleotide of which is linked to a 5 '-trans vinyl phosphonate ester group.

[0052] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5 '-trans vinyl phosphonate ester group.

[0053] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5 '-trans vinyl phosphonate ester group.

[0054] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5 '-trans vinyl phosphonate ester group.

[0055] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5 '-trans vinyl phosphonate ester group.

[0056] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5 '-trans vinyl phosphonate ester group.

[0057] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group.

[0058] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group.

[0059] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 3, 7, 11, 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 at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group.

[0060] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group.

[0061] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 3, 4, 5, 7, 10, and 14, with position 6 being a GNA modified nucleotide, and the remaining positions being 2'-methoxy modified nucleotides, in the 5' to 3' direction, of the antisense strand, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group.

[0062] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5 '-trans vinyl phosphonate ester group.

[0063] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 5, 7, and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5 '-trans vinyl phosphonate ester group.

[0064] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5 '-trans vinyl phosphonate ester group.

[0065] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5 '-trans vinyl phosphonate ester group.

[0066] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, the 5' terminal nucleotide of the antisense strand is linked to a 5 '-trans vinyl phosphonate ester group.

[0067] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand, and the 6thposition is a GNA modified nucleotide, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group.

[0068] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 7, and 14 of the antisense strand, and the 5thand 12thpositions of the antisense strand are 2'-deoxy modified nucleotides, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group.

[0069] In a preferred embodiment, the 2'-fluoro modified nucleotides are 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 at positions 2, 4, 6, 12, 14, 16, and 18 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group.

[0070] In a preferred embodiment, the 2'-fluoro modified nucleotides are at positions 7 and 9 of the sense strand, and the 11thposition of the sense strand is a 2'-deoxy modified nucleotide, and the remaining positions are 2'-methoxy modified nucleotides, in the 5' to 3' direction; the 2'-fluoro modified nucleotides are 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, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group.

[0071] In this document, in the description of the modification methods of the sense strand or the antisense strand of siRNA, when the base is "T", the person skilled in the art understands that the base "T" means deoxyribonucleotide, without 2'-F, 2'-O-CH3, nucleotide analogues, and the like modification of the base "T".

[0072] In one embodiment, the application provides an siRNA selected from Table 1; preferably, the siRNA is selected from N-ER-FY025096M44, N-ER-FY025102M44, N-ER-FY025175M44, N-ER-FY025177M44, N-ER-FY025096M45, N-ER-FY025102M45, N-ER-FY025175M45, N-ER-FY025177M45, N-ER-FY025096M46, N-ER-FY025102M46, N-ER-FY025175M46, N-ER-FY025177M46, N-ER-FY025096M47, N-ER-FY025102M47, N-ER-FY025175M47, N-ER-FY025177M47, N-ER-FY025096M48, N-ER-FY025102M48, N-ER-FY025175M48, N-ER-FY025177M48, N-ER-FY025102M49, N-ER-FY025175M49, N-ER-FY025096M50, N-ER-FY025102M50, N-ER-FY025175M50, N-ER-FY025177M50.

[0073] The application also provides an siRNA conjugate comprising an siRNA of the application and a conjugate group conjugated to the siRNA (as shown below, the double helix structure represents the siRNA and the conjugate group is attached to the 3' end of the sense strand of the siRNA):

[0074] In the conjugate structure described above, X can be selected from O or S, and in some embodiments, X is O.

[0075] In some embodiments, in the siRNA conjugate, the sense strand of the siRNA is complementary to the antisense strand 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 overhanging nucleotides extending out of the double-stranded region;

[0076] or,

[0077] In the siRNA conjugate, the sense strand of the siRNA is complementary to the antisense strand 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 forms a blunt end.

[0078] In some embodiments, the conjugate group is selected from:

[0079] In some embodiments, the siRNA conjugate is selected from the group consisting of siRNA conjugates of Table 2; preferably, the siRNA conjugate is selected from the group consisting of N-ER-FY025096M44L96, N-ER-FY025102M44L96, N-ER-FY025175M44L96, N-ER-FY025177M44L96, N-ER-FY025096M45L96, N-ER-FY025102M45L96, N-ER-FY025175M45L96, N-ER-FY025177M45L96, N-ER-FY025096M46L96, N-ER-FY025102M46L96, N-ER-FY025175M46L96, N-ER-FY025177M46L96, N-ER-FY025096M47L96, N-ER-FY025102M47L96, N-ER-FY025175M47L96, N-ER-FY025177M47L96, N-ER-FY025096M48L96, N-ER-FY025102M48L96, N-ER-FY025175M48L96, N-ER-FY025177M48L96, N-ER-FY025102M49L96, N-ER-FY025175M49L96, N-ER-FY025096M50L96, N-ER-FY025102M50L96, N-ER-FY025175M50L96, N-ER-FY025177M50L96.

[0080] The present application also provides a pharmaceutical composition comprising the siRNA of the present application, or the siRNA conjugate of the present application, and a pharmaceutically acceptable carrier.

[0081] The present application also provides a kit comprising the siRNA of the present application, or the siRNA conjugate of the present application, or the pharmaceutical composition of the present application.

[0082] The present application also provides use of the siRNA of the present application, or the siRNA conjugate of the present application, or the pharmaceutical composition of the present application, for the manufacture of a medicament for inhibiting the expression of MARC1 gene.

[0083] The present application also provides use of the siRNA of the present application, or the siRNA conjugate of the present application, or the pharmaceutical composition of the present application, for the manufacture of a medicament for preventing and / or treating a disease associated with overexpression of MARC1 gene.

[0084] In some embodiments, the disease is obesity, non-alcoholic fatty liver disease, alcohol-related fatty liver disease, non-alcoholic steatohepatitis, liver cirrhosis, liver fibrosis, elevated liver enzyme levels (ALT, AST, ALP), hepatocellular carcinoma, hypercholesterolemia and related cardiovascular diseases, insulin resistance, impaired glucose tolerance, hyperglycemia, type II diabetes and metabolic syndrome.

[0085] The present application also provides a method for inhibiting MARC1 gene expression in vivo or in vitro, comprising contacting the siRNA of the present application, or the siRNA conjugate of the present application, or the pharmaceutical composition of the present application with a cell expressing MARC1 or administering to a subject in need thereof.

[0086] The present application also provides a method for treating and / or preventing a disease associated with overexpression of MARC1 gene, comprising administering to a subject in need thereof a therapeutically effective amount or a prophylactically effective amount of the siRNA of the present application, or the siRNA conjugate of the present application, or the pharmaceutical composition of the present application.

[0087] In some embodiments, the disease is obesity, non-alcoholic fatty liver disease, alcohol-related fatty liver disease, non-alcoholic steatohepatitis, liver cirrhosis, liver fibrosis, elevated liver enzyme levels (ALT, AST, ALP), hepatocellular carcinoma, hypercholesterolemia and related cardiovascular diseases, insulin resistance, impaired glucose tolerance, hyperglycemia, type II diabetes and metabolic syndrome.

[0088] The present application also provides the siRNA of the present application, or the siRNA conjugate of the present application, or the pharmaceutical composition of the present application for use in therapy or as a medicament. Advantages

[0089] The siRNA, the pharmaceutical composition and the siRNA conjugate provided by the present application show excellent MARC1 gene expression inhibition activity in in vitro cell experiments, and have good potential for treating diseases associated with overexpression of MARC1 gene. For example, the siRNA and its conjugate disclosed by the present application can reduce the expression of MARC1 mRNA in the liver, have low toxic and side effects, good plasma stability, and good clinical application prospect.

[0090] The siRNA provided by the present application shows good inhibition effect on MARC1 gene in HepG2 cells. BRIEF DESCRIPTION OF DRAWINGS

[0091] Figure 1 shows the results of off-target analysis of the concentration of the siRNA conjugate of the present application at its IC 50 value of 0.625 nM.

[0092] Figure 2 shows the off-target analysis results of the siRNA conjugate of the present application at its IC 50 value of 0.15625 nM.

[0093] Figure 3 shows the off-target analysis results of the siRNA conjugate of the present application at its IC 50 value of 0.0390625 nM. DETAILED DESCRIPTION

[0094] DEFINITIONS

[0095] Throughout the specification, unless otherwise specifically identified, in the art, "G", "C", "A", "T", and "U" generally represent the bases of guanine, cytosine, adenine, thymine, and uracil, respectively, but it is also generally known in the art that "G", "C", "A", "T", and "U" each generally also represent the nucleotides containing guanine, cytosine, adenine, thymine, and uracil as the base, respectively, which is a common way of representation in indicating deoxyribonucleic acid sequences and / or ribonucleic acid sequences, thus in the context of the present application, the meaning of "G", "C", "A", "T", "U" includes all the above possible cases, and in the present application, "nucleotides", "ribonucleic acid", and "ribonucleotides" are used interchangeably, and herein "deoxyribonucleotides" and "2'-deoxyribonucleotides" are used interchangeably. Lowercase a, u, c, g: represent 2'-methoxy-modified nucleotides; Af, Gf, Cf, Uf: represent 2'-fluoro-modified nucleotides; dA, dG, dC, dU, dT: represent 2'-deoxy-modified ribonucleotides at the corresponding positions in the motif sequence; (invAb) is an inverted abasic deoxyribose residue; Lowercase s represents a phosphorothioate linkage between the two nucleotides adjacent to the letter s on the left and right; P1: represents a 5'-phosphonucleotide adjacent to the P1 on the right; EVP: represents a 5'-trans- vinylphosphonate nucleotide adjacent to the EVP on the right; (underlined + bold + italic): represents a GNA-modified nucleotide; Base represents a base, such as A, U, G, C, or T.

[0096] In the foregoing and hereinafter, 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 with a fluorine. The "non-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 with a non-fluorine group or a "non-fluoro modified nucleotide" is a nucleotide analog. In some embodiments, each non-fluoro modified nucleotide is independently selected from one of a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced with a non-fluorine group or a nucleotide analog. These nucleotides in which the hydroxyl group at the 2' position of the ribose group is replaced with a non-fluorine group are well known to those skilled in the art, and the nucleotides can be selected from one of 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, a 2'-deoxy modified nucleotide.

[0097] "Alkyl" includes straight-chain, branched-chain, or cyclic saturated alkyl groups. For example, alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, n-pentyl, cyclohexyl, and the like. By way of example, "C 1-6 "Alkyl" includes straight-chain, branched-chain, or cyclic saturated alkyl groups. For example, alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, n-pentyl, cyclohexyl, and the like. By way of example, "C 1-6 "Alkyl" includes straight-chain, branched-chain, or cyclic saturated alkyl groups. For example, alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, n-pentyl, cyclohexyl, and the like. By way of example, "C

[0098] "Alkoxy" refers herein to an alkyl group attached to the remainder of the molecule through an oxygen atom (-O-alkyl), wherein the alkyl group is as defined herein. Non-limiting examples of alkoxy groups include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, n-pentoxy, and the like.

[0099] "Nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid, but differs in structure from an adenine ribonucleotide, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide, or a thymine deoxyribonucleotide. Such as pseudouracil (Ψ), an isonucleotide, a bridged nucleic acid (BNA), or an acyclic nucleotide.

[0100] Pseudouracil (Ψ) refers to: a natural structural analog of uracil nucleoside, ribose is not connected to uracil N1, but to C5 of the pyrimidine ring, and its structure is as follows:

[0101] BNA refers to a constrained or inaccessible nucleotide. A BNA can contain a five-, six-, or seven-membered ring with a "fixed" C3'-endo sugar conformation bridged structure. The bridge is typically incorporated at the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET BNA, and the like, wherein LNA is represented by Formula (1), ENA is represented by Formula (2), and cET BNA is represented by Formula (3):

[0102] Base represents a base, such as A, U, G, C, or T.

[0103] Acyclic nucleotides are a class of nucleotides in which the sugar ring of the nucleotide is opened, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), wherein UNA is represented by Formula (4) and GNA is represented by Formula (5):

[0104] In the above Formula (4) and Formula (5), Base represents a base, such as A, U, G, C, or T, and R is selected from H, OH, or alkoxy (O-alkyl).

[0105] Iso-nucleotides refer to compounds in which the position of the base on the ribose ring is altered, such as compounds in which the base is moved from the 1'-position to the 2'-position or 3'-position of the ribose ring, such as represented by Formula (6) or (7):

[0106] In the above Formula (6) to Formula (7) compounds, Base represents a base, such as A, U, G, C, or T; and R is selected from H, OH, F, or a non-fluoro group as described above.

[0107] In some embodiments, the nucleotide analog is selected from one of pseudouracil, iso-nucleotide, LNA, ENA, cET BNA, UNA, and GNA. In some embodiments, each non-fluoro 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-fluoro modified nucleotide is a 2'-methoxy modified nucleotide, and herein and hereafter, the 2'-methoxy modified nucleotide refers to a nucleotide in which the 2'-hydroxyl of the ribosyl group is replaced with a methoxy group.

[0108] The "phosphorothioate group" refers to a phosphorothioate group in which one of the oxygen atoms in the phosphodiester linkage of the phosphate group is replaced with a sulfur atom.

[0109] The "phosphorothioate group" refers to the following formula:

[0110] The "5'-phosphonate nucleotide" refers to the following structure:

[0111] In the context of the present specification, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art, i.e. in a double-stranded nucleic acid molecule, the bases of one strand each pair with a base on the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or in RNA, uracil (U)); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair comprises one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be deduced from the sequence of its complementary strand. Correspondingly, "mismatch" in the art means that the bases at the corresponding positions in a double-stranded nucleic acid do not pair in a complementary manner.

[0112] In the above and below, unless specifically stated otherwise, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "essentially reverse complementary" means that there are no more than 1 base mismatch between the two nucleotide sequences; "completely reverse complementary" means that there are no base mismatches between the two nucleotide sequences.

[0113] In the above and below, a nucleotide sequence has a "nucleotide difference" with another nucleotide sequence if the base type of the nucleotide at the same position is changed in the former compared to the latter, e.g. if in the latter a nucleotide base is A, and in the former the corresponding nucleotide base at the same position is U, C, G or T, the nucleotide difference between the two nucleotide sequences at that position is recognized. In some embodiments, a nucleotide difference at a position is also recognized when an abasic nucleotide or an equivalent thereof is substituted for the nucleotide at that position.

[0114] In the above and below, "overhang" means one or more unpaired nucleotides that protrude from a duplex structure of an siRNA when a 3' end of one strand of the siRNA extends beyond the 5' end of the other strand, or vice versa. "Blunt" or "blunt end" means that there are no unpaired nucleotides at that end of the siRNA, i.e. there is no nucleotide overhang. A "blunt ended" siRNA is an siRNA that is double-stranded over its entire length, i.e. there is no nucleotide overhang at either end of the molecule. "Double-stranded region" means the region of an siRNA where the sense strand and the antisense strand are complementary to form a double-stranded region of the siRNA.

[0115] In the foregoing and hereafter, "5'-nucleotide" refers to a nucleotide in which the phosphate group is attached to the 5' carbon of the pentose sugar, which is the predominant type of nucleotide found free in organisms. "3'-nucleotide" refers to a nucleotide in which the phosphate group is attached to the 3' carbon of the pentose sugar, which can include, for example, adenosine-3'-phosphate, guanosine-3'-phosphate, cytidine-3'-phosphate, uridine-3'-phosphate, 2'-deoxythymidine-3'-phosphate, 2'-O-methyladenosine-3'-phosphate, 2'-O-methyladenosine-3'-phosphorothioate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroadenosine-3'-phosphorothioate, 2'-O-methylguanosine-3'-phosphate, 2'-O-methylguanosine-3'-phosphorothioate, 2'-fluoroguanosine-3'-phosphate, 2'-fluoroguanosine-3'-phosphorothioate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylcytidine-3'-phosphorothioate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'-phosphorothioate, 2'-O-methyluridine-3'-phosphate, 2'-O-methyluridine-3'-phosphorothioate, 2'-fluorouridine-3'-phosphate, 2'-fluorouridine-3'-phosphorothioate, 2'-deoxythymidine-3'-phosphorothioate. This definition can apply to modified or unmodified nucleotide phosphoramidite monomers.

[0116] The terms "iRNA," "RNAi agent," "iRNA agent," "RNA interference agent," used in the context of the present disclosure are used interchangeably herein to refer to the term as defined herein includes siRNA and mediates the targeted cleavage of RNA transcripts by the RNA-induced silencing complex (RISC) pathway. iRNAs direct the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNAs modulate, e.g., inhibit, expression of a target gene in a cell, such as a cell of a subject, e.g., a mammalian subject.

[0117] As used herein, "2'-deoxy-modified nucleotide" or "2'-deoxy-modification" refers to the replacement of a ribonucleotide A, U, C, G with the corresponding deoxyribonucleotide dA, dT, dC, dG in the process of modification. Herein, "2'-deoxy-modified nucleotide" or "2'-deoxy-modification" is also referred to as "replacement of a nucleotide with a deoxyribonucleotide."

[0118] In the context of the present specification, unless otherwise indicated, "conjugate" refers to the covalent linkage between two or more chemical moieties, each having a specific function; correspondingly, "conjugate" refers to the compound formed by the covalent linkage between the individual chemical moieties. Further, "siRNA conjugate" denotes the compound formed by the covalent linkage of one or more chemical moieties having a specific function to an siRNA. The siRNA conjugate should be understood in the context of the present specification as a general term for a plurality of siRNA conjugates or an siRNA conjugate represented by a certain chemical formula. In the context of the present specification, "conjugate molecule" should be understood as a specific compound that can be conjugated to an siRNA by a reaction to ultimately form an siRNA conjugate of the present application.

[0119] In the context of the present application, unless otherwise indicated, "conjugate" refers to the covalent linkage between two or more chemical moieties, each having a specific function; correspondingly, "conjugate" refers to the compound formed by the covalent linkage between the individual chemical moieties. Further, "siRNA conjugate" denotes the compound formed by the covalent linkage of one or more chemical moieties having a specific function to an siRNA. The siRNA conjugate should be understood in the context of the present specification as a general term for a plurality of siRNA conjugates or an siRNA conjugate represented by a certain chemical formula. In the context of the present specification, "conjugate molecule" should be understood as a specific compound that can be conjugated to an siRNA by a reaction to ultimately form an siRNA conjugate of the present application.

[0120] Various hydroxyl protecting groups can be used in the present application. In general, a protecting group renders a chemical functional group insensitive to particular reaction conditions, and can be added to and removed from the functional group in a molecule without substantially damaging the rest of the molecule. In some embodiments, the protecting group is stable under basic conditions, but can be removed under acidic conditions. In some embodiments, nonexclusive examples of hydroxyl 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, nonexclusive examples of hydroxyl 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).

[0121] The term "disease associated with MARC1 gene overexpression" is a disease or disorder associated with the involvement of complement MARC1. The term "disease associated with MARC1 gene overexpression" includes a disease, disorder or condition that would benefit from a reduction in MARC1 (i.e., "MARC1 -associated disease") expression. In some embodiments, the disease associated with abnormal MARC1 gene expression is selected from the group consisting of obesity, non-alcoholic fatty liver disease, alcohol-related fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, liver fibrosis, elevated liver enzyme levels (ALT, AST, ALP), hepatocellular carcinoma, hypercholesterolemia and related cardiovascular diseases, insulin resistance, glucose intolerance, hyperglycemia, type II diabetes and metabolic syndrome. Illustratively, reference is made to the following scientific literature, without limitation: Emdin C.A., Haas M.E., Khera A.V., et al. A missense variant in Mitochondrial Amidoxime Reducing Component 1 gene and protection against liver disease. PLoS Genet, 2020, 16(4): e1008629.; Ott G., Reichmann D., Boerger C., et al. Functional characterization of protein variants encoded by nonsynonymous single nucleotide polymorphisms in MARC1 and MARC2 in healthy Caucasians. Drug Metab Dispos, 2014, 42: 718-725.; Meroni M., Longo M., Tria G., et al. Genetics is of the essence to face NAFLD. Biomedicines, 2021, 9: 1359.; Parisinos C.A., Wilman H.R., Thomas E.L., et al. Genome-wide and Mendelian randomisation studies of liver MRI yield insights into the pathogenesis of steatohepatitis. Journal of Hepatology, 2020, 73: 241-251.; Du X., DeForest N., Majithia A.R.Human genetics to identify therapeutic targets for NAFLD: challenges and opportunities. Front. Endocrinol. 2021, 12: 777075.; Bence K.K., Birnbaum M.J. Metabolic drivers of non-alcoholic fatty liver disease. Journal of molmet, 2020, 101143.; Luukkonen P.K., Juuti A., Sammalkorpi H., et al. MARC1 variant rs2642438 increases hepatic phosphatidylcholines and decreases severity of non-alcoholic fatty liver disease in humans. Journal of Hepatology, 2020, 73:696-739.; Trépo E., Valenti L. Update on NAFLD genetics: from new variants to the clinic. Journal of Hepatology, 2020, 7634.; Sparacino-Watkins C.E., Tejero J., Sun B., et al. Nitrite reductase and nitric-oxide synthase activity of the mitochondrial molybdopterin enzymes mARC1 and mARC2. The Journal of Biological Chemistry, 2014, 289(15): 10345-10358.; Luukkonen, Panu K et al. “Distinct contributions of metabolic dysfunction and genetic risk factors in the pathogenesis of non-alcoholic fatty liver disease.” Journal of hepatology vol. 76, 3 (2022): 526-535. doi:10.1016 / j.jhep.2021.10.013; Romeo, Stefano. "MARC1 and HNRNPUL1: Two Novel Players in Alcohol-related Liver Disease." Gastroenterology vol. 159, 4 (2020): 1231-1232. doi:10.1053 / j.gastro.2020.08.009; Rivera-Paredez, Berenice et al. "Association of MARC1, ADCY5, and BCO1 Variants with the Lipid Profile, Suggests an Additive Effect for Hypertriglyceridemia in Mexican Adult Men." International journal of molecular sciences vol. 23, 1911815. 5 Oct. 2022, doi:10.3390 / ijms231911815; Gao, Chuan et al. "Genome-wide association analysis of serum alanine and aspartate aminotransferase, and the modifying effects of BMI in 388k European individuals." Genetic epidemiology vol. 45, 6 (2021): 664-681. doi:10.1002 / gepi.22392.

[0122] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0123] The term "subject," as used herein, refers to any animal, such as a mammal or a marsupial. Subjects of the present application include, but are not limited to, humans, non-human primates (e.g., rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, rabbits, or any species of poultry.

[0124] As used herein, "treatment" refers to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding the fact that the subject can still be afflicted with the disorder.

[0125] As used herein, "prevention" refers to an approach for obtaining beneficial or desired results, including but not limited to prophylactic benefit. To achieve "prophylactic benefit," the siRNA, siRNA conjugate, or pharmaceutical composition can be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though the subject can not yet be diagnosed with the disease.

[0126] siRNA

[0127] The present application relates to an siRNA capable of inhibiting the expression of a MARC1 gene. The siRNA of the present application contains nucleotide groups as a basic structural unit, which are known to those skilled in the art to contain a phosphate group, a ribose group, and a base. The siRNA is generally active, i.e., functional, and has a length of about 12 to 40 nucleotides, and in some embodiments, about 15 to 30 nucleotides.

[0128] The siRNA of the present application contains a sense strand and an antisense strand, each of the nucleotides in the siRNA being independently modified or unmodified, wherein the sense strand contains a nucleotide sequence I and the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse-complementary to form a double-stranded region. In some embodiments, the double-stranded region has a length of 15 to 30 nucleotide pairs. In other embodiments, the double-stranded region has a length of 17 to 23 nucleotide pairs. In still other embodiments, the double-stranded region has a length of 19 to 21 nucleotide pairs. In yet other embodiments, the double-stranded region has a length of 19 or 21 nucleotide pairs.

[0129] In some embodiments, the sense strand further comprises a nucleotide sequence V and / or the antisense strand further comprises a nucleotide sequence VI, each of the nucleotide sequences V and VI has a length of 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. In some embodiments, each of the nucleotide sequences V and VI has a length of 2 nucleotides. In other embodiments, the nucleotide sequence V is identical to or has nucleotide difference with the nucleotide at the corresponding position of the target mRNA, or VI is mismatched or complementary to the nucleotide at the corresponding position of the target mRNA. In other embodiments, each of the nucleotide sequences V and VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides.

[0130] In some embodiments, the siRNA provided herein has a sense strand and an antisense strand with the same length, or different length. In some embodiments, the sense strand or the antisense strand has 15-30 nucleotides. In other embodiments, the sense strand or the antisense strand has 19-25 nucleotides. In other embodiments, the sense strand or the antisense strand has 19-23 nucleotides. The siRNA provided herein can have a length ratio of the sense strand to the antisense strand of 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 siRNA has a length ratio of the sense strand to the antisense strand of 19 / 19, 19 / 21, 21 / 23, 21 / 21, or 23 / 23, which can result in a siRNA with better mRNA silencing activity in cells.

[0131] It has been found that different modification strategies can have a significant impact on the stability, biological activity, and cytotoxicity of siRNA. For example, CN201010106762.1 discloses several chemical modification strategies for siRNA, and confirms that seven of them are effective. Compared with the unmodified siRNA, the siRNA modified by one of the seven strategies has improved blood stability while maintaining an inhibitory activity comparable to that of the unmodified siRNA.

[0132] The nucleotides in the siRNA of the present application are each independently modified or unmodified nucleotides. In some embodiments, each of the nucleotides in the siRNA of the present application is an unmodified nucleotide; in some embodiments, some or all of the nucleotides in the siRNA of the present application are modified nucleotides, and these modifications on the nucleotide groups do not cause the siRNA of the present application to lose or substantially weaken its function of inhibiting the expression of the MARC1 gene.

[0133] 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" is used to refer to a nucleotide or a nucleotide analogue in which the ribosyl 2' position hydroxyl group is replaced with another group, or a nucleotide with a modified base. The modified nucleotide does not cause the siRNA to lose or substantially weaken its function of inhibiting the expression of a gene. For example, the modified nucleotides disclosed in J. K. Watts, G. F. Deleavey, and M. J. Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20):842-55 can be selected.

[0134] In some embodiments, at least one of the nucleotides in the sense strand or the antisense strand of the siRNA provided by the present application is a modified nucleotide, and / or at least one of the phosphate groups is a phosphate group with a modification group; in other words, at least one of the phosphate groups and / or at least one of the ribosyl groups in the phosphate-sugar backbone of at least one of the single strands of the sense strand and the antisense strand is a phosphate group with a modification group and / or a ribosyl group with a modification group. In some embodiments, the phosphate group with a modification group is a phosphorothioate group in which one of the oxygen atoms in the phosphodiester bond is replaced with a sulfur atom.

[0135] In some embodiments, when the nucleotide sequences of the sense strand and the antisense strand are complementary to each other to form a double-stranded region, and there is no overhanging nucleotide 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. Then, after the nucleotide sequence formed by the addition of the nucleotide sequence V to the 3' end of the sense strand is chemically modified, the nucleotide sequence V is removed, and accordingly, the sense strand of the siRNA forms a blunt end.

[0136] In some embodiments, when the nucleotide sequences of the sense strand and the antisense strand are complementary to each other to form a double-stranded region, and the 3' end of the sense strand has an overhanging nucleotide extending out of the double-stranded region, the overhanging nucleotide at the 3' end of the sense strand is removed as the nucleotide sequence of the sense strand, and accordingly, the sense strand of the siRNA forms a blunt end.

[0137] In some embodiments, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group.

[0138] After the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group, the following structures are formed:

[0139] wherein formula (8) shows the structure formed by the 5' terminal nucleotide linked to a 5' phosphate group, formula (9) shows the structure formed by the 5' terminal nucleotide linked to a 5' phosphate derivative group (EVP), and formula (10) shows the structure formed by the 5' terminal nucleotide linked to a 5' phosphate derivative group (5' methylene phosphate group), wherein Base represents a base, such as A, U, G, C, or T. R' is a hydroxyl group or substituted with various groups known to those skilled in the art, for example, the modified nucleotide after substitution 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, a 2'-deoxy modified nucleotide.

[0140] In some embodiments, the 5' terminal nucleotide of the sense strand or the antisense strand is not linked to a 5' phosphate group, a 5' phosphate derivative group, or (invAb) (i.e., the ribose group of the 5' terminal nucleotide of the sense strand or the antisense strand is a 5' hydroxyl group), which has the following structure:

[0141] wherein Base represents a base, such as A, U, G, C, or T. R is a hydroxyl group or hydrogen or substituted with various groups known to those skilled in the art, for example, R can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino.

[0142] An exemplary modified nucleotide has the following structure:

[0143] wherein Base represents a base, e.g., A, U, G, C, or T. The 2' position of the ribose group is substituted with R. The 2' position of the ribose group can be substituted with a variety of groups known to one of skill in the art, e.g., the 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, a 2'-deoxy modified nucleotide.

[0144] In some embodiments, the sense strand can comprise one or more capping residues or moieties, sometimes referred to in the art as "caps," "terminal caps," or "capping residues." As used herein, a "capping residue" is a non-nucleotide compound or other moiety that can be incorporated at one or more of the terminal ends of the nucleotide sequence of an siRNA disclosed herein. In some cases, a capping residue can provide certain beneficial properties to the siRNA, such as protection against exonuclease degradation. In some embodiments, an inverted abasic deoxyribose residue (invAb) is added as a capping residue. In some embodiments, the capping residue occurs at the 5' terminal end, the 3' terminal end, or both the 5' and 3' terminal ends of the sense strand.

[0145] In some embodiments, one or more inverted abasic deoxyribose residues (invAb) are added to the 3' terminal end of the sense strand. In some embodiments, one or more inverted abasic deoxyribose residues (invAb) are added to the 5' terminal end of the sense strand. In some embodiments, one or more inverted abasic deoxyribose residues (invAb) are added to the 5' terminal end of the sense strand and to the 3' terminal end of the sense strand. The inverted abasic deoxyribose residue can be linked via a phosphoester linkage, a phosphorothioate linkage, or other internucleoside linkage. In describing the site of modification in a method of modification, the (invAb) is not counted as the 1st site of the sequence, and the chemical structure of the inverted abasic deoxyribose residue is shown below:

[0146] Formula B when the (invAb) is at the 3' terminal end of the siRNA; and Formula C when the (invAb) is at the 5' terminal end of the siRNA:

[0147] In some embodiments, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe, 2'-O-CH3) modified nucleotide, and the like.

[0148] In some embodiments, the 2'-substituted alkoxy modified nucleotide is a 2'-methoxyethoxy (2'-O-CH2-CH2-O-CH3) modified nucleotide, a 2'-O-CH2-CH=CH2 modified nucleotide, and the like.

[0149] In some embodiments, the 2'-substituted alkyl modified nucleotide is a 2'-CH2-CH2-CH=CH2 modified nucleotide, and the like.

[0150] In some embodiments, all of the nucleotides in the sense strand and / or the antisense strand are modified nucleotides. In some embodiments, the siRNA provided herein has each of the nucleotides in the sense strand and the antisense strand independently being a 2'-fluoro modified nucleotide or a non-fluoro modified nucleotide.

[0151] In some embodiments, each of the non-fluoro modified nucleotides is a 2'-methoxy modified nucleotide, a GNA modified nucleotide, or a combination of any two or more thereof.

[0152] siRNA conjugate

[0153] The present application relates to a siRNA conjugate, which contains the siRNA described above and a conjugate group conjugated to the siRNA.

[0154] 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 the 3' end of the sense strand of the siRNA conjugate forms a blunt end. 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 overhanging 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. In the present disclosure, "the 3' end of the sense strand forms a blunt end" includes the case where the 3' end of the sense strand originally has a blunt end, and the case where the 3' end of the sense strand originally has an overhanging end, and the overhanging end is excluded to form a blunt end.

[0155] In some preferred embodiments, the siRNA conjugate is obtained by conjugating the siRNA with the conjugate group. In this case, the sense strand and the 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. The conjugate group is conjugated to the 3' end of the sense strand having a blunt end to form the siRNA conjugate.

[0156] In some preferred embodiments, the 3' end of the sense strand of the siRNA has overhanging nucleotides extending out of the double-stranded region. The sequence having a 3' blunt end formed by excluding the overhanging nucleotides at the 3' end of the sense strand is used as the nucleotide sequence for conjugating the conjugate group. The conjugate group is conjugated to the 3' blunt end of the sense strand to form the siRNA conjugate.

[0157] 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, the 3' end of the sense strand has no overhanging nucleotide, and a nucleotide sequence V is added to the 3' end of the sense strand as an overhanging nucleotide. The sequence formed by excluding the overhanging nucleotide at the 3' end of the sense strand to form a 3' blunt end is used as a nucleotide sequence for linking the conjugating group, and the conjugating group is linked to the 3' blunt end of the sense strand to form the siRNA conjugate.

[0158] 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, the 3' end of the sense strand has an overhanging nucleotide extending out of the double-stranded region, and the sequence formed by excluding the overhanging nucleotide at the 3' end of the sense strand to form a 3' blunt end is used as a nucleotide sequence for linking the conjugating group, and the conjugating group is linked to the 3' blunt end of the sense strand to form the siRNA conjugate.

[0159] Generally, the conjugating group comprises at least one targeting group which is pharmaceutically acceptable, or further comprises a linker, and the siRNA, the linker and the targeting group are sequentially linked. 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 conjugating group, for example, can be covalently conjugated to the conjugating group. The conjugation site of the siRNA to the conjugating group can be at the 3' end or the 5' end of the sense strand of the siRNA, or at the 5' end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA to the conjugating group is at the 3' end of the sense strand of the siRNA.

[0160] In some embodiments, the conjugate group can be attached to the phosphate group, 2'-position hydroxyl group, or base of the nucleotide. In some embodiments, the conjugate group can also be attached to the 3'-position hydroxyl group, in which case the nucleotides are linked by 2'-5' phosphodiester bonds. When the conjugate group is attached to the end of the siRNA strand, the conjugate group is usually attached to the phosphate group of the nucleotide; when the conjugate group is attached to the internal sequence of the siRNA, the conjugate group is usually attached to the ribose sugar ring or base. Various attachment 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.

[0161] In some embodiments, the siRNA and the conjugate group can be connected by acid-labile or reducible chemical bonds, which can be degraded in the acidic environment of the intracellular endosome, thereby allowing the siRNA to become free. For non-degradable conjugation, the conjugate group can be attached to the sense strand of the siRNA, thereby minimizing the impact of conjugation on the activity of the siRNA.

[0162] In some embodiments, the pharmaceutically acceptable targeting group can be a ligand commonly used in the field of siRNA administration, such as various ligands described in WO2009082607A2, which is incorporated herein by reference in its entirety.

[0163] In some embodiments, the pharmaceutically acceptable targeting group can be selected from one or more of the following ligands formed by targeting molecules or their derivatives: lipophilic molecules such as cholesterol, bile acids, vitamins (e.g., vitamin E), lipid molecules of different chain lengths; polymers such as polyethylene glycol; polypeptides such as transmembrane peptides; aptamers; antibodies; quantum dots; sugars such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folate; receptor ligands expressed by liver parenchymal cells such as asialoglycoproteins, asialo sugar residues, lipoproteins (such as high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin, etc.

[0164] In some embodiments, each ligand is independently selected from a ligand that binds to a cell surface receptor. In some embodiments, at least one ligand is a ligand that binds to a liver cell surface receptor. In some embodiments, at least one ligand is a ligand that binds to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand that binds to a human liver cell surface receptor. In some embodiments, at least one ligand is a ligand that binds to a liver surface asialoglycoprotein receptor (ASGPR). The classes of such ligands are known to those skilled in the art and generally function to bind to a specific receptor on the surface of a target cell, mediating delivery of the siRNA linked to the ligand to the target cell.

[0165] In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to an asialoglycoprotein receptor (ASGPR) on the surface of a mammalian liver cell. In some embodiments, each ligand is independently an asialoglycoprotein, such as asialoorosomucoid (ASOR) or asialofetuin (ASF). In some embodiments, the ligand is a sugar or a derivative of a sugar.

[0166] 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 can 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 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 derivatives thereof, mannans and derivatives thereof, galactose and derivatives thereof, xylose and derivatives thereof, ribose and derivatives thereof, fucose and derivatives thereof, lactose and derivatives thereof, maltose and derivatives thereof, arabinose and derivatives thereof, fructose and derivatives thereof, and sialic acid.

[0167] 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, a-D-mannofuranose, β-D-mannofuranose, a-D-mannopyranose, β-D-mannopyranose, a-D-glucopyranose, β-D-glucopyranose, a-D-glucoruranose, β-D-glucoruranose, a-D-fructofuranose, a-D-fructopyranose, a-D-galactopyranose, β-D-galactopyranose, a-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-0-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L- glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-0-methyl-D-mannopyranose, 2-deoxy-2- sulfonamido-D-glucopyranose, N-glycolyl-a-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4-tri-0-acetyl-1-thio-6-0-trityl-a-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-0-acetyl-2-deoxy-1,5-dithio-a-D-glucoheptoside ethyl ester, 2,5-anhydro-D-allosonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, or L-4-thioribose. Other choices of the ligands can be found, for example, in CN105378082A, which is incorporated by reference in its entirety.

[0168] 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 monovalent, divalent, trivalent, and tetravalent as used herein refer to the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate formed by the conjugation of the siRNA molecule to the conjugation group containing galactose or N-acetylgalactosamine as the targeting group is 1 : 1, 1:2, 1:3, or 1:4, respectively. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA of the present application is conjugated to the conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA of the present application is conjugated to the conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0169] The targeting group can be linked to the siRNA molecule via a suitable linker, which can be selected by a person skilled in the art according to the specific type of the targeting group. The linkers, the types of the targeting groups and the connection modes to the siRNA can be found in the disclosure of WO2015006740A2, which is incorporated herein by reference in its entirety.

[0170] Synthesis method of siRNA

[0171] The nucleotide monomers are connected one by one in the order of nucleotide arrangement from 3'-5' direction by the conventional solid-phase phosphoramidite method in the art. Each connection of a nucleotide monomer includes four steps of deprotection, coupling, oxidation or sulfurization, and capping. When the phosphate linkage is used between two nucleotides, the connection of the next nucleotide monomer includes four steps of deprotection, coupling, oxidation, and capping. When the phosphorothioate linkage is used between two nucleotides, the connection of the next nucleotide monomer includes four steps of deprotection, coupling, sulfurization, and capping. The nucleotide monomers are selected according to the target sequence in the present application, and the selected nucleotide monomers are the nucleotide monomers commonly used by a person skilled in the art, for example, the nucleotide monomers for synthesizing A can be but are not limited to selecting adenosine-3-phosphate. It should be understood that these monomers are connected to each other by 5'-3' phosphodiester bonds or 5'-3' phosphorothioate groups when they exist in an oligonucleotide, and when the last nucleotide at the 3' position is hydroxyl, for example, in the 5' to 3' direction, it is realized according to the conventional means in the art.

[0172] For example, the synthesis conditions of the siRNA of the present application can be as follows:

[0173] The deprotection conditions include: the reaction temperature is 25°C, the reaction time is 70 seconds, and the deprotection reagent is selected from dichloroacetic acid in dichloromethane solution (3% V / V), and the molar ratio of the deprotection reagent to the 4,4'-dimethoxytrityl protecting group on the solid support is 5:1.

[0174] The coupling reaction conditions include: the reaction temperature is 25°C, the reaction time is 600 seconds, the coupling reagent is selected from 5-ethylthio-1H-tetrazole (ETT) in 0.25M acetonitrile solution, the molar ratio of the nucleic acid sequence connected on the solid support to the nucleotide monomer is 1:10, and the molar ratio of the nucleic acid sequence connected on the solid support to the coupling reagent is 1:65.

[0175] The oxidation reaction conditions include: the reaction temperature is 25°C, the reaction time is 15 seconds, the oxidation reagent is selected from 0.05M iodine tetrahydrofuran solution, and the molar ratio of the oxidation reagent to the nucleic acid sequence connected on the solid support in the coupling step is 30:1. The reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine=3:1:1.

[0176] The sulfuration reaction conditions include: the reaction temperature is 25°C, the reaction time is 300 seconds, the sulfuration reagent is selected from the group consisting of hydroxylamine, and the molar ratio of the sulfuration reagent to the nucleic acid sequence connected on the solid carrier in the coupling step is 120:1. The reaction is carried out in a mixed solvent of acetonitrile:pyridine = 1:1.

[0177] The capping reaction conditions include: the reaction temperature is 25°C, the reaction time is 15 seconds, the capping reagent is selected from a mixed solution of CapA (10% acetic anhydride acetonitrile solution) and CapB (10% N-methyl imidazole pyridine / acetonitrile solution) with a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence connected on the solid carrier is the molar ratio of acetic anhydride:N-methyl imidazole:the nucleic acid sequence connected on the solid carrier is 1:1:1.

[0178] After all the nucleotide monomers are connected, the nucleic acid sequence connected on the solid carrier is subjected to amination, purification, desalination in sequence to obtain siRNA sense and antisense strands, and finally the two strands are heated to anneal to obtain the product.

[0179] The methods of amination, purification, desalination and annealing are well known in the art. For example, amination is carried out by contacting the nucleotide sequence connected with the solid carrier with concentrated ammonia water; purification is carried out by chromatography; desalination is carried out by reverse phase chromatography; and annealing is carried out by gradually cooling the mixture of the sense and antisense strands in equimolar ratio under different stringent conditions.

[0180] The synthesized siRNA is shown in Table 1.

[0181] siRNA conjugate synthesis method

[0182] Take the synthesis of L96 as an example:

[0183] In the first step, compound L96-A is obtained by reacting DMTr-L96 and succinic anhydride:

[0184] Preparation process: DMTr-L96, succinic anhydride, 4-dimethylaminopyridine and diisopropylethylamine are added to dichloromethane, stirred at 25°C for 24 hours, then the reaction solution is washed with 0.5M triethylamine phosphate, the aqueous phase is washed with dichloromethane three times, the organic phases are combined and evaporated under reduced pressure to obtain the crude product. Then column chromatography is used to purify the pure product L96-A.

[0185] In the second step, L96-A is reacted with NH2-SPS to obtain L96-B:

[0186] Preparation process: L96-A, O-benzotriazol-tetramethyl urea hexafluorophosphate (HBTU) and diisopropyl ethylamine (DIPEA) were mixed and dissolved in acetonitrile, stirred at room temperature for 5 minutes to obtain a uniform solution, and then added to the reaction solution. Ammonia methyl resin (NH2-SPS, 100-200 mesh) was added to the reaction solution, and the shaking table reaction was started at 25°C. After 18 hours of reaction, the filter cake was washed with dichloromethane and acetonitrile in sequence to obtain the filter cake. The obtained filter cake was subjected to cap reaction with CapA / CapB mixed solution to obtain L96-B, which is a solid phase carrier containing a conjugated molecule.

[0187] Third step, preparation of siRNA conjugate

[0188] L96-B was used as a solid phase carrier to synthesize siRNA according to the siRNA synthesis method described above, and the siRNA antisense chain was synthesized according to the siRNA synthesis method described above, and annealing generated the siRNA conjugate of the present application.

[0189] The synthesized siRNA conjugate is shown in Table 2.

[0190] Pharmaceutical composition

[0191] The present application provides a pharmaceutical composition containing siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.

[0192] The pharmaceutically acceptable carrier can be one or more of the carriers conventionally used in the field of siRNA administration, such as, but not limited to, a lipid nanoparticle (LNP), a magnetic nanoparticle (e.g., a nanoparticle based on Fe3O4 or Fe2O3), a carbon nanotube, mesoporous silicon, a calcium phosphate nanoparticle, polyethylenimine (PEI), a polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA), and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA), and derivatives thereof.

[0193] The content of the siRNA and the pharmaceutically acceptable carrier in the pharmaceutical composition is not particularly limited and can be a conventional content of each component.

[0194] In some embodiments, the pharmaceutical composition can further include a pharmaceutically acceptable additional excipient, which can be one or more of various formulations or compounds conventionally used in the art. For example, the pharmaceutically acceptable additional excipient can include at least one of a pH buffer, a protective agent, and an osmotic pressure adjusting agent.

[0195] The pH buffer can be a tris-hydroxymethyl aminomethane hydrochloride buffer having a pH of 7.5 to 8.5 and / or a phosphate buffer having a pH of 5.5 to 8.5, for example, a phosphate buffer having a pH of 5.5 to 8.5.

[0196] The protective agent can be at least one of myo-inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The content of the protective agent can be 0.01 to 30% by weight, based on the total weight of the pharmaceutical composition.

[0197] The osmotic pressure adjusting agent can be sodium chloride and / or potassium chloride. The content of the osmotic pressure adjusting agent is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmoles per kilogram (mOsm / kg). The content of the osmotic pressure adjusting agent can be readily determined by one skilled in the art depending on the desired osmotic pressure.

[0198] In some embodiments, the pharmaceutical composition can be a liquid formulation, such as an injection solution, or a lyophilized powder formulation that is mixed with a liquid excipient to prepare a liquid formulation for administration. The liquid formulation can be, but is not limited to, for subcutaneous, intramuscular, or intravenous injection, or can be, but is not limited to, for administration to the lung by nebulization, or for administration to other organ tissues (e.g., liver) by nebulization through the lung. In some embodiments, the pharmaceutical composition is for intravenous injection.

[0199] 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 can also be referred to as an organic amine), a helper lipid, and / or a pegylated lipid.

[0200] The following examples are intended to further illustrate the present application, but are not to be construed as limiting the application in any way.

[0201] Example

[0202] Other objects, features, and advantages of the present application will become apparent from the following detailed description, but it is to be understood that both the detailed description and the specific examples, while indicating specific embodiments of the application, are given by way of illustration only, since various changes and modifications within the spirit and scope of the application will become apparent to those skilled in the art from this detailed description.

[0203] The experimental techniques and experimental methods used in the present example are all conventional techniques and methods, unless otherwise specified, for example, the experimental methods not specifically indicated in the following examples are generally performed according to the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained through regular commercial channels.

[0204] Example 1 Preparation of siRNA

[0205] The siRNA molecules with the following sequences were synthesized by Tianlin Biotech (Shanghai) Co., Ltd.

[0206] Table 1 siRNAs and their sequences

[0207] wherein, capital letters "G", "C", "A", "T" and "U" each generally represent a nucleotide containing guanine, cytosine, adenine, thymine and uracil as a base, respectively; lowercase letters a, u, c, g: represent 2'-methoxy-modified nucleotides; Af, Gf, Cf, Uf: represent 2'-fluoro-modified nucleotides; dA, dG, dC, dU, dT: represent 2'-deoxy-modified ribonucleotides at the corresponding positions in the motif sequence; lowercase letter s represents a phosphorothioate linkage between the two nucleotides adjacent to the left and right of the letter s; EVP: represents that the nucleotide adjacent to the right of the EVP is a 5'-trans-vinyl phosphonate nucleotide; (underlined + bold + italic): represents a GNA-modified nucleotide; "(invAb)": represents an inverted abasic deoxyribose residue.

[0208] The siRNA conjugates with the following sequences were synthesized by Tianlin Biotech (Shanghai) Co., Ltd.:

[0209] Table 2 siRNA conjugates and their sequences:

[0210] wherein, L96 is connected to the 3' end of the sense strand in Table 1 or the 3' end of the sense strand forms a blunt end by a phosphodiester bond, and L96 is:

[0211] In Table 1 and Table 2, if the 5' terminal nucleotide of the sense strand, the modified sense strand and the modified sense strand connected with the conjugate group is not marked with P1, EVP or (invAb) on the left side, it represents that the 5' terminal nucleotide is not connected with a 5' phosphate group or a 5' phosphate derivative group or (invAb) (i.e. the ribose group of the 5' terminal nucleotide is 5' hydroxyl), and its structure is shown as formula X:

[0212] wherein, Base represents a base, for example, A, U, G, C or T; R is hydroxyl or hydrogen or various groups known to those skilled in the art, for example, R can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino.

[0213] In Tables 1 and 2, if the 5' terminal nucleotide of the antisense strand and the modified antisense strand is not marked with P1 or EVP to the left, it means that the ribosome of the 5' terminal nucleotide is a 5' hydroxyl group, and its structure is as shown in Formula X.

[0214] In Tables 1 and 2, when the 3' ends of the sense strand and the modified sense strand are not connected (invAb), the 3' position of the 3' terminal nucleotides of the sense strand and the modified sense strand is a hydroxyl group; in Tables 1 and 2, the 3' terminal nucleotides of the antisense strand and the modified antisense strand are a hydroxyl group.

[0215] Example 2: siRNA and siRNA conjugates inhibit MARC1 gene expression

[0216] 2.1 Experimental Materials:

[0217] HepG2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number SCSP-510.

[0218] RNA extraction kit 96 Kit, item number QIAGEN-74182;

[0219] Transfection reagent, purchased from Invitrogen, catalog number 13778-150;

[0220] MEM medium, purchased from Gibco, catalog number 41090036;

[0221] Reverse transcription kit ( 1st Strand cDNA Synthesis Kit (+gDNA wiper), purchased from Vazyme, catalog number R312-02;

[0222] TaqMan TM Gene expression premix, purchased from Applied Biosystems, catalog number 4369016;

[0223] Opti-MEM: serum-reduced culture medium, purchased from Gibco, catalog number 31985070;

[0224] Target MARC1 primer and probe set, purchased from Thermo, Hs00224227_m1;

[0225] TaqMan Gene Expression Assay (GAPDH), purchased from Thermo, ID-Hs99999905_m1.

[0226] 2.2 Experimental method:

[0227] 2.2.1 HepG2 cells were cultured in fresh MEM medium in 96-well plates for 48 hours, and the cultured cells were resuspended in MEM medium without PS (penicillin-streptomycin mixture) to prepare a cell suspension with a density of 1.11 x 10 5 cells / mL, and 90 μL of the cell suspension was added to each well of a 96-well plate, i.e. 10,000 cells per well.

[0228] 2.2.2 The dry powder of the siRNA and siRNA conjugate (collectively referred to as siRNA in the description of the experimental process of this example for ease of description) to be tested was centrifuged at low temperature and high speed, and then dissolved in ultra-pure distilled water (ULtra Pure Distilled Water) to prepare a 100 μM siRNA stock solution.

[0229] 2.2.3 Preparation of 200 nM siRNA transfection diluent Z and 2 nM siRNA diluent W

[0230] (1) Preparation of 10 μM siRNA stock solution Q and 0.1 μM siRNA stock solution E:

[0231] a. Take 2 μL of the 100 μM siRNA stock solution prepared in step 2.2.2 above, add 18 μL of ultra-pure distilled water, and obtain a 10 μM siRNA stock solution Q;

[0232] b. Take 2 μL of the 10 μM siRNA stock solution Q prepared in step a), add 18 μL of ultra-pure distilled water, and obtain a 1 μM siRNA stock solution Y;

[0233] c. Take 2 μL of the 1 μM siRNA stock solution Y prepared in step b), add 18 μL of ultra-pure distilled water, and obtain a 0.1 μM siRNA stock solution E;

[0234] (2) Take 2 μL of the prepared siRNA stock solution E, add 98 μL of Opti-MEM, and obtain a 2 nM siRNA diluent W; take 2 μL of the prepared siRNA stock solution Q, add 98 μL of Opti-MEM, and obtain a 200 nM siRNA diluent Z;

[0235] 2.2.4 Transfection of HepG2 cells

[0236] (1) Take transfection reagent 3 μL, add 97 μL of Opti-MEM, and obtain transfection reagent diluent; and take Mix the transfection reagent dilution with the 2 nM siRNA dilution W prepared in step 2.2.3 at a 1 : 1 volume ratio, stand for 5 minutes, and add 10 μL of the transfection mixture to the HepG2 cells cultured in step 2.2.1 in the 96-well plate (the final volume is 100 μL, and the concentration of siRNA in the system is 0.1 nM).

[0237] (2) Take Mix the transfection reagent dilution with the 2 nM siRNA dilution W prepared in step 2.2.3 at a 1 : 1 volume ratio, stand for 5 minutes, and add 10 μL of the transfection mixture to the HepG2 cells cultured in step 2.2.1 in the 96-well plate (the final volume is 100 μL, and the concentration of siRNA in the system is 0.1 nM). Mix the transfection reagent dilution with the 2 nM siRNA dilution W prepared in step 2.2.3 at a 1 : 1 volume ratio, stand for 5 minutes, and add 10 μL of the transfection mixture to the HepG2 cells cultured in step 2.2.1 in the 96-well plate (the final volume is 100 μL, and the concentration of siRNA in the system is 0.1 nM). Mix the transfection reagent dilution with the 2 nM siRNA dilution W prepared in step 2.2.3 at a 1 : 1 volume ratio, stand for 5 minutes, and add 10 μL of the transfection mixture to the HepG2 cells cultured in step 2.2.1 in the 96-well plate (the final volume is 100 μL, and the concentration of siRNA in the system is 0.1 nM).

[0238] Incubate for 48 hours after the above transfection; set 2 repeats for each concentration (10 nM and 0.1 nM).

[0239] 2.2.5 Extract the total RNA from the HepG2 cells obtained in step 2.2.4 according to the instructions of the RNA extraction kit.

[0240] 2.2.6 Reverse transcribe the total RNA obtained in step 2.2.5 to cDNA using the reverse transcription kit according to the following steps:

[0241] a) Remove gDNA according to the following table using gDNA enzyme;

[0242] 42°C, 2 min; 4°C, stand

[0243] b) Perform the reverse transcription program as follows:

[0244] 50°C, 15 min; 85°C, 5 s.

[0245] c) Store the reverse transcription product obtained in step b) at 4°C for real-time PCR analysis.

[0246] 2.2.7 Perform real-time PCR analysis

[0247] a) Prepare the qPCR reaction mixture as shown in the following table, and keep all reagents on ice during the entire operation;

[0248] b) Perform the qPCR program as follows

[0249] 50°C, 2 min, 95°C, 10 min;

[0250] 95°C, 15 s, 60°C, 1 min (40 cycles of this operation).

[0251] 2.2.8 Result analysis

[0252] a) The Ct value was automatically calculated using the Quant Studio 6 Flex software with default settings;

[0253] b) The relative expression of the gene was calculated using the following formula:

[0254] ΔCt = Ct (MARC1 gene) - Ct (GAPDH)

[0255] ΔΔCt = ΔCt (test sample group) - ΔCt (Mock group)

[0256] mRNA expression relative to the Mock group = 2 -ΔΔCt .

[0257] Mock group: a group without siRNA added compared with the test sample group

[0258] Inhibition rate (%) = (Mock group mRNA relative expression - test sample group mRNA relative expression) / Mock group mRNA relative expression x 100%

[0259] 2.3 Silencing experiment results

[0260] The concentrations of 0.1 nM and 10 nM were selected for testing, and the results are shown in Tables 3 and 4.

[0261] Table 3 Note: “--” indicates that the data is not shown.

[0262] Table 4

[0263] As can be seen from Tables 3 and 4, the siRNA of the present application can significantly inhibit the expression of the MARC1 gene at 10 nM and 0.1 nM.

[0264] Example 3

[0265] The determination concentration range of the following siRNA conjugate to be tested is set as follows: 10 nM, 4-fold dilution, 8 concentration gradients; siRNA conjugate (siRNA conjugate final concentration is 10 nM, 2.5 nM, 0.625 nM, 0.156 nM, 0.039 nM, 0.0098 nM, 0.0024 nM and 0.0006 nM, respectively, in a duplicate well) is transfected into HepG2 cells, and then IC 50 determination is carried out in a manner similar to that of Example 2.

[0266] Result analysis:

[0267] a) The Ct value is automatically calculated using the default settings of Quant Studio 6 Flex software;

[0268] b) The relative expression of the gene is calculated using the following formula:

[0269] ΔCt = Ct (MARC1 gene) - Ct (GAPDH)

[0270] ΔΔCt = ΔCt (test sample group) - ΔCt (Mock group), wherein the Mock group represents a group without the addition of siRNA conjugate compared with the test sample group;

[0271] mRNA expression relative to the Mock group = 2 -ΔΔCt

[0272] Inhibition rate (%) = (Mock group mRNA relative expression - test sample group mRNA relative expression) / Mock group mRNA relative expression x 100%

[0273] Calculation process: taking the log value of siRNA concentration as the X axis and the percentage inhibition rate as the Y axis, using the "log (inhibitor) vs. response - variable slope" of the analysis software GraphPad Prism 8 to fit the dose-effect curve, so as to obtain the IC 50 value of each siRNA.

[0274] The fitting formula is: Y = Bottom + (Top - Bottom) / (1 + 10^((logIC 50 -X) x HillSlope))

[0275] Wherein: Top represents the percentage inhibition rate at the top platform, and the Top of the curve is generally between 80% and 120%; Bottom represents the percentage inhibition rate at the bottom platform, and the Bottom of the curve is generally between -20% and 20%;

[0276] HillSlope represents the slope of the percentage inhibition rate curve.

[0277] The results are shown in Table 5.

[0278] Table 5

[0279] Example 4: Rat liver homogenate in vitro stability experiment

[0280] 4.1 Experimental reagents and consumables

[0281] 4.2 Experimental steps

[0282] 4.2.1 Preparation of liver homogenate

[0283] 4.2.1.1 Preparation of grinding fluid

[0284] 4.2.1.2 Tissue homogenate

[0285] The rat liver tissue (from SD rats, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) was prepared into liver homogenate (concentration of 20 mg / mL) at a ratio of 100 mg:5 mL of grinding fluid. After preparation, the grinding beads were added and placed in the homogenizer. The grinding parameters were set as follows.

[0286] 4.2.2 Sample preparation

[0287] The siRNA conjugate sample was prepared into a 1 mg / mL solution with enzyme-free water and was ready for use. The internal standard sample was prepared into a 0.125 mg / mL solution with enzyme-free water.

[0288] 4.2.3 Sample incubation

[0289] (1) Add 250 μL of prepared liver homogenate to a 2 mL enzyme-free tube;

[0290] (2) Add 50 μL of siRNA conjugate sample solution to the tube of step (1);

[0291] (3) The system was 300 μL of biological sample solution, vortexed, and stood for 5 min;

[0292] (4) Divide into 2 tubes, each containing 100 μL;

[0293] (5) Incubate the system at 37°C for 48 h.

[0294] 4.2.4 Biological sample processing

[0295] Each 100 μL of biological sample system vortex, mix, add 300 μL clarity OTX lysis liquid (Clarity OTX Lysis-loading Buffer, purchased from Agilent- Finnigan, item number AL0-8579) vortex, stand for 30 min, add 100 μL of internal standard solution, vortex, low speed centrifugation for 5 min, speed 1500 rpm, ready for use (total sample volume is about 500 μL).

[0296] 4.2.5 Solid phase extraction:

[0297] (1) Preparation of solid phase extraction reagent

[0298] Activator: take 200 mL of methanol into the mobile phase bottle, labeled as activator;

[0299] Equilibrium liquid: prepare 1M phosphate buffer solution 【877 mL of sodium dihydrogen phosphate (1.56 g / L) + 123 mL of disodium hydrogen phosphate (3.58 g / L) 】, dilute 100 times, adjust pH to 5.5 with phosphoric acid, labeled as equilibrium liquid;

[0300] Rinse liquid: take 500 mL of equilibrium liquid into a 1L mobile phase bottle, add 500 mL of acetonitrile, mix well, and label as rinse liquid;

[0301] Eluent: weigh 7.9 g of ammonium bicarbonate into a 1L mobile phase bottle, add 1L of water, adjust pH to 9.5 with sodium hydroxide, take 500 mL of the adjusted ammonium bicarbonate solution into a 1L mobile phase bottle, add 500 mL of acetonitrile, mix well, and label as eluent;

[0302] (2) The extraction steps are as follows:

[0303] 4.2.6 Post-processing

[0304] Take the eluent (in three times, 600 μL each, a total of 1800 μL) into a 2 mL EP tube, vacuum concentrate (or nitrogen 40℃ blow dry) for 10 hours, speed 1800 rpm; add 100 μL of mobile phase (initial ratio) to the concentrated sample for reconstitution, high speed centrifugation at 15℃ for 20 min at 12000 rpm, take 10 μL of supernatant and inject into high resolution mass spectrometry. The antisense strand ratio of siRNA conjugate of the application is detected by LC-MS / MS method semi-quantitatively, and the calculation formula is: AS strand remaining percentage % = AS MS intensity / AS strand and all related degradation products MS intensity × 100%. Wherein MS intensity is the mass spectrometry intensity signal value. The metabolic results after in vitro incubation in rat liver homogenate for 48 hours are shown in Table 6.

[0305] Table 6 Remaining percentage of antisense strand of siRNA conjugate

[0306] wherein the sequences of N-ER-FY025096M8L96 and N-ER-FY025177M8L96 are shown in Table 7.

[0307] Table 7

[0308] In Table 6, AS represents the antisense strand of the siRNA conjugate, and the greater the remaining amount of AS, the better the stability of the drug, and the better the long-acting property of the drug. As can be seen from Table 6, the siRNA conjugate of the present application has excellent in vitro stability in rat liver homogenate.

[0309] Example 5: Silencing effect of siRNA conjugate in mice expressing human MARC1 (hMARC1) gene

[0310] 5.1 AAV constructs overexpress hMARC1 gene mouse model

[0311] 6-8 week old C57BL / 6 male mice (provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.) enter the facility, and a single injection of adeno-associated virus AAV8 of hMARC1 gene is performed via the tail vein

[0312] (pAAV[Exp]-CBh>SEAP:{MARC1 part 3'UTR} (virus provided by Yunzhou Biotech (Guangzhou) Co., Ltd.) to target gene overexpression modeling, and the administration volume is 100 μL (6 x 10 11 vg) per mouse, and ordinary feed is provided.

[0313] 5.2 In vivo silencing siRNA efficacy investigation for hMARC1 mouse model

[0314] Fourteen days after AAV8 virus injection, grouping (5 mice per group) is performed, and a single 3 mg / kg (mpk) dose of siRNA conjugate of the present application is administered to the mice subcutaneously, and the administration volume is 5 μL / g, and the solvent is RNase-free sterile PBS, and the blank group is injected with the same volume of RNase-free sterile PBS. The SEAP protein expression amount (i.e., the hMARC1 protein expression amount) is detected at 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, 56 days and 63 days after administration, and the calculation formula is: % inhibition rate = (1- average protein expression of administration group / average protein expression of blank group) * 100%, and the inhibition rate (%) of the siRNA conjugate in the mice expressing the human MARC1 (hMARC1) gene is obtained, as shown in Table 8.

[0315] Table 8

[0316] As can be seen from Table 8, the siRNA conjugate of the present application has high inhibitory activity on the hMARC1 gene in vivo, and can reduce the expression level of hMARC1 for a long time, having a long-acting inhibitory effect.

[0317] Example 6: In vitro off-target experiment

[0318] The materials used in this experiment are referred to Example 2.

[0319] 6.1 Extraction of RNA samples after transfection of siRNA conjugate in HepG2 cells

[0320] 6.1.1 Sample preparation

[0321] 1) Dry powder of the test substance was centrifuged at low temperature and high speed.

[0322] 2) Dissolved with DEPC Water to prepare a 10 μM stock solution.

[0323] 6.1.2 Cell transfection

[0324] 1) siRNA dilution

[0325] 2) Mix the above dilutions B-G with transfection reagent dilution (same preparation method as in Example 2) at a volume ratio of 1:1, and stand for 5 minutes. Add the above transfection mixture to the 24-well plate at a volume of 50 μL per well, and add 450 μL of cell suspension to the well containing the above mixture, so that the final concentration of siRNA drug is 10, 2.5, 0.625, 0.15625, 0.0390625, 0.009765625 nM.

[0326] 3) After transfection for 48 hours without changing the liquid, perform qPCR detection.

[0327] 6.1.3 RNA extraction (VAMNE Magnetic Cell / Tissue Total RNA Kit (Cat. No.: RMA101-C2))

[0328] 1) Instrument sterilization: Turn on the instrument and irradiate the ultraviolet lamp for 30 minutes in advance.

[0329] 2) 96-channel reagent distribution map

[0330] 3) Preparation of lysis mixture:

[0331] 4) Add 110 μL lysis mix to each well of the 96-well plate; place the 96-well plate with lysis mix in a metal bath and incubate at room temperature for 5 minutes at 300 cycles / minute, then mix by pipetting 20 times using the automated workstation, and add to the 96-well deep well plate with binding mix in station 1.

[0332] 5) Pre-sealed reagent processing

[0333] Remove the pre-sealed reagent from the kit and centrifuge the reagent and magnetic beads to the bottom of the empty plate using a plate centrifuge at 500 rpm for 1 minute.

[0334] 6) DNase I reaction preparation and dispensing

[0335] Use the empty 96-well deep well plate as station 4 and add 100 μL DNase I reaction to each well.

[0336] 7) Machine loading

[0337] Carefully tear off the seal of the centrifuged pre-dispensed reagent (to avoid liquid splashing), and place the deep well plate in one direction A-H. Place the 96-well deep well plate in the nucleic acid extractor according to the 96-channel reagent distribution map, insert the magnetic rod sleeve into the magnetic rod holder of the machine, and confirm that it is securely installed. Open the program named No. 1; the name is cells; select the program No. NMR0211 for extraction.

[0338] 6.2 Detection of the multi-concentration point inhibition rate of siRNA conjugate in HepG2 cells

[0339] Reverse transcribe the RNA extracted in step 6.1, and the subsequent steps and inhibition rate calculation are the same as in Example 2. The multi-concentration point inhibition rate of siRNA conjugate N-ER-FY025175M46L96 in HepG2 cells is shown in Table 9 below.

[0340] Table 9

[0341] According to the inhibition rate test results, RNA samples at concentrations of 0.625 nM, 0.15625 nM and 0.0390625 nM were selected for sequencing.

[0342] 6.3 Second-generation sequencing

[0343] 1) Purify the total RNA extracted in step 6.1 according to the manufacturer's (Qiagen-217604_miRNeasy Tissue / Cells Advanced Mini Kit) guidelines.

[0344] 2) The concentration of total RNA was detected using NanoDrop One, and the RNA integrity was analyzed according to the manufacturer's (Agilent 2100 Bioanalyzer\Agilent-5067-1511_Agilent RNA 6000 Nano Kit) guidelines.

[0345] 3) Library preparation was performed according to the manufacturer's (Kapa Biosystems - KIT1004_Kapa HyperPlus\Kapa Biosystems - 7792- 0140_Kapa HyperPlus Kit) guidelines.

[0346] 4) The library concentration was detected according to the manufacturer's (Thermo Fisher Scientific - Qubit 4 fluorometer\Thermo Fisher Scientific - Q33231_Qubit 1X dsDNA HS Assay Kit) guidelines, and the library fragment size was analyzed according to the manufacturer's (Agilent 4200 TapeStation\Agilent-5067-5584_High Sensitivity D1000 ScreenTape) guidelines.

[0347] 5) Library sequencing was performed according to the manufacturer's (Illumina - NovaSeq 6000\Illumina-20028312_NovaSeq 6000 S4 Reagent Kit v1.5(300 cycles)) guidelines.

[0348] This experiment is based on the second-generation sequencing technology to study the effect of HepG2 cell transfection of the test substance N-ER-FY025175M46L96 on the transcriptome expression. The screening criteria for differential genes are set as |log2(FoldChange)|>1 & padj.<0.05. The results are shown in Figures 1-3 (MTARC1 in the figure is the target gene MARC1).

[0349] The results show that, in addition to the target gene MARC1, the differential genes knocked down at three concentrations (blue) do not overlap, indicating that there is no dose-dependent effect of differential genes, i.e., no off-target genes are found in the in vitro HepG2 cell experiment of N-ER-FY025175M46L96.

[0350] In addition, the inhibition rate IC 50 of N-ER-FY025175M46L96 in HepG2 cells is 0.0187 nM. When the concentration of N-ER-FY025175M46L96 is at its IC 50 ​No significant difference genes were found with log2(FoldChange) and padj. values close to the target gene MARC1 at 2-fold, 8-fold and 33-fold values (as shown in Figures 1-3), indicating that N-ER-FY025175M46L96 has low off-target risk, good safety and good drugability.

[0351] Example 7: Repeat Dose Toxicity Test of N-ER-FY025175M46L96 in SD Rats by Subcutaneous Injection

[0352] The experiment had 3 groups, and each group of female and male SD rats (from Guokecai Fuhebei Pharmaceutical Technology Co., Ltd.) had 3 rats. The main test group was subcutaneously injected with vehicle control (PBS), 100 or 300 mg / kg of N-ER-FY025175M46L96, respectively, once every two weeks for a total of 3 times, and the volume of the drug was 5 mL / kg. On D30, each group of female and male animals was planned for autopsy at the end of the dosing period. The satellite group (with the same dose and administration method as the main test group) had 3 female and 3 male rats for cytokine and TK blood collection.

[0353] The toxicity indicators of this experiment included: death / near-death, general observation, body weight, food intake, clinical pathology indicators (hematology and coagulation indicators, serum biochemical indicators), gross necropsy, organ weight, histopathological examination, and tissue distribution. The satellite group animals collected blood samples at 4h and 24h after dosing during the adaptation period, D1 and D29 for cytokine analysis. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8 and 24 hours before and after dosing on D1 and D29 for toxicokinetic analysis.

[0354] During the experiment, all animals survived to the planned autopsy, and all animals had no obvious abnormalities related to the test product in general observation, body weight, food intake, cytokines, clinical pathology indicators (hematology, coagulation, serum biochemistry), gross necropsy.

[0355] Example 8: Silencing Effect of siRNA Conjugate in Mice Expressing Human MARC1 (hMARC1) Gene

[0356] 8.1 AAV8 Constructed Overexpression hMARC1 Gene Mouse Model

[0357] 6-8 week old C57BL / 6 male mice (provided by Beijing Vivotec Animal Technology Co., Ltd.) were introduced into the facility, and a single injection of hMARC1 gene adeno-associated virus AAV8 (pAAV[Exp]-CBh>SEAP:{MARC1 part 3'UTR} (virus provided by Yunzhou Biotechnology (Guangzhou) Co., Ltd.) was performed via the tail vein to target gene overexpression modeling, and the volume of the drug was 100 μL (6 x 1011 vg) per mouse, normal chow diet.

[0358] 8.2 In vivo efficacy of silencing siRNA in hMARC1 mouse model

[0359] After 14 days of AAV8 virus injection, the mice were grouped (5 mice per group) and administered a single 3 mg / kg (mpk) dose of siRNA conjugate of the present disclosure subcutaneously, with a volume of 5 μL / g and a solvent of RNase-free sterile PBS. The same volume of RNase-free sterile PBS was injected into the blank group. After administration, the expression of SEAP protein (i.e., the expression of hMARC1 protein) was detected, and the calculation formula was: % inhibition = (1 - average protein expression of the administration group / average protein expression of the blank group) * 100%, to obtain the inhibition rate (%) of the siRNA conjugate in the mouse expressing the human MARC1 (hMARC1) gene.

[0360] From this experiment, it can be concluded that the siRNA conjugate of the present disclosure has high inhibitory activity on the protein expression of the hMARC1 gene in vivo, can reduce the level of hMARC1 protein for a long time, and has a high inhibition rate of more than 65% from the 35th day to the 63rd day of the experiment, and the in vivo inhibition effect has long-acting.

Claims

1. An siRNA for inhibiting the expression of MARC1 gene, the siRNA comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently modified or unmodified, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, 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 set forth in SEQ ID NO: 1, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 2; (2) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 3, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 4; (3) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 5, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 6; (4) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:

7. The sense strand further comprises a nucleotide sequence V and / or the antisense strand further comprises a nucleotide sequence VI, each of the nucleotide sequence V and the nucleotide sequence VI has a length of 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; preferably, each of the nucleotide sequence V or the nucleotide sequence VI has a length of 2 nucleotides; more preferably, the nucleotide sequence V is identical to or has nucleotide difference with the nucleotide at the corresponding position of the target mRNA, the nucleotide sequence VI is mismatched or complementary to the nucleotide at the corresponding position of the target mRNA; or, each of the nucleotide sequence V or the nucleotide sequence VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides.

3. The siRNA of claim 1 or 2, 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 with a modification group. Preferably, the modified nucleotide is selected from a 2'-fluoro-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'-deoxy nucleotide, a 2'-amino-modified nucleotide, a 2'-substituted amino-modified nucleotide, a nucleotide analogue, or a combination of any two or more thereof; preferably, the phosphate group with a modification group is a phosphorothioate group in which one of the oxygen atoms in the phosphodiester bond is replaced by a sulfur atom. ​ 2. The siRNA of claim 1, wherein, ​ ​ ​ More preferably, the modified nucleotides are selected from 2'-F modified nucleotides, 2'-O-CH3 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'-deoxy modified nucleotides, nucleotide analogs, or a combination of any two or more thereof.

4. The siRNA according to any one of claims 1-3, wherein, 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; and / or neither the 5' terminal nor the 3' terminal of the sense strand is linked to an inverted abasic deoxyribose residue, or only the 5' terminal of the sense strand is linked to one inverted abasic deoxyribose residue, or only the 3' terminal of the sense strand is linked to one inverted abasic deoxyribose residue, or the 5' terminal and the 3' terminal of the sense strand are respectively linked to one inverted abasic deoxyribose residue; Preferably, the inverted abasic deoxyribose residue is linked to the 3' terminal nucleotide and / or the 5' terminal nucleotide of the sense strand through a phosphodiester bond, a phosphorothioate group, or other internucleoside linkage.

5. The siRNA of any one of claims 1-4, 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, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 7, 9, 10 and 11, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 5, 7, 8 and 9, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 9, 10 and 11, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 8, 9 and 10, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 8, 9, 10 and 11, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7 and 9, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9 and 11, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 11, 13 and 15, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 11 and 13, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 8 and 9, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 9, 11 and 13, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 9 and 11, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 11 and 13, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 11, 12 and 13, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11 and 16, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11 and 17, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11, 16 and 17, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9 and 14, and the remaining positions are non-fluoro-modified nucleotides; and / or In the antisense strand, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 2, 6, 14, and 16, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 3, 5, 8, 10, 14, 16, and 18, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 3, 4, 5, 7, 10, and 14, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 4, 6, 12, 14, 16, 18, and 20, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2 and 14, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, 18, and 20, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 7, 10, and 14, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 5, 7, and 14; or, 2'-fluoro-modified nucleotides are at positions 2, 3, 5, 7, 10, 12, and 14, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 7, and 14, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 4, 6, 12, 14, 16, and 18, and the remaining positions are non-fluoro-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 18, and the remaining positions are non-fluoro-modified nucleotides. Preferably, each non-fluoro-modified nucleotide is independently selected from one of the group consisting of a nucleotide whose ribosyl 2' position hydroxyl is replaced by a non-fluoro group, or a nucleotide analog selected from one of the group consisting of pseudouracil, anisouracil, LNA, ENA, cET BNA, UNA, and GNA.

6. The siRNA of any one of claims 1-5, 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'-deoxy-modified nucleotide, a GNA-modified nucleotide, or a combination of any two or more thereof. Preferably, in the sense strand, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are at positions 7, 9, 10 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 5, 7, 8 and 9, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 9, 10 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 8, 9 and 10, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 8, 9, 10 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7 and 9, and the nucleotide at position 11 of the sense strand is a 2'-deoxy-modified nucleotide, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 11, 13 and 15, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9, 11 and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 8 and 9, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 9, 11 and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 9 and 11, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 11 and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 11, 12 and 13, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11 and 16, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11 and 17, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 3, 7, 11, 16 and 17, and the remaining positions are 2'-methoxy-modified nucleotides; or, 2'-fluoro-modified nucleotides are at positions 7, 9 and 14, and the remaining positions are 2'-methoxy-modified nucleotides; and / or In the antisense strand, 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 2, 3, 5, 8, 10, 14, 16, and 18, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified nucleotides are located at positions 2, 3, 4, 5, 7, 10, and 14, with the remaining positions being 2'-methoxymodified nucleotides; or, 2'-fluoromodified... Nucleotides located at positions 2, 4, 6, 12, 14, 16, 18, and 20, with the remaining positions being nucleotides modified by 2'-methoxy groups; or, nucleotides modified by 2'-fluoride groups located at positions 2 and 14, with the remaining positions being nucleotides modified by 2'-methoxy groups; or, nucleotides modified by 2'-fluoride groups located at positions 2, 3, 4, 5, 7, 10, and 14, with position 6 being a nucleotide modified by GNA, and the remaining positions being nucleotides modified by 2'-methoxy groups; or, nucleotides modified by 2'-fluoride groups located at positions 2, 4, 5, 6, 8, 19, 10, 11, 12, 14, 16, 18, and 20, with the remaining positions being nucleotides modified by 2'-methoxy groups. Positions 0, 12, 14, 16, 18, and 20, with the remaining positions being nucleotides modified with 2'-methoxy groups; or, positions 2, 7, 10, and 14, with the remaining positions being nucleotides modified with 2'-methoxy groups; or, positions 2, 5, 7, and 14, with the remaining positions being nucleotides modified with 2'-methoxy groups; or, positions 2, 3, 5, 7, 10, 12, and 14, with position 6 being a nucleotide modified with GNA, and the remaining positions being nucleotides modified with 2'-methoxy groups; or, 2 The 2'-fluorinated nucleotides are located at positions 2, 7, and 14, and the nucleotides at positions 5 and 12 of the antisense strand are 2'-deoxy nucleotides, with the remaining positions being 2'-methoxy nucleotides; or, the 2'-fluorinated nucleotides are located at positions 2, 4, 6, 12, 14, 16, and 18, with the remaining positions being 2'-methoxy nucleotides; or, the 2'-fluorinated nucleotides are located at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 18, with the remaining positions being 2'-methoxy nucleotides.

7. The siRNA according to any one of claims 1-6, wherein the positive strand comprises a phosphate thioester group located at the following positions, oriented from the 5' end to the 3' end: (1) Between the first nucleotide and the second nucleotide starting at the 5' end of the positive strand; and Between the second and third nucleotides starting at the 5' end of the positive strand; or, (2) Between the first nucleotide and the second nucleotide starting at the 5' end of the positive strand; and Between the second and third nucleotides starting at the 5' end of the positive strand; and Between the reverse debased deoxyribose residue starting at the 3' end of the positive strand and the first nucleotide; or, (3) Between the first nucleotide and the second nucleotide starting at the 5' end of the positive strand; and / or In the direction from the 5' end to the 3' end, the antisense strand comprises a phosphorothioate group at a position selected from the group consisting of: between the 1st and 2nd nucleotides from the 5' end of the antisense strand; and between the 2nd and 3rd nucleotides from the 5' end of the antisense strand; and between the 1st and 2nd nucleotides from the 3' end of the antisense strand; and between the 2nd and 3rd nucleotides from the 3' end of the antisense strand.

8. The siRNA of any one of claims 1-7, 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'-deoxy-modified nucleotide, a GNA-modified nucleotide, or a combination of any two or more thereof; preferably, in the direction from 5' to 3', 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 direction from 5' to 3', 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, the 5' terminal nucleotide of the antisense strand is linked with a 5'-trans-vinyl phosphonate group; or, in the direction from 5' to 3', 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 direction from 5' to 3', 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, the 5' terminal nucleotide of the antisense strand is linked with a 5'-trans-vinyl phosphonate group; or, in the direction from 5' to 3', 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 direction from 5' to 3', 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, the 5' terminal nucleotide of the antisense strand is linked with a 5'-trans-vinyl phosphonate group; or, in the direction from 5' to 3', 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 direction from 5' to 3', 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, the 5' terminal nucleotide of the antisense strand is linked with a 5'-trans-vinyl phosphonate group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11, and 2'-methoxy modified nucleotides in the remaining positions of the sense strand in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 3, 5, 8, 10, 14, 16, and 18, and 2'-methoxy modified nucleotides in the remaining positions of the antisense strand in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11, and 2'-methoxy modified nucleotides in the remaining positions of the sense strand in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 3, 4, 5, 7, 10, and 14, and 2'-methoxy modified nucleotides in the remaining positions of the antisense strand in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 8, 9, 10, and 11, and 2'-methoxy modified nucleotides in the remaining positions of the sense strand in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 3, 4, 5, 7, 10, and 14, and 2'-methoxy modified nucleotides in the remaining positions of the antisense strand in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11, and 2'-methoxy modified nucleotides in the remaining positions of the sense strand in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 4, 6, 12, 14, 16, 18, and 20, and 2'-methoxy modified nucleotides in the remaining positions of the antisense strand in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11, and 2'-methoxy modified nucleotides in the remaining positions of the sense strand in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2 and 14, and 2'-methoxy modified nucleotides in the remaining positions of the antisense strand in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7 and 9, with the nucleotide at position 11 of the sense strand being a 2'-deoxy modified nucleotide, and 2'-methoxy modified nucleotides in the remaining positions of the sense strand in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, 18, and 20, and 2'-methoxy modified nucleotides in the remaining positions of the antisense strand in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, and 11 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 11, 13, and 15 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 11, 13, and 15 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 11, 13, and 15 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 11, 13, and 15 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 11, 13, and 15 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 11, and 13 of the sense strand, and 2'- methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'- methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 11, and 13 of the sense strand, and 2'- methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'- methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 11, and 13 of the sense strand, and 2'- methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'- methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 11, and 13 of the sense strand, and 2'- methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'- methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 11, and 13 of the sense strand, and 2'- methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'- methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 11, and 13 of the sense strand, and 2'- methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'- methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 11, and 13 of the sense strand, and 2'- methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 of the antisense strand, and 2'- methoxy modified nucleotides in the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11, and 2'-methoxy modified nucleotides in the remaining positions of the sense strand, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 5, 7, and 14, and 2'-methoxy modified nucleotides in the remaining positions of the antisense strand, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11, and 2'-methoxy modified nucleotides in the remaining positions of the sense strand, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 5, 7, and 14, and 2'-methoxy modified nucleotides in the remaining positions of the antisense strand, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11, and 2'-methoxy modified nucleotides in the remaining positions of the sense strand, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 5, 7, and 14, and 2'-methoxy modified nucleotides in the remaining positions of the antisense strand, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11, and 2'-methoxy modified nucleotides in the remaining positions of the sense strand, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 5, 7, and 14, and 2'-methoxy modified nucleotides in the remaining positions of the antisense strand, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11, and 2'-methoxy modified nucleotides in the remaining positions of the sense strand, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 5, 7, and 14, and 2'-methoxy modified nucleotides in the remaining positions of the antisense strand, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11, and 2'-methoxy modified nucleotides in the remaining positions of the sense strand, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 5, 7, and 14, and 2'-methoxy modified nucleotides in the remaining positions of the antisense strand, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11, and 2'-methoxy modified nucleotides in the remaining positions of the sense strand, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 5, 7, and 14, and 2'-methoxy modified nucleotides in the remaining positions of the antisense strand, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 3, 7, 9, and 11 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 7, and 14 of the antisense strand, 2'-deoxy modified nucleotides at positions 5 and 12 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 4, 6, 12, 14, 16, and 18 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; or, 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 of the sense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction; 2'-fluoro modified nucleotides at positions 2, 4, 6, 12, 14, 16, and 18 of the antisense strand, and 2'-methoxy modified nucleotides at the remaining positions, in the 5' to 3' direction, with the 5' terminal nucleotide of the antisense strand linked to a 5 '-trans vinyl phosphonate ester group; 9. The siRNA of claims 1-8 selected from the siRNAs of Table 1; preferably, the siRNA is selected from N-ER-FY025096M44, N-ER-FY025102M44, N-ER-FY025175M44, N-ER-FY025177M44, N-ER-FY025096M45, N-ER-FY025102M45, N-ER-FY025175M45, N-ER-FY025177M45, N-ER-FY025096M46, N-ER-FY025102M46, N-ER-FY025175M46, N-ER-FY025177M46, N-ER-FY025096M47, N-ER-FY025102M47, N-ER-FY025175M47, N-ER-FY025177M47, N-ER-FY025096M48, N-ER-FY025102M48, N-ER-FY025175M48, N-ER-FY025177M48, N-ER-FY025102M49, N-ER-FY025175M49, N-ER-FY025096M50, N-ER-FY025102M50, N-ER-FY025175M50, N-ER-FY025177M50.

10. An siRNA conjugate comprising the siRNA of any one of claims 1-9 and a conjugate group conjugated to the siRNA.

11. The siRNA conjugate of claim 10, wherein, In the siRNA conjugate, the sense strand of the siRNA is complementary to the antisense strand to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, the 3' end of the antisense strand has 1-3 overhanging nucleotides extending out of the double-stranded region; or, In the siRNA conjugate, the sense strand of the siRNA is complementary to the antisense strand to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, the 3' end of the antisense strand forms a blunt end.

12. The siRNA conjugate of claim 10 or 11, wherein the conjugate group is selected from:

13. The siRNA conjugate of any one of claims 10-12, wherein the siRNA conjugate is selected from the siRNA conjugates of Table 2; preferably, the siRNA conjugate is selected from N-ER-FY025096M44L96, N-ER-FY025102M44L96, N-ER-FY025175M44L96, N-ER-FY025177M44L96, N-ER-FY025096M45L96, N-ER-FY025102M45L96, N-ER-FY025175M45L96, N-ER-FY025177M45L96, N-ER-FY025096M46L96, N-ER-FY025102M46L96, N-ER-FY025175M46L96, N-ER-FY025177M46L96, N-ER-FY025096M47L96, N-ER-FY025102M47L96, N-ER-FY025175M47L96, N-ER-FY025177M47L96, N-ER-FY025096M48L96, N-ER-FY025102M48L96, N-ER-FY025175M48L96, N-ER-FY025177M48L96, N-ER-FY025102M49L96, N-ER-FY025175M49L96, N-ER-FY025096M50L96, N-ER-FY025102M50L96, N-ER-FY025175M50L96, N-ER-FY025177M50L96.

14. A pharmaceutical composition comprising the siRNA of any one of claims 1-9, or the siRNA conjugate of any one of claims 10-13, and a pharmaceutically acceptable carrier.

15. A kit comprising the siRNA of any one of claims 1-9, or the siRNA conjugate of any one of claims 10-13, or the pharmaceutical composition of claim 14.

16. Use of the siRNA of any one of claims 1-9, or the siRNA conjugate of any one of claims 10-13, or the pharmaceutical composition of claim 14 in (a) or (b): (a) for the manufacture of a medicament for inhibiting expression of MARC1 gene; (b) for the manufacture of a medicament for preventing and / or treating a disease associated with overexpression of MARC1 gene.

17. The use according to claim 16, wherein the disease is obesity, non-alcoholic fatty liver disease, alcohol-related fatty liver disease, non-alcoholic steatohepatitis, liver cirrhosis, liver fibrosis, elevated liver enzyme levels (ALT, AST, ALP), hepatocellular carcinoma, hypercholesterolemia and related cardiovascular diseases, insulin resistance, impaired glucose tolerance, hyperglycemia, type II diabetes and metabolic syndrome.

18. A method of inhibiting expression of MARC1 gene in vivo or in vitro, comprising contacting a cell expressing MARC1 with the siRNA of any one of claims 1-9, or the siRNA conjugate of any one of claims 10-13, or the pharmaceutical composition of claim 14, or administering to a subject in need thereof.

19. A method of treating and / or preventing a disease associated with overexpression of MARC1 gene, comprising administering to a subject in need thereof a therapeutically effective amount or a prophylactically effective amount of the siRNA of any one of claims 1-9, or the siRNA conjugate of any one of claims 10-13, or the pharmaceutical composition of claim 14.

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