Sirna targeting inhbe gene expression, and conjugate and use thereof

Abstract

Provided in the present invention are an siRNA inhibiting inhibin subunit βE (INHBE) gene expression and a conjugate thereof. The siRNA comprises a sense strand and an antisense strand. The antisense strand comprises at least 17 consecutive nucleotides that differ from nucleotide sequences as set forth in SEQ ID NO: 2 to SEQ ID NO: 277 by no more than 4 nucleotides, and has a length of 17-30 nucleotides. The sense strand has a length of 17-30 nucleotides, and is at least partially complementary to the antisense strand. The siRNA, siRNA conjugate and pharmaceutical composition provided by the present invention have good stability, INHBE gene inhibitory activity, cytotoxicity and immunostimulatory activity.

Description

A siRNA targeting INHBE gene expression and its conjugate and use Technical Field

[0001] The present invention relates to an siRNA for inhibiting the expression of inhibin subunit beta E (INHBE) gene, a conjugate thereof, a pharmaceutical composition, and use thereof in treating metabolic diseases and / or cardiovascular diseases. Background Art

[0002] Inhibin subunit beta E (INHBE), encoded by the INHBE gene, is a member of the transforming growth factor beta (TGF-β) superprotein family. The preproprotein, translated from the gene, undergoes proteolytic processing to produce the inhibin βE subunit. Different inhibin subunits can combine to form inhibins or activins, which are involved in regulating numerous cellular processes, including cell proliferation, apoptosis, immune responses, and hormone secretion. INHBE is closely associated with the regulation of lipid metabolism. INHBE protein expression is tissue-specific. After expression in the liver, it is secreted into the circulation, where it binds to activin receptor-like kinase 7 (ALK7) on adipocytes, activates the transcription factor Smad2 / 3, and initiates the expression of a series of genes, promoting lipid storage and accumulation.

[0003] There is increasing evidence that the biological function and high expression of INHBE play a role in diseases such as obesity, type 2 diabetes mellitus (T2DM), insulin resistance, and lipid metabolism disorders. A study that included exome sequences of 360,000 people to identify genetic variants associated with lower abdominal obesity levels (Nat Commun 2022; 13, 4319) showed that INHBE loss-of-function mutations were associated with improved fat distribution, manifested as a lower weight-adjusted waist-to-hip ratio (WHRadjBMI). People with INHBE loss-of-function mutations had increased levels of 3-hydroxybutyrate and ketone bodies in their blood, indicating increased lipid breakdown. INHBE loss-of-function mutations were associated with a reduced risk of T2DM and coronary heart disease (CHD), suggesting that INHBE inactivation contributes to improved glucose and lipid metabolism. Another study of liver biopsy samples from humans with varying degrees of insulin resistance found that the liver expression level of INHBE mRNA was positively correlated with human insulin resistance and body mass index (PLos One 2018; 13(3): e0194798). The level of INHBE protein in the blood of obese people is three times that of normal people. Higher INHBE expression levels are also associated with high liver triglyceride levels. In addition, animal studies have shown that compared with wild-type mice, INHBE systemic knockout mice are resistant to weight gain induced by a high-fat diet (HFD), the level of β-hydroxybutyric acid, a fat decomposition product in the blood, is significantly increased, and the expression levels of lipid decomposition-related genes (including Atgl, Cgi-58) in visceral adipose tissue are upregulated. Reducing the expression of INHBE by siRNA can also reduce the weight of HFD-induced mice and increase the proportion of muscle volume, while food intake does not change. Therefore, INHBE is a potential therapeutic target for obesity and metabolic disorder complications and chronic diseases such as T2DM and CHD.

[0004] A statistical study covering 15.8 million Chinese adult subjects showed that according to the body mass index (BMI) classification standard, overweight people (BMI = 24-28) accounted for about 34.8%, and obese people (BMI ≥ 28) accounted for about 14.1% (Diabetes Obes Metab. 2023; 25(11): 3390-3399). Compared with people with normal BMI, overweight / obese people have a higher prevalence of metabolic complications. Common complications include fatty liver, prediabetes, dyslipidemia, and hypertension; body fat levels, especially visceral fat levels, are positively correlated with the risk of T2DM and coronary artery disease (CAD). In the elderly population, overweight and obesity also increase the risk of aging-related diseases. Clinically, it is recommended to treat obesity through changing unhealthy lifestyles, drug therapy, and weight loss surgery. In recent years, newly approved incretin-based drugs for the treatment of T2DM and obesity, such as semaglutide, have demonstrated clear weight loss and blood sugar lowering effects, while also bringing about multiple metabolic-related clinical benefits such as reduced liver fat and improved liver fatty lesions. They have increasingly become cornerstone drugs for the treatment of metabolic diseases such as obesity and T2DM. However, they also have shortcomings such as causing a significant reduction in muscle mass during weight loss, limiting central dopamine secretion levels, triggering gastrointestinal adverse reactions, and even increasing the risk of intestinal obstruction. There are also reports of weight rebound after discontinuation of the drug, which have created new unmet clinical needs in the treatment of obesity and related metabolic complications. The development of new weight-loss drugs that can maintain muscle mass and prevent muscle loss during rapid weight loss, while maintaining the body's nutritional support without affecting food intake, and that can achieve long-term sustained weight loss and prevent rebound will be key to the future clinical management of obesity and related metabolic complications.

[0005] The present application provides a small interfering RNA (siRNA) preparation targeting INHBE, which can specifically bind to INHBE mRNA, destroy the normal translation template function of INHBE mRNA, thereby preventing its translation of INHBE protein, inhibiting the secretion of hepatic INHBE protein and its regulatory effect on adipose tissue lipid metabolism, and thereby achieving the purpose of treating obesity and related metabolic complications by increasing lipid decomposition.

[0006] Compared to traditional drugs, siRNA has poor stability and is susceptible to nuclease degradation when administered systemically. Furthermore, efforts are needed to further enhance its activity while avoiding side effects such as off-target effects, immune stimulation, and cytotoxicity. Therefore, developing more candidate siRNAs that are stable in the blood, have good biological activity, low cytotoxicity, and can inhibit INHBE gene expression over a long period of time has become an urgent issue. Furthermore, the development of highly effective and long-lasting weight loss drugs using these candidate siRNAs that inhibit INHBE gene expression requires clinical research and is commercially viable. Summary of the Invention

[0007] The present invention provides an siRNA for inhibiting INHBE gene expression, the siRNA comprising a sense strand and an antisense strand; wherein the antisense strand comprises at least 17 consecutive nucleotides that differ by no more than 4 nucleotides from the nucleotide sequence shown in any one of SEQ ID NO: 2 to SEQ ID NO: 277, and the antisense strand is 17 to 30 nucleotides in length; the sense strand is 17 to 30 nucleotides in length and is at least partially complementary to the antisense strand.

[0008] The term "at least partially complementary" means that the two sequences are completely complementary, or have no more than 5, 4, 3 or 2 mismatched base pairs overall, while retaining the ability to hybridize under relevant conditions.

[0009] In some embodiments of the present invention, the antisense strand differs from any one of the nucleotide sequences shown in SEQ ID NO:2 to SEQ ID NO:277 by no more than 4 nucleotides; in some embodiments of the present invention, the antisense strand differs from any one of the nucleotide sequences shown in SEQ ID NO:2 to SEQ ID NO:277 by no more than 3 nucleotides; in some embodiments of the present invention, the antisense strand differs from any one of the nucleotide sequences shown in SEQ ID NO:2 to SEQ ID NO:277 by no more than 2 nucleotides; in some embodiments of the present invention, the antisense strand differs from any one of the nucleotide sequences shown in SEQ ID NO:2 to SEQ ID NO:277 by no more than 1 nucleotide; in some embodiments of the present invention, the antisense strand is any one of the nucleotide sequences shown in SEQ ID NO:2 to SEQ ID NO:277.

[0010] In some embodiments of the present invention, the sense strand and the antisense strand have no more than 3 nucleotide mismatches; in some embodiments of the present invention, the sense strand and the antisense strand have no more than 2 nucleotide mismatches; in some embodiments of the present invention, the sense strand and the antisense strand have no more than 1 nucleotide mismatch; in some embodiments of the present invention, the sense strand and the antisense strand are fully complementary.

[0011] Preferably, the sense strand has at least 15, 16, 17, 18, 19, 20 or 21 nucleotides of complementarity with the antisense strand.

[0012] In some embodiments of the present invention, the antisense strand is 19 to 27 nucleotides in length; and the sense strand is 19 to 25 nucleotides in length.

[0013] In some embodiments of the present invention, the antisense strand is 19 to 23 nucleotides in length; and the sense strand is 19 to 21 nucleotides in length.

[0014] In some embodiments of the present invention, the antisense strand is 23 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments of the present invention, the antisense strand is 22 nucleotides in length and the sense strand is 20 nucleotides in length. In some embodiments of the present invention, the antisense strand is 21 nucleotides in length and the sense strand is 21 nucleotides in length. In some embodiments of the present invention, the antisense strand is 21 nucleotides in length and the sense strand is 19 nucleotides in length. In some embodiments of the present invention, the antisense strand is 19 nucleotides in length and the sense strand is 19 nucleotides in length.

[0015] In some embodiments of the present invention, the siRNA comprises one or more single-stranded nucleotide overhangs. For example, an overhang of 1, 2, 3, or 4 nucleotides. In some embodiments of the present invention, the overhang can be on the sense strand, the antisense strand, or any combination thereof. In some embodiments of the present invention, the overhang is present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the siRNA.

[0016] In some embodiments of the present invention, the 3' end of the antisense strand of the siRNA has an overhang of 2 nucleotides.

[0017] In some embodiments of the invention, the siRNA has blunt ends. In some embodiments of the invention, the siRNA has at least one blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand).

[0018] In some embodiments of the invention, the siRNA has two blunt ends.

[0019] In some embodiments of the present invention, the nucleotide sequence (5'→3') of the siRNA is selected from duplex 1 to duplex 276:

[0020] Table 1. Sense and antisense strand sequences of unmodified siRNA duplexes

[0021] In some embodiments of the present invention, the siRNA contains at least one modified nucleotide.

[0022] In some embodiments of the present invention, all nucleotides in the sense strand and / or antisense strand of the siRNA are modified nucleotides or nucleotide analogs.

[0023] In some embodiments of the invention, the modified nucleotide is selected from 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-split nucleotide analogs, 2'-fluoroarabino nucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 3'-methoxy nucleotides, 2'-allyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, diol-modified nucleotides, abasic nucleotides, morpholino nucleotides, threose nucleotides, locked nucleotides, unlocked nucleotides, glycerol nucleotides or base-modified nucleotides. In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0024] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0025] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0026] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0027] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0028] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 3, 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0029] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0030] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 3, 4, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein position 2 at the 5' end of the sense strand is a 2'-fluoro nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0031] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 4, 5, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein position 2 at the 5' end of the sense strand is a 2'-fluoro nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0032] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 4, 6, 7, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein position 2 at the 5' end of the sense strand is a 2'-fluoro nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0033] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 1, 3, 5, 7, 9, 10, 11, 13, 15, 17, 19, and 21 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0034] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 4, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 1, 3, 5, 7, 9, 10, 11, 13, 15, 17, 19, and 21 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0035] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, position 7 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0036] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, position 6 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0037] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, position 4 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0038] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, position 5 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0039] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 8, 9, 10, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, position 7 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, 11, and 15 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0040] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, position 6 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0041] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 5, 7, and 12 of the 5' end of the antisense strand are 2'-deoxynucleotides, position 14 is a 2'-fluoro nucleotide, and the remaining positions are 2'-methoxy nucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, and 11 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0042] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 5, 7, and 12 of the 5' end of the antisense strand are 2'-deoxynucleotides, positions 6, 8, 9, 10, 14, and 16 are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0043] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 5, 7, and 9 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0044] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0045] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0046] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0047] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0048] In some embodiments of the present invention, the antisense strand of the siRNA is 19 nucleotides in length, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0049] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein the 14th position at the 5' end of the antisense strand is a 2'-fluoro nucleotide, the 2nd, 5th, and 7th positions are 2'-deoxy nucleotides, the 12th position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0050] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, the 1st position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0051] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 22nd position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0052] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 23rd position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 21 nucleotides in length, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0053] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, the 1st position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0054] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 21st position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0055] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 22nd position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0056] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0057] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 6th, 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0058] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, the 6th position is a 2'-deoxy nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0059] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 6th, 8th, 9th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 6th, 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0060] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, the 1st position is a threose nucleotide, and the remaining positions are 2'-methoxy nucleotides.

[0061] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 20th position is a threose nucleoside, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0062] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 21st position is a threose nucleoside, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0063] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 5th, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0064] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 5th, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 9th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0065] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 7th, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0066] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 7th, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 9th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0067] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 5th position is DNA, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0068] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 7th position is DNA, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0069] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 5th position is DNA, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 9th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0070] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 5th position is a threose nucleic acid or a modified threose nucleic acid, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 9th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0071] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 5th position is a glycerol nucleic acid, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 9th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0072] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 7th position is DNA, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 9th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0073] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 5th, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0074] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 7th, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0075] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 5th, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 7th, 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0076] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 7th, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 7th, 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0077] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 5th position is DNA, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0078] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 7th position is DNA, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 8th, 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0079] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 5th position is DNA, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 7th, 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0080] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides in length, wherein the 2nd, 7th, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, the 7th position is DNA, and the remaining positions are 2'-methoxy nucleotides; the sense strand of the siRNA is 20 nucleotides in length, wherein the 7th, 8th, 9th, and 10th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides.

[0081] In some embodiments of the present invention, the modified nucleotide is a nucleotide in which the phosphate group is modified with a phosphorothioate group, that is, a non-bridging oxygen atom in the phosphodiester bond is replaced with a sulfur atom, thereby replacing the phosphodiester bond with a phosphorothioate diester bond.

[0082] In some embodiments of the invention, the 5' and 3' ends of the sense strand independently contain 0, 1 or 2 phosphorothioate linkages; and / or the 5' and 3' ends of the antisense strand independently contain 1 or 2 phosphorothioate linkages.

[0083] In some embodiments of the present invention, at least one of the nucleotides between the 1st and 2nd positions at the 5' end of the sense chain, between the 2nd and 3rd positions at the 5' end of the sense chain, between the 1st and 2nd positions at the 3' end of the sense chain, between the 2nd and 3rd positions at the 3' end of the sense chain, between the 1st and 2nd positions at the 3' end of the antisense chain, between the 2nd and 3rd positions at the 3' end of the antisense chain, between the 1st and 2nd positions at the 5' end of the antisense chain, and between the 2nd and 3rd positions at the 5' end of the antisense chain is connected by a thiophosphate group; preferably, at least four of the nucleotides are connected by thiophosphate groups; in some embodiments of the present invention, at least six of the nucleotides are connected by thiophosphate groups; in some embodiments of the present invention, all eight of the nucleotides are connected by thiophosphate groups.

[0084] In some embodiments of the present invention, the nucleotides at positions 1 and 2, and the nucleotides at positions 2 and 3 at the 5' end of the sense strand are linked by phosphorothioate groups.

[0085] In some embodiments of the present invention, the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 of the 5' end of the sense strand are linked by phosphorothioate groups, and the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 of the 3' end are linked by phosphorothioate groups.

[0086] In some embodiments of the present invention, the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 at the 3' end of the antisense strand are linked by phosphorothioate groups, and the nucleotides at positions 1 and 2 and the nucleotides at positions 2 and 3 at the 5' end are linked by phosphorothioate groups.

[0087] In some embodiments of the present invention, the nucleotides between the 1st and 2nd positions at the 5' end of the sense chain, the nucleotides between the 2nd and 3rd positions at the 5' end of the sense chain, the nucleotides between the 1st and 2nd positions at the 3' end of the sense chain, the nucleotides between the 2nd and 3rd positions at the 3' end of the sense chain, the nucleotides between the 1st and 2nd positions at the 3' end of the antisense chain, the nucleotides between the 2nd and 3rd positions at the 3' end of the antisense chain, the nucleotides between the 1st and 2nd positions at the 5' end of the antisense chain, and the nucleotides between the 2nd and 3rd positions at the 5' end of the antisense chain are all linked by phosphorothioate groups.

[0088] In some embodiments of the present invention, the sense strand may include one or more blocking residues or moieties, referred to as "blocking residues." A "blocking residue" is a non-nucleotide compound or other moiety that can be incorporated into one or more ends of the nucleotide sequence of the siRNA. In some embodiments of the present invention, the blocking residue is present at the 5' end, the 3' end, or both the 5' end and the 3' end of the sense strand.

[0089] In some embodiments of the present invention, an inverted abasic residue (invAb) is added as a capping residue. See F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16. In some embodiments of the present invention, the 5' end and / or 3' end of the sense strand may contain more than one inverted abasic deoxyribose moiety as a capping residue.

[0090] In some embodiments of the present invention, one or more reverse abasic residues (invAb) are added to the 3' end of the sense strand. In some embodiments of the present invention, one or more reverse abasic residues (invAb) are added to the 5' end of the sense strand. In some embodiments of the present invention, one or more reverse abasic residues can be inserted between the nucleotide sequence of the delivery vector and the siRNA sense strand. In some embodiments of the present invention, one or more reverse abasic residues are included near or at one or more ends of the siRNA sense strand.

[0091] In some embodiments of the invention, one or more inverted abasic residues (invAb) are added to the 5' end of the sense strand. In some embodiments of the invention, one or more inverted abasic residues may be inserted between the delivery vector and the nucleotide sequence of the siRNA sense strand.

[0092] The inverted abasic residue can be linked to the nucleic acid via a phosphodiester bond, a phosphorothioate diester bond, or the like.

[0093] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is selected from the following structures:

[0094] Wherein, Base is base A, U, G, C, T or other modified bases.

[0095] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is a nucleotide modified with (E)-vinyl phosphate.

[0096] In some embodiments of the present invention, the siRNA contains at least one modified nucleotide.

[0097] In some embodiments of the present invention, the base of the base-modified nucleotide is selected from the following structures:

[0098] In some embodiments of the present invention, the base-modified nucleotides may be located at positions 5, 6, 7, or 8 of the antisense strand of the siRNA.

[0099] In some embodiments of the present invention, the base-modified nucleotides may be located at single-stranded nucleotide overhangs in the siRNA.

[0100] Preferably, the siRNA antisense strand contains an overhang of 2 nucleotides, and the base-modified nucleotide is the first nucleotide of the overhang of the siRNA antisense strand.

[0101] Preferably, the siRNA antisense strand contains an overhang of 2 nucleotides, and the base-modified nucleotide is the second nucleotide of the overhang of the siRNA antisense strand.

[0102] For the modified siRNA duplexes listed in the table below, the basic sequence corresponding to the antisense strand of each modified siRNA duplex before modification corresponds to one of SEQ ID NO: 2 to SEQ ID NO: 277, SEQ ID NO: 575 listed above, and the basic sequence corresponding to the positive strand before modification corresponds to one of SEQ ID NO: 278 to SEQ ID NO: 554 listed above.

[0103] As used herein, "modified siRNA duplex" refers to a siRNA duplex comprising modifications. It should be noted that there is no necessary correspondence between the number X in "modified siRNA duplex X" and "unmodified siRNA duplex X" in the present invention. For example, there is no necessary correspondence between "modified siRNA duplex 224" and "unmodified siRNA duplex 224." In other words, "modified siRNA duplex 224" does not refer to the duplex obtained by modifying "unmodified siRNA duplex 224." Correspondingly, herein, unless otherwise specified, the modified siRNA duplexes provided by the present invention are selected from, for example, modified siRNA duplexes 1 to modified siRNA duplex 329 provided in Table 2, and the unmodified siRNA duplexes provided by the present invention are selected from, for example, duplexes 1 to duplex 277 provided in Table 1.

[0104] In some embodiments of the present invention, the nucleotide sequence (5'→3') of the modified siRNA is selected from modified duplex 1 to duplex 329:

[0105] Table 2. Sense and antisense strand sequences of modified siRNA duplexes

[0106] The present invention also provides a siRNA conjugate obtained by conjugating the above siRNA with a conjugation molecule.

[0107] In the present invention, unless otherwise specified, "conjugation" refers to the covalent attachment of two or more chemical moieties; "conjugate" refers to a compound formed by covalent attachment of chemical moieties; and "siRNA conjugate" refers to a compound formed by covalent attachment of one or more chemical moieties to an siRNA. It should be noted that each chemical moiety can be attached to the siRNA directly or via a linker.

[0108] In the present invention, unless otherwise specified, the “-” in “linker-targeting ligand” means that the linker and the targeting ligand are covalently linked.

[0109] In some embodiments of the invention, the targeting ligand is attached to the siRNA via a linker to form a conjugate molecule.

[0110] In some embodiments of the present invention, the targeting ligand is linked to the 3' end or 5' end of the siRNA sense strand independently or simultaneously through a linker to form a conjugated molecule.

[0111] In some embodiments of the present invention, the siRNA of the present invention can be conjugated with a pharmaceutically acceptable conjugate molecule to obtain an siRNA conjugate. In some embodiments of the present invention, the siRNA is covalently conjugated to the conjugate molecule. To reduce the potential impact of conjugation on siRNA activity, the conjugation site of the siRNA to the conjugate molecule can be at the 3' end or 5' end of the siRNA sense strand, or at the 5' end of the antisense strand. In some embodiments, the conjugation site of the siRNA to the conjugate molecule can also be within the internal sequence of the siRNA.

[0112] The pharmaceutically acceptable targeting ligand can be a targeting ligand conventionally used in the field of siRNA administration, such as, but not limited to, one or more of the following targeting ligands or their derivatives: lipophilic molecules, such as cholesterol, bile acid, vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-permeable peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); or receptor ligands expressed by hepatocytes, such as asialoglycoproteins, asialoglycosylated residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as epinephrine), growth factors, transferrin, etc.

[0113] In some embodiments of the present invention, the targeting ligand is N-acetylgalactosamine.

[0114] In some embodiments of the invention, the targeting ligand is directly linked to the 3' end of the sense strand of the siRNA. In some embodiments of the invention, the targeting ligand is directly linked to the 5' end of the sense strand of the siRNA.

[0115] In some embodiments of the present invention, the targeting ligand is linked to the 3' end of the sense strand of the siRNA via a linker.

[0116] In some embodiments of the present invention, the targeting ligand is linked to the 5' end of the sense strand of the siRNA via a linker.

[0117] In some embodiments of the present invention, the targeting ligand is N-acetylgalactosamine, which is linked to the 3' end of the siRNA sense strand via a linker.

[0118] In some embodiments of the present invention, the delivery vector is GalNAc(L96) having the following structure:

[0119] In some embodiments of the invention, GalNAc(L96) is linked to the 3' end of the sense strand of the siRNA.

[0120] In some embodiments of the invention, GalNAc(L96) is linked to the 5' end of the sense strand of the siRNA.

[0121] In some embodiments of the invention, GalNAc(L96) is linked to the inverted abasic residue (invAb) at the 3' end of the siRNA sense strand.

[0122] In some embodiments of the invention, GalNAc(L96) is linked to the inverted abasic residue (invAb) at the 5' end of the sense strand of the siRNA.

[0123] In some embodiments of the present invention, the delivery vector is Ser(GN) having the following structure:

[0124] In some embodiments of the invention, Ser(GN) is linked to the 3' end of the sense strand of the siRNA. In some embodiments of the invention, Ser(GN) is linked to the 5' end of the sense strand of the siRNA.

[0125] In some embodiments of the present invention, Ser(GN) is attached to both the 3' end and the 5' end of the sense strand of the siRNA.

[0126] In some embodiments of the present invention, the structure of the GalNAc delivery vector LP-GalNAc (attached to the 5'-end of the sense strand) is as follows:

[0127] In some embodiments of the present invention, the GalNAc delivery vector structure XY-GalNAc (attached to the 3' end of the sense strand) is as follows:

[0128] or

[0129] In some embodiments of the present invention, other GalNAc delivery vector structures used (attached to the 5'-end of the sense strand) are as follows:

[0130] In some embodiments of the present invention, other GalNAc delivery vector structures used (attached to the 3'-end of the sense strand) are as follows:

[0131] For the modified siRNA conjugates listed in the table below, the basic sequence corresponding to the antisense strand of each modified siRNA conjugate before modification corresponds to one of SEQ ID NO: 2 to SEQ ID NO: 277, SEQ ID NO: 575 to SEQ ID NO: 606 listed above, and the basic sequence corresponding to the positive strand before modification corresponds to one of SEQ ID NO: 278 to SEQ ID NO: 574 listed above.

[0132] As used herein, "modified siRNA conjugate" refers to an siRNA conjugate comprising a modified siRNA duplex conjugated to a conjugation molecule. It should be noted that "modified siRNA conjugate X" herein does not necessarily correspond to the number X in "modified siRNA duplex X" or "unmodified siRNA duplex X." For example, "modified siRNA conjugate 237" does not necessarily correspond to "modified siRNA duplex 237" or "unmodified siRNA duplex 237." In other words, "modified siRNA conjugate 237" does not refer to the duplex obtained by modifying and conjugating "modified siRNA duplex 237" or "unmodified siRNA duplex 237." Accordingly, herein, unless otherwise specified, the modified siRNA conjugates provided herein are selected from, for example, modified siRNA conjugates 1 to 685 provided in Table 3.

[0133] In some embodiments of the present invention, the siRNA conjugate is selected from Conjugate 1 to Conjugate 685:

[0134] Table 3. Sequences of modified siRNA conjugates

[0135] The structural characterization methods and results of the modified duplexes and conjugates are detailed in Tables 3b and 3c:

[0136] Representative LC-MS testing method: When the test sample is subjected to denaturing IP·RP-LC, the complementary double strands are unwound into single strands (sense and antisense strands). The positive and antisense strand precursor ions are then fragmented by gas phase tandem mass spectrometry. All detected fragment ions are analyzed and resolved using the CONFIRM Sequence software. The sequence of the test sample is consistent with the theoretical sequence, i.e., the deviation between the actual molecular weight (MW) and the theoretical molecular weight (MW) is less than 0.5 parts per million. The results are shown in Table 3b.

[0137] Table 3b Molecular weight (MW) of modified duplexes

[0138] Table 3c Molecular weight (MW) of the conjugates

[0139] The present invention also provides a pharmaceutical composition comprising any of the above-mentioned siRNAs and / or any of the above-mentioned siRNA conjugates and a pharmaceutically acceptable carrier.

[0140] In some embodiments of the present invention, the pharmaceutical composition contains one siRNA as described in the first aspect. In other embodiments of the present disclosure, the pharmaceutical composition contains at least two siRNAs as described in the first aspect (for example, but not limited to, two, three, four, five, six, seven, eight, nine, ten or more) as active ingredients. Preferably, the at least two siRNAs as described in the first aspect each target a different target sequence in the INHBE gene, thereby being expected to act simultaneously against different target sequences and bring about a synergistic effect. Here, the so-called "different target sequences" means that there is no overlap between the target sequences, or the number of overlapping consecutive nucleotides between the target sequences is less than 5 (for example, the number of overlapping consecutive nucleotides is 4, 3, 2, 1, or 0). In this case, the at least two siRNAs as described in the first aspect can be present in any different ratios. Preferably, the at least two siRNAs as described in the first aspect are present in a molar ratio of 1:100 to 100:1; more preferably, the at least two siRNAs as described in the first aspect are present in a molar ratio of 1:10 to 10:1, 1:5 to 5:1, or 1:2 to 2:1. In some embodiments of the present invention, the at least two siRNAs as described in the first aspect are present in the same molar ratio.

[0141] The present invention also provides use of any of the above siRNAs and / or any of the above siRNA conjugates and / or the above pharmaceutical compositions in the preparation of a medicament for treating a pathological condition or disease associated with INHBE gene expression.

[0142] Furthermore, the pathological condition or disease is a metabolic disease and / or a cardiovascular disease. Further preferably, the metabolic disease is obesity, type 2 diabetes mellitus (T2DM), insulin resistance, lipodystrophy, fatty liver disease, non-alcoholic hepatic steatosis (NAFLD) or metabolic dysfunction-associated hepatic steatosis (MAFLD), non-alcoholic steatohepatitis (NASH) or metabolic dysfunction-associated steatohepatitis (MASH); and the cardiovascular disease is hyperlipidemia or dyslipidemia, atherosclerosis, cardiomyopathy, heart failure or coronary heart disease (CHD).

[0143] The siRNA, siRNA conjugate and pharmaceutical composition provided by the present invention have good stability, excellent INHBE gene inhibition activity, satisfactory cytotoxicity and immunostimulation, and can significantly reduce body fat levels and body weight and improve metabolic abnormalities.

[0144] The sequence of the INHBE gene targeted by the siRNA of the present invention is shown in SEQ ID NO: 1:

[0145] SEQ ID NO: 1 (INHBE gene)

[0146] In the present invention, "siRNA" refers to an oligonucleotide molecule containing RNA or RNA-like (e.g., chemically modified RNA) that can reduce or inhibit the translation of messenger RNA (mRNA) in a sequence-specific manner. siRNA can act through an RNA interference mechanism (e.g., by inducing mRNA degradation through interaction with the mRNA interference pathway mechanism (RNA-induced silencing complex RISC) of mammalian cells), or other arbitrary mechanisms or pathways. Although it is believed that the term siRNA drug used in the present invention mainly acts through the RNA interference mechanism, the siRNA drug is not limited to or restricted to any specific pathway or mechanism of action. siRNA drugs include, but are not limited to, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer enzyme substrates. The siRNA drug of the present invention is composed of an oligonucleotide chain that is at least partially complementary to the target mRNA. In some embodiments, the siRNA drug of the present invention is double-stranded and consists of an antisense chain and a sense chain that is at least partially complementary to the antisense chain.

[0147] The term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed in alphabetical order using standard nucleotide nomenclature.

[0148] In the present invention, unless otherwise specified, capital letters C, G, U, A, T, and I represent the base composition of nucleotides: C (cytosine), G (guanine), U (uracil), A (adenine), T (thymine), and I (hypoxanthine), including modified and unmodified nucleotides; lowercase letters c, g, u, a, t, and i represent that a nucleotide is a 2'-methoxy nucleotide, or in other words, lowercase letters c, g, u, a, t, and i represent that the nucleotides C, G, U, A, T, and I are 2'-methoxy nucleotides, respectively; lowercase letter f represents that the nucleotide adjacent to the right of the identifier f is a 2'-fluoro nucleotide; lowercase letter d represents that the nucleotide adjacent to the right of the identifier d is a 2'-deoxy nucleotide; gn represents that the nucleotide adjacent to the right of the identifier gn is a glycerol nucleotide (GNA); tn represents that the nucleotide adjacent to the right of the identifier tn is a threose nucleotide (TNA); cptn represents that the nucleotide adjacent to the right of the identifier tn is a threose nucleotide (TNA); The symbol "mΨ" (Ψ is Psi, indicated by "Psi" in the sequence table) indicates a pseudouridine with methoxy modification at the 2' position of the ribose sugar; "ln" indicates a locked nucleic acid; "una" indicates an unlocked nucleic acid; "moe" indicates a 2'-O-methoxyethyl modified nucleotide; "FD" indicates a 2'-F,2'-D modified nucleotide; "*" indicates a phosphorothioate linkage between the two nucleotides immediately to the left and right of the symbol "*" (or between the nucleotide and the delivery vector); "eVP" indicates a (E)-vinyl phosphate-modified nucleotide; "invAb" indicates an inverted abasic residue; "L96" indicates a site conjugated with the delivery vector GalNAc (L96). "Ser(GN)" indicates a site conjugated with the delivery vector Ser(GN). "A1GN" indicates a site conjugated with the delivery vector A1GN.

[0149] In the present invention, unless otherwise specified, the structures of ln, cEt, and bna used are as follows:

[0150] Wherein, Base is the base A, U, G, 5mC (5-methylcytosine), T or other modified bases.

[0151] In the present invention, unless otherwise specified, the bases of other base-modified nucleotides used are described as follows:

[0152] in It indicates that it is connected to the rest of the oligonucleotide through a phosphodiester bond or a phosphorothioate diester bond.

[0153] In the present invention, unless otherwise specified, the term "complementary" refers to the ability of an oligonucleotide of a first sequence to hybridize with an oligonucleotide of a second sequence under certain conditions and form a double-stranded structure. "At least partially complementary" means that the two sequences can be completely complementary, or generally have no more than 5, 4, 3, or 2 mismatched base pairs, while retaining the ability to hybridize under relevant conditions. In addition, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches for the purpose of determining complementarity. In the present invention, when meeting the above hybridization ability requirements, "complementary" sequences may also include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairs or Hoogstein base pairs. Correspondingly, in the present invention, unless otherwise specified, "mismatch" means that the bases at corresponding positions in the siRNA duplex molecule are not paired in a complementary form.

[0154] In the present invention, unless otherwise specified, "difference in nucleotide sequence" refers to a change in the base type of the nucleotide at the same or corresponding position compared to the original nucleotide sequence. For example, when a nucleotide base in the original nucleotide sequence is A, the nucleotide base at the same or corresponding position is changed to U, C, G or dT, dC, dG, etc., it is considered that there is a difference in nucleotide sequence at this position. It should be noted here that, when compared to the original nucleotide sequence, the nucleotide at the same or corresponding position is only different in terms of whether there is a modification or modification type, it is not considered that there is a difference in nucleotide sequence at this position. For example, when a nucleotide base in the original nucleotide sequence is U, the nucleotide at the same or corresponding position is dT or other base-modified nucleotides (such as I, m6A, X, B), it is not considered that there is a difference in nucleotide sequence at this position.

[0155] In the present invention, unless otherwise specified, the term "pharmaceutically acceptable" means that the carrier, vehicle, diluent, excipient and / or the salt / ester / hydrate formed therefrom are generally chemically or physically compatible with other ingredients constituting a pharmaceutical dosage form and physiologically compatible with the receptor.

[0156] In the present invention, unless otherwise specified, the term "inhibit" refers to the down-regulation of target gene expression due to siRNA-mediated degradation of target gene mRNA. The "down-regulation" refers to a decrease in target gene expression by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more, or even 100%, relative to the absence of siRNA treatment. A 100% decrease in target gene expression means no detectable level of target gene expression.

[0157] In the present invention, the siRNA may further contain modified nucleotides as needed, provided that the modified nucleotides do not significantly weaken or abolish the siRNA's ability to inhibit INHBE gene expression. Currently, there are a variety of methods available in the art for modifying siRNA, including, for example, backbone modifications (e.g., phosphate group modifications), ribose group modifications, and base modifications (Watts, JK, GF Deleavey, and MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008. 13(19-20): p. 842-55).

[0158] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0159] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. DETAILED DESCRIPTION

[0160] It is known to those skilled in the art that the siRNA of the present invention can be obtained by conventional siRNA preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis), wherein both solid phase synthesis and liquid phase synthesis are commercially available. It is also clear to those skilled in the art that modified nucleotide groups can be introduced into the siRNA of the present invention by using nucleotide monomers with corresponding modifications. Methods for preparing nucleotide monomers with corresponding modifications are well known to those skilled in the art, and commercial monomers are also available on the market.

[0161] Example 1: siRNA synthesis

[0162] For the sense and antisense strands of the siRNA sequence of the present invention and the sense and antisense strands of the modified duplex, deoxynucleoside CPG is used as a solid support; the sense strand is synthesized using the solid support, and the antisense strand is synthesized using universal CPG.

[0163] Sequence synthesis was performed on a 48-channel synthesizer at a 0.2 μmol scale. The phosphoramidite monomer was used at a concentration of 0.05 M, and the activator was 0.3 M BTT.

[0164] The cutting and deprotection of the sequence were carried out in a 1.5 ml tube. The first step was to use AMA and the second step was to use triethylamine trihydrofluoride to remove the two protecting groups. For sequences comprising two fully modified sequences, ammoniacal liquor was used for aminolysis. Acetone: ethanol (80:20) mixture was used to precipitate the sequence after cutting and deprotection and to dissolve it in RNase-free water. Each sequence was analyzed by LC-MS to determine sequence accuracy, quantitatively measured by spectrophotometer and determined purity by HPLC.

[0165] After HPLC purification, lyophilization and quality inspection, the salt was replaced by sodium acetate alcohol precipitation and desalted using a 3KD ultrafiltration tube. After desalting, the sense chain and antisense chain were quantitatively determined by spectrophotometer and mixed in a 1:1 ratio and annealed to form siRNA duplexes.

[0166] Example 2: In vitro activity assay - Hep3B cell transfection

[0167] Cell culture and transfection

[0168] Cell culture: Hep3B cells (ATCC) were cultured in MEM complete medium (Gibco, supplemented with 10% FBS) at 37°C in a 5% CO2 environment until nearly confluent. The cells were then trypsinized and plated. 2.0 × 10 5 Hep3B cells and 1.0 mL of MEM complete medium (Gibco, supplemented with 10% FBS) were cultured at 37° C. in a 5% CO 2 environment for 16-24 h before transfection.

[0169] Cell transfection: Add 1.5 μL of lipofectamine RNAiMax (Invitrogen) to 48.5 μL of opti-MEM per well, then add 50 μL of siRNA and mix. Add to a PCR tube and incubate at room temperature for 5 minutes. Finally, add this siRNA mixture to the cells and continue incubation for 24 hours before RNA extraction. Single-dose experiments were performed at 10 nM and 1 nM or 1 nM and 0.1 nM siRNA duplex concentrations.

[0170] RNA extraction

[0171] Use the total RNA isolation kit (Omega, cat: R6834-02): collect cells, wash with 1% PBS, then add 400 μL of lysis buffer (containing 2% β-mercaptoethanol) to lyse the cells. Follow the subsequent steps according to the instructions of the RNA isolation kit. Finally, add 30 μL of RNase-free water, let it stand for 2 minutes, and then centrifuge at 14000g for 2 minutes to collect RNA.

[0172] cDNA synthesis

[0173] cDNA synthesis was performed using the Quanshijin gDNA removal cDNA synthesis kit (Beijing Quanshijin Biotechnology Co., Ltd., Beijing, China, Cat#AE311-03). 1 μg of total RNA was added to each sample, and cDNA synthesis was performed using a gradient thermal cycler (LongGene, A600) according to the manufacturer's instructions.

[0174] Real-time fluorescence quantitative PCR

[0175] The synthesized cDNA and mixed master solution (including primers, qPCR premix and ultrapure water) were added to a 384-well plate (Biocon Biotechnology Cat# PC-0040-9U). The final real-time fluorescence quantitative PCR system contained 0.25 μM upstream and downstream primers of the target gene (INHBE) or internal reference gene (GADPH) and 1× SYBR Green premix (Applied Biosystem Cat# A25742).

[0176] The ΔΔCt assay was performed in ABI QuantStudio TM Real-time fluorescence PCR was performed in a 6-well real-time fluorescence PCR system. Each duplex was tested for 3-4 independent transfections, and each transfection was performed in triplicate.

[0177] Table 4. Modified duplex Hep3B transfection test results

[0178] Table 5. Modified duplex Hep3B transfection test results

[0179] Table 6. Conjugate Hep3B transfection test results

[0180] Table 7. Conjugate Hep3B transfection test results

[0181] Table 8. IC50 of the conjugates tested in Hep3B

[0182] Table 9. Conjugate Hep3B transfection test results

[0183] Example 3. In vitro activity assay - free uptake by primary human liver cells

[0184] After the human primary liver cells were revived, the cells were diluted with culture medium in proportion to adjust the density to 600,000 cells / mL. Different concentrations of conjugate were added to a 96-well collagen plate in an amount of 10 μL / well, and 90 μL / well of human primary liver cells (54,000 cells / well) were added to the plate. A PBS control group was also set up. After plating, the plates were placed in a 5% CO2, 37°C incubator and cultured for 48 hours. After 48 hours, the culture medium was removed and the cells were collected for RNA extraction. Total RNA was extracted using the RNeasy 96 Kit (QIAGEN-74182) according to the kit instructions. Subsequently, cDNA was synthesized using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme-R323-01) according to the instructions. Real-time fluorescence PCR (Tables 10-11) was performed using the ΔΔCt assay in an Applied Biosystems-QuantStudio 7Flex real-time fluorescence PCR system. Among them, a reference conjugate PC c, which is known to have an INHBE gene inhibitory effect, was used as a positive control. The information of the reference conjugate PC c is as follows:

[0185] Sense strand (5'→3'): c*u*gucafCafGfAfCuccacuucauGalNAc(L96)

[0186] Antisense strand (5'→3'): a*fU*gadAggnTggagucfUgfUgacag*u*a.

[0187] Table 10. Free uptake test results of human primary liver cells

[0188] Table 11. IC50 of the conjugates tested in free uptake by human primary liver cells

[0189] Example 4: In vivo activity test of hINHBE overexpressing mice by high-pressure tail vein injection of HDI

[0190] Six- to seven-week-old mice were randomly divided into groups based on body weight, with 3 to 5 mice per group. On Day 1, the conjugate was subcutaneously injected at a dose of 1 mp / kilogram (mpk), along with an equal volume of PBS as a control. On Day 4 or Day 21, all mice were injected via the tail vein with a hINHBE plasmid DNA solution (8% of their body weight) over 5 seconds. On Day 5 or Day 22 (24 hours after INHBE plasmid injection), all mice were euthanized by CO2 inhalation. Liver samples were collected, and liver hINHBE mRNA levels were measured by qPCR to evaluate the knockdown effect of the different conjugates on the target gene (Tables 12 and 13).

[0191] Table 12. Inhibition of liver hINHBE mRNA by the conjugate in HDI-hINHBE mice

[0192] Table 13. Inhibition of liver hINHBE mRNA by the conjugate in HDI-hINHBE mice

[0193] Example 5: Activity test of the conjugate in cynomolgus monkeys

[0194] Twelve healthy male cynomolgus macaques were screened during the acclimation period based on body weight, hematology, and blood biochemistry levels and randomly divided into groups of three. Baseline (pre-dose) liver samples were collected by liver puncture. After the animals recovered for 1 to 2 weeks, subcutaneous injections were administered. Conjugate 279 received a single subcutaneous dose of 10 mg / kg on Day 0; the conjugate 590 single-dose group received a subcutaneous dose of 4.5 mg / kg on Day 0; the conjugate 590QM*2 group received a subcutaneous dose of 4.5 mg / kg on Day 0 and Day 28, for a total of two doses; and the PBS group received a single subcutaneous injection on Day 0. Liver samples were collected by liver puncture on Days 14, 28, 42, 56, and 84. The relative expression levels of INHBE mRNA in the liver at different time points were determined by qPCR (Table 14).

[0195] Table 14. Inhibition of liver INHBE mRNA by the conjugate in cynomolgus monkeys

[0196] NA means not detected. One animal in the conjugate 279 10 mg / kg group did not respond and the data were excluded, so the data included two animals.

[0197] The results showed that a single dose of 10 mg / kg of conjugate 279 significantly reduced INHBE mRNA expression levels in the liver of cynomolgus monkeys, with an inhibition rate exceeding 70%, which was sustained until Day 84 after administration. A single dose of 4.5 mg / kg of conjugate 590 inhibited INHBE mRNA in cynomolgus monkeys by exceeding 70%, which was sustained until Day 42 after administration. When conjugate 590 was injected at a dose of 4.5 mg / kg on Day 0 and Day 28, respectively, the inhibition of liver INHBE mRNA reached 85% on Day 84 after administration.

Claims

1. An siRNA for inhibiting INHBE gene expression, characterized in that: The siRNA comprises a sense strand and an antisense strand; wherein the antisense strand comprises at least 17 consecutive nucleotides that differ by no more than 4 nucleotides from the nucleotide sequence shown in any one of SEQ ID NO: 2 to SEQ ID NO: 277, and the antisense strand is 17 to 30 nucleotides in length; the sense strand is 17 to 30 nucleotides in length and is at least partially complementary to the antisense strand.

2. The siRNA according to claim 1, wherein: The antisense strand is 19 to 27 nucleotides long; the sense strand is 19 to 25 nucleotides long.

3. The siRNA according to claim 1, wherein: The antisense strand differs from any one of the nucleotide sequences shown in SEQ ID NO: 2 to SEQ ID NO: 277 by no more than 4 nucleotides.

4. The siRNA according to claim 1, wherein: The sense strand and the antisense strand have no more than 3 nucleotide mismatches.

5. The siRNA according to claim 1, wherein: The sequence of the siRNA is selected from duplex 1 to duplex 276.

6. The siRNA according to any one of claims 1 to 5, characterized in that: The siRNA contains at least one modified nucleotide.

7. The siRNA according to claim 6, wherein: All nucleotides in the sense strand and / or antisense strand of the siRNA are modified nucleotides or nucleotide analogs.

8. The siRNA according to claim 7, wherein: The modified nucleotide is selected from 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-split nucleotide analogs, 2'-fluoro-arabino nucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 3'-methoxy nucleotides, 2'-allyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, diol-modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides, unlocked nucleotides, threose nucleotides or glycerol nucleotides.

9. The siRNA according to any one of claims 1 to 8, characterized in that: The 5' and 3' ends of the sense strand independently contain 0, 1 or 2 phosphorothioate linkages; and / or the 5' and 3' ends of the antisense strand independently contain 1 or 2 phosphorothioate linkages.

10. The siRNA according to any one of claims 1 to 9, characterized in that: The first nucleotide at the 5' end of the antisense strand is a nucleotide modified with (E)-vinyl phosphate.

11. The siRNA according to any one of claims 1 to 10, characterized in that: The siRNA contains at least one base-modified nucleotide.

12. The siRNA according to any one of claims 1 to 11, characterized in that: The antisense strand of the siRNA is 21 nucleotides in length, wherein the 2nd, 5th, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 19 nucleotides in length, wherein the 7th, 9th, and 11th positions at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 5, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9 and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, position 10 is an FD-modified nucleotide, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9 and 11 of the 5' end of the sense strand are 2'-fluoro nucleotides, position 10 is an FD-modified nucleotide, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 22 nucleotides in length, wherein positions 2, 5, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 20 nucleotides in length, wherein positions 8, 9, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, position 10 is an FD-modified nucleotide, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, position 1 is XY-EVP-007-2, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9 and 11 of the 5' end of the sense strand are 2'-fluoro nucleotides, position 10 is XY-024-2, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9 and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, position 10 is XY-024-2, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, position 5 is DNA, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9 and 11 of the 5' end of the sense strand are 2'-fluoro nucleotides, position 10 is XY-024-2, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, position 5 is DNA, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 10 and 11 of the 5' end of the sense strand are 2'-fluoro nucleotides, position 9 is a cEt-modified nucleotide, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 11, and 12 at the 5' end of the sense strand are 2'-fluoro nucleotides, position 10 is XY-024-2, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9 and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, position 10 is XY-024-9, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 10 and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, position 9 is XY-024-13, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9 and 11 at the 5' end of the sense strand are 2'-fluoro nucleotides, position 10 is XY-024-13, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, position 22 is XY-016-4, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9 and 11 of the 5' end of the sense strand are 2'-fluoro nucleotides, position 10 is XY-024-13, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 5, 9, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, 11, and 12 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 9, 12, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, 11, and 12 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 12, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, position 5 is DNA, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, 11, and 12 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 12, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, position 7 is DNA, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, 11, and 12 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 22 nucleotides in length, wherein positions 2, 12, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, position 5 is DNA, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 20 nucleotides in length, wherein positions 8, 9, 10, and 11 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 23 nucleotides in length, wherein positions 2, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, positions 5 and 7 are DNA, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 21 nucleotides in length, wherein positions 9, 10, and 11 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 22 nucleotides in length, wherein positions 2, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 20 nucleotides in length, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 19 nucleotides in length, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 12, 14, and 16 of the 5' end of the antisense strand are 2'-fluoro nucleotides, position 7 is DNA, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 19 nucleotides in length, wherein positions 7, 9, and 11 of the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; or The antisense strand of the siRNA is 21 nucleotides in length, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; and the sense strand of the siRNA is 19 nucleotides in length, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoro nucleotides, and the remaining positions are 2'-methoxy nucleotides; Optionally, the first nucleotide at the 5' end of the antisense strand is a (E)-vinyl phosphate-modified nucleotide.

13. The siRNA according to any one of claims 11 or 12, characterized in that: The siRNA is selected from modified siRNA duplex 1 to modified siRNA duplex 329.

14. An siRNA conjugate obtained by conjugating the siRNA according to any one of claims 1 to 13 to a conjugating molecule.

15. The siRNA conjugate according to claim 14, characterized in that: The targeting ligand forms a conjugated molecule through a linker and is independently or simultaneously connected to the 3' end or 5' end of the siRNA sense strand.

16. The siRNA conjugate according to claim 15, characterized in that: Optionally further comprises a delivery carrier, which is GalNAc (L96) or Ser (GN), or GalNAc (Ser1), GalNAc (Ser2), GalNAc (Ser3), GalNAc (Ser4), or LP-GalNAc, XY-GalNAc, GalNAc (A1GN), GalNAc (A1dGN), GalNAc (A3GN), GalNAc (A3dGN), GalNAc (A5GN), GAlNAc (NAG25), GAlNAc (NAG37), Among them, GalNAc(L96) has the following structure: Ser(GN) may have the following structure: GalNAc(Ser1), GalNAc(Ser2), GalNAc(Ser3), and GalNAc(Ser4) have the following structures respectively: LP-GalNAc has the following structure: XY-GalNAc has the following structure: or GalNAc(A1GN), GalNAc(A1dGN), GalNAc(A3GN), GalNAc(A3dGN), GalNAc(A5GN), GAlNAc(NAG25), and GAlNAc(NAG37) have the following structures, respectively: The delivery vehicle portion is linked to the 3' end of the siRNA sense strand, or to the 5' end of the siRNA sense strand, or to the inverted abasic residue (iab) at the 3' end of the siRNA sense strand, or to the inverted abasic residue (iab) at the 5' end of the siRNA sense strand, or one or more of the delivery vehicle portions are simultaneously linked to the 3' end of the siRNA sense strand, the 5' end of the siRNA sense strand, the inverted abasic residue (iab) at the 3' end of the siRNA sense strand, and the inverted abasic residue (iab) at the 5' end of the siRNA sense strand; Preferably, the delivery vector GalNAc (L96) or Ser (GN) is linked to the 3' end of the siRNA sense strand or to the 5' end of the siRNA sense strand; one or more Ser (GN) can be linked to the 3' end and the 5' end of the siRNA sense strand simultaneously; More preferably, the delivery carrier GalNAc(L96) is linked to the 3' end of the sense strand of the siRNA.

17. The siRNA conjugate according to claim 15 or 16, characterized in that: The siRNA conjugates are selected from conjugate 1 to conjugate 604, and conjugate 619 to conjugate 685.

18. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the siRNA according to any one of claims 1 to 14 and / or the siRNA conjugate according to any one of claims 15 to 17 and a pharmaceutically acceptable carrier.

19. Use of the siRNA according to any one of claims 1 to 14 and / or the siRNA conjugate according to any one of claims 15 to 17 and / or the pharmaceutical composition according to claim 18 in the preparation of a medicament for treating and / or preventing a pathological condition or disease associated with overexpression of the INHBE gene.

20. The use according to claim 19, characterized in that: The pathological condition or disease is a metabolic disease and / or a cardiovascular disease; further preferably, the metabolic disease is obesity, type 2 diabetes mellitus (T2DM), insulin resistance, lipodystrophy, fatty liver disease, non-alcoholic hepatic fatty disease (NAFLD) or metabolic dysfunction-associated hepatic fatty disease (MAFLD), non-alcoholic steatohepatitis (NASH) or metabolic dysfunction-associated fatty liver disease (MASH); the cardiovascular disease is hyperlipidemia or dyslipidemia, atherosclerosis, cardiomyopathy, heart failure or coronary heart disease (CHD).

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