Sirna targeting expression of activin a receptor type 1c (ACVR1c) gene, and conjugate thereof and use thereof

Abstract

Provided in the present invention are an siRNA for inhibiting the expression of the activin A receptor type 1C (ACVR1C) gene, 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 set forth in SEQ ID NO: 1236-SEQ ID NO: 2470 by no more than 4 nucleotides. The antisense strand is 17-30 nucleotides in length; and the sense strand is 17-30 nucleotides in length, and is at least partially or completely complementary to the antisense strand. The siRNA, siRNA conjugate and pharmaceutical composition provided in the present invention exhibit good stability, great ACVR1C gene inhibitory activity, and satisfactory cytotoxicity and immunostimulatory activity.

Description

A siRNA targeting activin A receptor type 1C (ACVR1C) gene expression, its conjugates, and applications.

[0001] This application is based on and claims priority to Chinese patent applications No. 202411622273.X, filed on November 14, 2024, and No. 202510417143.0, filed on April 3, 2025, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to an siRNA and its conjugates that inhibit the expression of the activin A receptor type 1C (ACVR1C) gene through a process mediated by an RNA-induced silencing complex (RISC), and to siRNA compositions comprising the same, and their use in the treatment of metabolic and / or cardiovascular diseases. Background Technology

[0003] Activin A receptor type 1C refers to a type I receptor of the TGF-β family of signal transduction molecules. ACVR1C possesses intrinsic serine / threonine kinase activity in its cytoplasmic domain, thereby inducing phosphorylation and activation of the SMAD2 / 3 / 4 complex translocated to the nucleus, where it binds to the SMAD-binding element (SBE) to activate gene transcription. ACVR1C expression levels vary considerably across tissues, but are highest in white and brown adipose tissue. In addition to the full-length protein, variants of ACVR1C are also expressed in adipose tissue, the brain, and the ovary (Murakami M et al., Biochem Genet. 2013; 51(3-4):202-210). ACVR1C is also known as activin receptor-like kinase 7 (ALK-7). Polymorphisms in ACVR1C have been found to be associated with an increased risk of metabolic syndrome in Chinese women and may be involved in cardiovascular remodeling in patients with metabolic syndrome (Zhang, W et al., Arq Bras Cardiol. 2013:101(2):134-140). Additionally, variants predicted to cause loss of ACVR1C gene function are thought to affect body fat distribution and prevent type 2 diabetes (Emdin CA et al., Diabetes. 2019:68(1):226-234). Studies in adipocytes of obese mouse strains have shown that ACVR1C dysfunction (due to nonsense mutations) leads to increased lipolysis in adipocytes and reduced fat accumulation, while conversely, ACVR1C activation inhibits lipolysis by suppressing the expression of lipopeptidase. In addition, lower-weight ACVR1C-deficient mice exhibited enhanced glucose tolerance and insulin sensitivity, and measurements of metabolic rate in these mice revealed increased O2 consumption, decreased respiratory quotient, and increased energy expenditure (Yogosawa, S et al., Diabetes 2013:62(1):115-123).

[0004] A statistical study covering 15.8 million Chinese adults showed that, according to the body mass index (BMI) classification criteria, overweight individuals (BMI = 24-28) accounted for approximately 34.8%, and obese individuals (BMI ≥ 28) accounted for approximately 14.1% (Diabetes Obes Metab. 2023; 25(11):3390-3399). Compared with people with normal BMI, overweight / obese individuals have a higher prevalence of metabolic complications, with common complications including fatty liver, prediabetes, dyslipidemia, and hypertension; body fat levels, especially visceral fat levels, are positively correlated with the risk of type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD). In the elderly population, overweight and obesity also increase the risk of age-related diseases. Clinical recommendations include treatment of obesity through lifestyle modifications, drug therapy, and bariatric surgery. In recent years, newly approved incretin-based drugs for the treatment of type 2 diabetes mellitus (T2DM) and obesity, such as semaglutide, have shown clear efficacy in weight loss and blood glucose control, while also providing multiple metabolic-related clinical benefits such as reduced liver lipids and improvement of hepatic steatosis. They have increasingly become cornerstone drugs for the treatment of obesity and T2DM and other metabolic diseases. However, they also have drawbacks, including significant muscle loss during weight loss, restriction of central dopamine secretion, gastrointestinal adverse reactions, and even an increased risk of intestinal obstruction. Furthermore, there are reports of weight rebound after discontinuation of these drugs. These issues represent new unmet clinical needs in the treatment of obesity and related metabolic complications. Developing novel weight-loss drugs that can maintain muscle mass and prevent muscle loss during rapid weight loss, while maintaining nutritional support through food intake, and providing long-term, sustained weight loss to prevent rebound, will be key to the future clinical management of obesity and related metabolic complications.

[0005] Compared to traditional drugs, siRNAs exhibit poor stability and are easily degraded by nucleases when administered systemically. Furthermore, it is necessary to explore ways to further enhance activity while avoiding off-target effects, immune stimulation, cytotoxicity, and other side effects. Therefore, developing more candidate siRNAs that are stable in the blood, possess good biological activity, exhibit low cytotoxicity, and can effectively inhibit ACVR1C gene expression is an urgent problem to be solved. Simultaneously, developing highly effective and long-acting weight-loss drugs using these candidate siRNAs that inhibit ACVR1C gene expression is both necessary for clinical research and a realistic possibility for commercialization. Summary of the Invention

[0006] This application provides a siRNA that targets ACVR1C, which can specifically bind to ACVR1C mRNA, disrupt the normal translation template function of ACVR1C mRNA, increase lipolysis in adipocytes, and reduce fat accumulation, thereby achieving the purpose of treating obesity and related metabolic complications.

[0007] This invention provides an siRNA for inhibiting ACVR1C gene expression, the siRNA comprising a sense strand and an antisense strand; wherein the antisense strand comprises at least 17 consecutive nucleotides differing from the nucleotide sequences shown in any of SEQ ID NO: 1236 to SEQ ID NO: 2470 by no more than 4 nucleotides, the antisense strand being 17 to 30 nucleotides in length; the sense strand being 17 to 30 nucleotides in length and at least partially complementary to the antisense strand.

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

[0009] In some embodiments of the present invention, the antisense strand differs from any nucleotide sequence shown in SEQ ID NO: 1236 to SEQ ID NO: 2470 by no more than 4 nucleotides; in some embodiments of the present invention, the antisense strand differs from any nucleotide sequence shown in SEQ ID NO: 1236 to SEQ ID NO: 2470 by no more than 3 nucleotides; in some embodiments of the present invention, the antisense strand differs from any nucleotide sequence shown in SEQ ID NO: 1236 to SEQ ID NO: 2470 by no more than 2 nucleotides; in some embodiments of the present invention, the antisense strand differs from any nucleotide sequence shown in SEQ ID NO: 1236 to SEQ ID NO: 2470 by no more than 1 nucleotide; in some embodiments of the present invention, the antisense strand is any nucleotide sequence shown in SEQ ID NO: 1236 to SEQ ID NO: 2470.

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

[0011] Preferably, the sense strand and the antisense strand are complementary to each other by at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides.

[0012] In some embodiments of the present invention, the antisense strand is 19 to 27 nucleotides in length; 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; the sense strand is 19 to 21 nucleotides in length.

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

[0015] In some embodiments of the invention, the siRNA comprises one or more single-stranded nucleotide overhangs, such as 1, 2, 3, or 4 nucleotide overhangs. In some embodiments of the invention, the overhangs may be on the sense strand, the antisense strand, or any combination thereof. In some embodiments of the invention, the overhangs are located at 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 siRNA has a two-nucleotide overhang at the 3' end of the antisense strand.

[0017] In some embodiments of the present invention, the siRNA has a blunt end. In some embodiments of the present 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 present 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 the following duplexes shown in Table 1.

[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 modifying 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 present invention, the modified nucleotide is selected from 2'-methoxynucleotides, 2'-fluoronucleotides, 2'-deoxynucleotides, 2',3'-cleaved nucleotide analogs, 2'-fluoroarabinonucleotides, 2'-methoxyethylnucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, 3'-methoxynucleotides, 2'-allyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate groups, nucleotides containing 5'-phosphate mimics, diol-modified nucleotides, debased nucleotides, morpholinonucleotides, threonucleotides, locked nucleotides, unlocked nucleotides, glycerol nucleotides, or base-modified nucleotides.

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

[0025] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0026] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 9th, 10th, and 11th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0027] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 5, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0028] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0029] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 7, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0030] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 5, 7, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0031] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 5, 8, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0032] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 5, 7, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0033] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 5, 7, 12, and 14 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0034] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0035] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0036] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 3, 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0037] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0038] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 13 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0039] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 3, 4, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein position 2 at the 5' end of the sense strand is a 2'-fluoronucleotide, and the remaining positions are 2'-methoxynucleotides.

[0040] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 4, 5, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein position 2 at the 5' end of the sense strand is a 2'-fluoronucleotide, and the remaining positions are 2'-methoxynucleotides.

[0041] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 4, 6, 7, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein position 2 at the 5' end of the sense strand is a 2'-fluoronucleotide, and the remaining positions are 2'-methoxynucleotides.

[0042] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, 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'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0043] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 4, 6, 8, 10, 14, 16, 18, 20, and 22 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, 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'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0044] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 7 is glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0045] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 7 is threonine (TNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0046] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 2nd, 12th, 14th, and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, the 7th position is threonine (TNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 7th, 9th, 10th, and 11th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0047] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 6 is threonine (TNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0048] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 2nd, 12th, 14th, and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, the 6th position is threonine (TNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 7th, 9th, 10th, and 11th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0049] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 6 is glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0050] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 4 is glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0051] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 5 is glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0052] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 7 is glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, 11, and 15 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0053] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 8, 9, 10, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 6 is a glycerol nucleotide (GNA), and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0054] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 5, 7, and 12 at the 5' end of the antisense strand are 2'-deoxynucleotides, position 14 is a 2'-fluoronucleotide, and the remaining positions are 2'-methoxynucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0055] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 5, 7, and 12 at the 5' end of the antisense strand are 2'-deoxynucleotides, positions 6, 8, 9, 10, 14, and 16 are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0056] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 5, 7, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 11, and 13 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0057] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 5, 7, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 11, and 12 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0058] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 5, 7, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides long, wherein positions 7, 9, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0059] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 5, 7, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides. In some embodiments of the present invention, the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, 11, and 12 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0060] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 5th, 7th, and 9th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0061] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 7th, 9th, 10th, and 11th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0062] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0063] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0064] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions of the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein the 7th, 8th, and 9th positions of the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0065] In some embodiments of the present invention, the antisense strand of the siRNA is 19 nucleotides long, wherein positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0066] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 14th position at the 5' end of the antisense strand is a 2'-fluoronucleotide, the 2nd, 5th, and 7th positions are 2'-deoxynucleotides, the 12th position is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0067] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein the 9th, 10th, and 11th positions at the 5' end of the sense strand are 2'-fluoronucleotides, the 1st position is a threonucleotide, and the remaining positions are 2'-methoxynucleotides.

[0068] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 22 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0069] In some embodiments of the present invention, the antisense strand of the siRNA is 23 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 23 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 21 nucleotides long, wherein positions 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0070] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, position 1 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides.

[0071] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 21 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0072] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 22 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0073] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0074] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 6, 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0075] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, position 6 is 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides.

[0076] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 6, 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0077] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 3, 5, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 8, 9, 10, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0078] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 5, 7, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 7, 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0079] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 3, 7, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 7, 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, position 6 is 2'-deoxynucleotide, and the remaining positions are 2'-methoxynucleotides.

[0080] In some embodiments of the present invention, the antisense strand of the siRNA is 22 nucleotides long, wherein positions 2, 6, 8, 9, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 20 nucleotides long, wherein positions 6, 8, 9, and 10 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0081] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoronucleotides, the 1st position is a threonucleotide, and the remaining positions are 2'-methoxynucleotides.

[0082] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 6th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, the 20th position is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein the 7th, 8th, and 9th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0083] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein positions 2, 6, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, position 21 is a threonucleotide, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein positions 7, 8, and 9 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0084] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein positions 2, 5, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein positions 7, 9, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0085] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein positions 2, 7, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein positions 7, 9, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0086] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, the 7th position is a DNA modification, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein the 7th, 9th, and 11th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0087] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein the 2nd, 12th, 14th, and 16th positions at the 5' end of the antisense strand are 2'-fluoronucleotides, the 5th position is a DNA modification, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein the 7th, 9th, and 11th positions at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0088] In some embodiments of the present invention, the antisense strand of the siRNA is 21 nucleotides long, wherein positions 2, 12, 14, and 16 at the 5' end of the antisense strand are 2'-fluoronucleotides, positions 5 and 7 are DNA modifications, and the remaining positions are 2'-methoxynucleotides; the sense strand of the siRNA is 19 nucleotides long, wherein positions 7, 9, and 11 at the 5' end of the sense strand are 2'-fluoronucleotides, and the remaining positions are 2'-methoxynucleotides.

[0089] In some embodiments of the present invention, the modified nucleotide is a nucleotide in which the phosphate group is modified by a thiophosphate group. That is, a sulfur atom replaces the non-bridging oxygen atom in the phosphodiester bond, thereby replacing the phosphodiester bond with a thiophosphate diester bond.

[0090] In some embodiments of the present invention, the 5' end and 3' end of the sense chain each independently contain 0, 1, or 2 thiophosphate groups; and / or the 5' end and 3' end of the antisense chain each independently contain 1 or 2 thiophosphate groups.

[0091] In some embodiments of the present invention, at least one of the nucleotides at positions 1 and 2 of the 5' end of the sense strand, the nucleotides at positions 2 and 3 of the 5' end of the sense strand, the nucleotides at positions 1 and 2 of the 3' end of the sense strand, the nucleotides at positions 2 and 3 of the 3' end of the sense strand, the nucleotides at positions 1 and 2 of the 3' end of the antisense strand, the nucleotides at positions 2 and 3 of the 3' end of the antisense strand, the nucleotides at positions 1 and 2 of the 5' end of the antisense strand, and the nucleotides at positions 2 and 3 of the 5' end of the antisense strand are linked by a thiophosphate group; preferably, at least four are linked by thiophosphate groups; in some embodiments of the present invention, at least six are linked by thiophosphate groups; in some embodiments of the present invention, all eight are linked by thiophosphate groups.

[0092] In some embodiments of the present invention, the nucleotides at positions 1 and 2, and at positions 2 and 3, of the 5' end of the positive strand are linked by phosphate thioester groups.

[0093] In some embodiments of the present invention, the nucleotides at positions 1 and 2, and positions 2 and 3 at the 5' end of the positive strand are linked by thiophosphate groups, and the nucleotides at positions 1 and 2, and positions 2 and 3 at the 3' end are linked by thiophosphate groups.

[0094] In some embodiments of the present invention, the nucleotides at positions 1 and 2 at the 3' end of the antisense strand are linked by thiophosphate groups, and the nucleotides at positions 2 and 3 at the 5' end are linked by thiophosphate groups.

[0095] In some embodiments of the present invention, the nucleotides at positions 1 and 2 of the 5' end of the sense strand, the nucleotides at positions 2 and 3 of the 5' end of the sense strand, the nucleotides at positions 1 and 2 of the 3' end of the sense strand, the nucleotides at positions 2 and 3 of the 3' end of the sense strand, the nucleotides at positions 1 and 2 of the 3' end of the antisense strand, the nucleotides at positions 2 and 3 of the 3' end of the antisense strand, the nucleotides at positions 1 and 2 of the 5' end of the antisense strand, and the nucleotides at positions 2 and 3 of the 5' end of the antisense strand are all linked by thiophosphate groups.

[0096] In some embodiments of the present invention, the positive strand may include one or more capping residues or portions, referred to as "capping residues". A "capping residue" is a non-nucleotide compound or other portion that can be incorporated into one or more ends of a nucleotide sequence of siRNA. In some embodiments of the present invention, the capping residues are present at the 5' end, the 3' end, or both the 5' end and the 3' end of the positive strand.

[0097] In some embodiments of the present invention, an inverse debasing 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 positive strand may contain more than one inverse debasing deoxyribose moiety as a capping residue.

[0098] In some embodiments of the invention, one or more inverse abase residues (invAb) are added to the 3' end of the positive strand. In some embodiments of the invention, one or more inverse abase residues (invAb) are added to the 5' end of the positive strand. In some embodiments of the invention, one or more inverse abase residues may be inserted between the delivery vector and the nucleotide sequence of the siRNA positive strand. In some embodiments of the invention, one or more inverse abase residues are included at or near one or more ends of the siRNA positive strand.

[0099] In some embodiments of the invention, one or more inverse abase residues (invAb) are added to the 5' end of the positive strand. In some embodiments of the invention, one or more inverse abase residues may be inserted between the delivery vector and the nucleotide sequence of the siRNA positive strand.

[0100] Reverse debasing residues can be linked to nucleic acids via phosphodiester bonds, thiophosphate diester bonds, etc.

[0101] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is selected from the following structures:

[0102] Wherein, Base is a base A, U, G, C, T or other modified bases.

[0103] In some embodiments of the present invention, the first nucleotide at the 5' end of the antisense strand is a (E)-vinyl phosphate modified nucleotide.

[0104] In some embodiments of the present invention, the siRNA contains at least one base-modified nucleotide.

[0105] In some embodiments of the present invention, the bases of the base-modified nucleotide are selected from the following structures:

[0106] In some embodiments of the present invention, the base-modified nucleotide may be located at positions 5, 6, 7, and 8 of the siRNA antisense strand.

[0107] In some embodiments of the present invention, the base-modified nucleotide may be located at the overhang of a single-stranded nucleotide in the siRNA.

[0108] Preferably, the siRNA antisense strand contains two nucleotide overhangs, and the base-modified nucleotide is the first nucleotide of the siRNA antisense strand overhang.

[0109] Preferably, the siRNA antisense strand contains two nucleotide overhangs, and the base-modified nucleotide is the second nucleotide of the siRNA antisense strand overhang.

[0110] In some embodiments of the present invention, the nucleotide sequence (5'→3') of the modified siRNA is selected from the modified duplexes listed in Table 2 below.

[0111] Table 2. Sensitive and antisense strand sequences of the modified siRNA duplex

[0112] The present invention also provides siRNA conjugates obtained by conjugating the above-mentioned siRNA with conjugating molecules.

[0113] In this invention, unless otherwise specified, "conjugation" refers to the covalent connection between two or more chemical parts; "conjugated compound" refers to a compound formed by the covalent connection between various chemical parts; and "siRNA conjugated compound" refers to a compound formed by the covalent attachment of one or more chemical parts to siRNA. It should be noted that the chemical parts can be directly attached to the siRNA or attached to the siRNA via a linker.

[0114] In this invention, unless otherwise specified, the "-" in "connector-targeting ligand" refers to the covalent connection between the connector and the targeting ligand.

[0115] In some embodiments of the present invention, the targeting ligand is linked to siRNA via a linker to form a conjugate molecule.

[0116] In some embodiments of the present invention, the targeting ligand forms a conjugated molecule via a linker, which is independently or simultaneously attached to the 3' or 5' end of the siRNA's positive strand.

[0117] In some embodiments of the present invention, the siRNA of the present invention can be conjugated with a pharmaceutically acceptable conjugating molecule to obtain an siRNA conjugate. In some embodiments of the present invention, the siRNA is covalently conjugated to the conjugating molecule. To reduce the potential impact of conjugation on siRNA activity, the conjugation site between the siRNA and the conjugating molecule can be at the 3' or 5' end of the siRNA's sense strand, or at the 5' end of the antisense strand. In some embodiments, the conjugation site between the siRNA and the conjugating molecule can also be within the internal sequence of the siRNA.

[0118] The pharmaceutically acceptable targeting ligand may be a targeting ligand commonly used in the field of siRNA drug delivery, such as, but not limited to, one or more of the following targeting ligands or their derivatives: lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g., vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as transmembrane peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid; or receptor ligands expressed by hepatocytes, such as desialyl glycoprotein, desialyl sugar residues, lipoproteins (e.g., high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, transferrin, etc.

[0119] In some embodiments of the present invention, the targeting ligand is N-acetylgalactosamine.

[0120] In some embodiments of the present invention, the targeting ligand is directly attached to the 3' end of the siRNA's positive strand.

[0121] In some embodiments of the present invention, the targeting ligand is directly attached to the 5' end of the siRNA positive strand.

[0122] In some embodiments of the present invention, the targeting ligand is attached to the 3' end of the siRNA positive strand via a adapter.

[0123] In some embodiments of the present invention, the targeting ligand is attached to the 5' end of the siRNA positive strand via a adapter.

[0124] In some embodiments of the present invention, the targeting ligand is N-acetylgalactosamine, which is attached to the 3' end of the siRNA sense strand via a linker.

[0125] In some embodiments of the present invention, the delivery carrier is GalNAc(L96), having the following structure:

[0126] In some embodiments of the present invention, GalNAc(L96) is linked to the 3' end of the siRNA positive strand.

[0127] In some embodiments of the present invention, GalNAc(L96) is linked to the 5' end of the siRNA positive strand.

[0128] In some embodiments of the present invention, GalNAc(L96) is linked to the inverse ablation residue (invAb) at the 3' end of the siRNA positive strand.

[0129] In some embodiments of the present invention, GalNAc(L96) is linked to the inverse ablation residue (invAb) at the 5' end of the siRNA's positive strand.

[0130] In some embodiments of the present invention, at least one long-chain hydrocarbon chain is conjugated to the end or non-end of any chain of siRNA to obtain an siRNA conjugate.

[0131] In some embodiments of the present invention, the long-chain hydrocarbon chain is selected from chain-like aliphatic compounds with a carbon chain length of 12 or more, alicyclic compounds containing one ring, or aliphatic compounds containing two or more rings.

[0132] In some embodiments of the invention, the long-chain hydrocarbon chain contains functional groups selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0133] In some embodiments of the present invention, the long-chain hydrocarbon chain is selected from the group consisting of: substituted or unsubstituted C12-C24 saturated alkanes, substituted or unsubstituted C12-C24 unsaturated hydrocarbons, substituted or unsubstituted C12-C24 fatty acids, and C12-C24 aliphatic amines.

[0134] In some embodiments of the present invention, the at least one long hydrocarbon chain is obtained by replacing the non-terminal nucleotide conjugation of any one chain of the modified siRNA.

[0135] In some embodiments of the present invention, the at least one long-chain hydrocarbon chain is conjugated to the modified siRNA via a linker containing an ether, thioether, urea, carbonate, amine, amide, hydroxylamine, maleimide-thioether, disulfide, phosphate diester, sulfonamide bond, click reaction product, or carbamate.

[0136] In some embodiments of the present invention, the at least one long-chain hydrocarbon chain is conjugated to the modified siRNA via a linker or via an internucleotide phosphate bond.

[0137] In some embodiments of the present invention, the at least one long hydrocarbon chain is conjugated to a nucleobase, sugar moiety, or internucleotide phosphate bond in the modified siRNA.

[0138] In some embodiments of the present invention, in the siRNA conjugate, a long hydrocarbon chain is conjugated to the 3' end, 5' end or non-terminus of the sense strand via L1, while the antisense strand is not conjugated with a long hydrocarbon chain.

[0139] Alternatively, in the siRNA conjugate, a long hydrocarbon chain is conjugated to the 3' end, 5' end, or non-terminus of the antisense strand via L2 conjugation, while the sense strand has no long hydrocarbon chain conjugation;

[0140] Alternatively, in the siRNA conjugate, one long hydrocarbon chain is conjugated to the 3' end, 5' end, or non-terminus of the antisense chain via L3, and another long hydrocarbon chain is conjugated to the 3' end, 5' end, or non-terminus of the antisense chain via L4;

[0141] Alternatively, in the siRNA conjugate, one long hydrocarbon chain is conjugated to the 5' end of the sense strand via M1-T1, and another long hydrocarbon chain is conjugated to the 3' end or non-terminus of the sense strand via L5, while the antisense strand has no long hydrocarbon chain conjugation.

[0142] Alternatively, in the siRNA conjugate, one long hydrocarbon chain is conjugated to the 3' end of the sense strand via M2-T2, and another long hydrocarbon chain is conjugated to the 5' end or non-terminus of the sense strand via L6, while the antisense strand has no long hydrocarbon chain conjugation.

[0143] Alternatively, in the siRNA conjugate, one long hydrocarbon chain is conjugated to the 5' end of the sense strand via M3-T3, one long hydrocarbon chain is conjugated to the 3' end of the sense strand via M4-T4, one long hydrocarbon chain is conjugated to the non-terminus of the sense strand via L7, and the antisense strand has no long hydrocarbon chain conjugation.

[0144] Alternatively, in the siRNA conjugate, one long hydrocarbon chain is conjugated to the 5' end of the sense strand via M5-T5, and another long hydrocarbon chain is conjugated to the 3' end of the sense strand via M6-T6, while the antisense strand has no long hydrocarbon chain conjugation.

[0145] Alternatively, in the siRNA conjugate, one long hydrocarbon chain is conjugated to the 5' end of the sense strand via M7-T7, and another long hydrocarbon chain is conjugated to the 3' end, 5' end, or non-terminus of the antisense strand via L8;

[0146] Alternatively, in the siRNA conjugate, one long hydrocarbon chain is conjugated to the 3' end of the sense strand via M8-T8, and another long hydrocarbon chain is conjugated to the 3' end, 5' end, or non-terminus of the antisense strand via L9;

[0147] Alternatively, in the siRNA conjugate, a long hydrocarbon chain passes through M... 10 -T 10 A long-chain hydrocarbon chain is coupled to the 5' end of the positive chain via M9-T9, and another long-chain hydrocarbon chain is coupled to the 3' end of the positive chain via L. 10 It is attached to the 3' end, 5' end, or non-end of the antisense chain.

[0148] The structural diagrams of siRNA conjugates are shown in general formulas 1 to 10:

[0149] The L1, L2, L3, L 4、L5, L6, L7, L8, L9, L 10 It can be independently selected from one or more of the following structural units: ether, thioether, urea, carbonate, amine, hydroxylamine, amide, maleimide-thioether, disulfide, phosphate diester, sulfonamide bond, click reaction product, carbamate;

[0150] The M1, M2, M3, M 4、 M5, M6, M7, M8, M9, M 10 T1, T2, T3, T 4、 T5, T6, T7, T8, T9, T 10 It can be independently selected from one or more of the following structural units: -N(R 1 )-, -O-, -S-, -C(O)-, -N(R 1 )C(O)-、-C(O)N(R 2 )-、-N(R 1 )C(O)N(R 2 -, -C(O)O-, -OC(O)-, -N(R) 1 )C(O)O-、-OC(O)N(R 2 )-, -OPO2-O-, -OP(O)(S)-O-, -OP(O)(R 3 )-O-、-OP(S)(R 3 )-O-、-OP(O)(NR 1 R 2 -N-、-OP(S)(NR) 1 R 2 )-N-、-OP(O)(NR 1 R 2 )-O-、-OP(S)(NR 1 R 2 )-O-、-P(O)(NR 1 R 2 -N-、-P(S)(NR) 1 R 2 -N-、-P(O)(NR) 1 R 2 )-O-、-P(S)(NR 1 R 2 -O-, -SS-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; wherein each R 1 R 2 and R3 It is independently hydrogen or an unsubstituted C1-C6 alkyl group; the substitution is preferably deuterated or halogenated.

[0151] The lipid is a long-chain hydrocarbon chain.

[0152] In this invention, "terminal" refers to the 3' terminal and the 5' terminal; "non-terminal" refers to sites other than the 3' terminal and the 5' terminal.

[0153] In the siRNA conjugates of this invention, the conjugation structures containing long-chain hydrocarbon chains include, but are not limited to, the structures shown below:

[0154] In some embodiments of the present invention, the siRNA conjugate is selected from the conjugates listed in Table 3 below.

[0155] Table 3. Sequences of modified siRNA conjugates

[0156] The present invention also provides a pharmaceutical composition comprising any of the above-described siRNAs and / or any of the above-described siRNA conjugates and a pharmaceutically acceptable carrier.

[0157] In some embodiments of the present invention, the pharmaceutical composition contains one siRNA as described in the first aspect. In other embodiments of this disclosure, the pharmaceutical composition contains at least two siRNAs as described in the first aspect (e.g., but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) as active ingredients. Preferably, the at least two siRNAs as described in the first aspect each target different target sequences in the ACVR1C gene, thereby expecting to exert synergistic effects by acting simultaneously on different target sequences. Here, "different target sequences" means that there is no overlap between target sequences, or the number of overlapping consecutive nucleotides between target sequences is less than 5 (e.g., 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 proportions. Preferably, the at least two siRNAs as described in the first aspect may exist in a molar ratio of 1:100 to 100:1; more preferably, the at least two siRNAs as described in the first aspect may exist 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 exist in the same molar ratio.

[0158] The present invention also provides the use of any of the above-described siRNAs and / or any of the above-described siRNA conjugates and / or the above-described pharmaceutical compositions in the preparation of a medicament for treating pathological conditions or diseases associated with ACVR1C gene expression.

[0159] Further, the pathological condition or disease is a metabolic disease and / or a cardiovascular disease. More preferably, the metabolic disease is obesity, type 2 diabetes mellitus (T2DM), insulin resistance, lipodystrophy, fatty liver disease, non-alcoholic fatty liver disease (NAFLD) or metabolic dysfunction-related fatty liver disease (MAFLD), non-alcoholic steatohepatitis (NASH) or metabolic dysfunction-related steatohepatitis (MASH); the cardiovascular disease is hyperlipidemia or dyslipidemia, atherosclerosis, cardiomyopathy, heart failure, or coronary heart disease (CHD).

[0160] The siRNA, siRNA conjugates, and pharmaceutical compositions provided by this invention have good stability, excellent ACVR1C gene inhibitory activity, satisfactory cytotoxicity and immunostimulatory properties, and can significantly reduce body fat levels, weight, and improve metabolic abnormalities.

[0161] The sequence of the ACVR1C gene targeted by siRNA in this invention (SEQ ID NO:2471) is shown below:

[0162] NM_145259.3 Homo sapiens activin A receptor 1C type (ACVR1C), transcript variant 1, mRNA:

[0163] In this invention, "siRNA" refers to an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that contains the ability to reduce or inhibit the translation of messenger RNA (mRNA) in a sequence-specific manner. siRNA can function through RNA interference mechanisms (e.g., by interacting with the mRNA interference pathway mechanism in mammalian cells (RNA-induced silencing complex RISC)) or any other mechanism or pathway. While the term siRNA drug as used in this invention is considered to function primarily through RNA interference mechanisms, the siRNA drug is not limited to any particular pathway or mechanism of action. siRNA drugs include, but are not limited to, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer enzyme substrates. The siRNA drug of this invention consists of an oligonucleotide chain having at least a partial complementarity to the mRNA that serves as the target. In some embodiments, the siRNA drug of this invention is double-stranded and consists of an antisense strand and a sense strand that is at least partially complementary to the antisense strand.

[0164] The term "length of the double-stranded region" refers to the number of base pairs in the base-pairing region of an siRNA molecule.

[0165] The term "protrusion" or "nucleotide protrusion" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of siRNA. For example, a protrusion exists when the 3' end of one strand of siRNA extends beyond the 5' end of the other strand (or vice versa). siRNA may contain a protrusion of at least one nucleotide, or the protrusion may contain at least 2 nt, at least 3 nt, at least 4 nt, at least 5 nt, or more. The protrusion may contain or be composed of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The protrusion may be on the sense strand, antisense strand, or any combination thereof. The nucleotide of the protrusion may be present at the 5' end, 3' end, or both ends of the antisense strand or sense strand of the siRNA.

[0166] The term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed alphabetically using standard nucleotide nomenclature.

[0167] In this invention, unless otherwise specified, uppercase letters C, G, U, A, T, I represent the base composition of nucleotides, including modified and unmodified nucleotides; lowercase letters c, g, u, a, t, i represent a nucleotide that is 2'-methoxynucleotide; lowercase letter f represents a nucleotide adjacent to the right of the identifier f that is 2'-fluoronucleotide; lowercase letter d represents a nucleotide adjacent to the right of the identifier d that is 2'-deoxynucleotide; gn represents a nucleotide adjacent to the right of the identifier gn that is glycerol nucleotide (GNA); tn represents a nucleotide adjacent to the right of the identifier tn that is threonucleotide (TNA); mΨ represents a pseudouridine modified with a methoxy group at the 2' position of the ribose; the identifier * represents a phosphate thioester linkage between two nucleotides adjacent to the identifier * (or between a nucleotide and a delivery carrier); eVP represents a nucleotide adjacent to its right that is (E)-vinyl phosphate modified; invAb represents a reverse debasement residue; L96 indicates that the delivery carrier GalNAc (L96) is conjugated at this location. Ser(GN) refers to the presence of the delivery carrier Ser(GN) at this location. A1GN refers to the presence of the delivery carrier A1GN at this location; LCT0, LCT1, LCT2, LCT3, LCT4, LCT5, LCT6, LCT7, LCT8, LCT9, and LCT10 are lipid-linked nucleoside compounds with different bases as described above.

[0168] In this invention, unless otherwise specified, ln refers to locked nucleic acid (LNA), and cEt refers to 2'-O-ethyl-bridged nucleic acid, the structure of which is described below:

[0169] Wherein, Base is a base A, U, G, 5mC, T or other modified bases.

[0170] Unless otherwise specified, the bases of other base-modified nucleotides used in this invention are described below:

[0171] in This indicates that the oligonucleotide is linked to the rest of the oligonucleotide at this location via a phosphodiester bond or a thiophosphate diester bond.

[0172] In this 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 have no more than 5, 4, 3, or 2 mismatched base pairs in total, while retaining the ability to hybridize under the relevant conditions. Furthermore, where the two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches for determining complementarity. In this invention, to satisfy the above hybridization ability requirements, the "complementary" sequence may also include or consist entirely of base pairs formed from non-Watson-Crick base pairs and / or from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U swing base pairs or Hoogstein base pairs. Correspondingly, in this invention, unless otherwise specified, "mismatch" means that in the siRNA double-stranded molecule, the bases at corresponding positions are not paired in a complementary manner.

[0173] In this invention, unless otherwise specified, "difference in nucleotide sequence" refers to a change in the type of bases of nucleotides at the same or corresponding positions compared to the original nucleotide sequence. For example, if a nucleotide base in the original nucleotide sequence is A, and the nucleotide base at the same or corresponding position is changed to U, C, G, or dT, dC, dG, etc., a difference in nucleotide sequence is considered to exist at that position. It should be noted that if, compared to the original nucleotide sequence, the nucleotides at the same or corresponding positions differ only in the presence or type of modification, a difference in nucleotide sequence is not considered to exist at that position. For example, if a nucleotide base in the original nucleotide sequence is U, and the nucleotide at the same or corresponding position is dT or a nucleotide modified with other bases (such as I, m6A, X, B), a difference in nucleotide sequence is not considered to exist at that position.

[0174] In this invention, unless otherwise specified, the term "pharmaceutical acceptable" means that the carrier, transporter, diluent, excipient and / or the salt / ester / hydrate formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with the receptor.

[0175] In this invention, unless otherwise specified, the term "inhibition" refers to the down-regulation of target gene expression due to siRNA-mediated mRNA degradation. "Down-regulation" refers to a decrease in target gene expression level of 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%, compared to the absence of siRNA treatment. A 100% decrease in target gene expression level means that there is no detectable level of target gene expression.

[0176] In this invention, the siRNA may also contain modified nucleotides as needed, and the modified nucleotides will not cause a significant weakening or loss of the siRNA's function in inhibiting ACVR1C gene expression. Currently, there are various ways to modify siRNA in the art, including, for example, backbone modification (such as phosphate group modification), ribose group modification, and base modification (Watts, JK, G.F. Deleavey, and M.J. Damha, Chemically Modified siRNA: Tools and Applications. Drug Discov Today, 2008, 13(19-20): p. 842-55).

[0177] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0178] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Detailed Implementation

[0179] Those skilled in the art will recognize that the siRNA described in this invention can be obtained using conventional siRNA preparation methods (e.g., solid-phase synthesis and liquid-phase synthesis), both of which are commercially available custom-made services. Those skilled in the art will also understand that modified nucleotide groups can be introduced into the siRNA described in this invention using appropriately modified nucleotide monomers. Methods for preparing appropriately modified nucleotide monomers are well known to those skilled in the art, and commercially available monomers are also available.

[0180] Example 1: siRNA Synthesis

[0181] The synthesis process is briefly described as follows: CPG is used as a solid-phase carrier for the sense and antisense strands of the siRNA sequence of the present invention and the sense and antisense strands of the modified double strands; CPG is used as a solid-phase carrier for the sense and antisense strands of the siRNA conjugates of the present invention, wherein the modified nucleosides LCT1-A, LCT1-U, LCT1-G, LCT1-C and LCT3-A, LCT3-U, LCT3-G, LCT3-C in the sense strand sequence are prepared using the corresponding phosphoramidite compounds as solid-phase synthesis raw materials.

[0182] Using a YB-192S synthesizer, a solid-phase synthesis method of phosphoramidite was employed. Starting with a solid support, nucleoside monomers were sequentially linked in the 3'-5' direction to achieve a synthesis scale of 0.2 μmol.

[0183] The phosphorus amide monomer is linked through a continuous cycle of four chemical reactions: deprotection, coupling, oxidation / sulfidation, and capping. The phosphorus amide monomer is prepared as a 0.05 M acetonitrile solution, with 0.3 M BTT in acetonitrile as the activator, a 3% trichloroacetic acid / dichloromethane solution as the deprotecting agent, a 0.05 M iodine / pyridine / tetrahydrofuran / aqueous solution (v / v / v = 2 / 1 / 7) as the oxidizing agent, acetic anhydride / acetonitrile solution as capping agent A (v / v = 2 / 8), pyridine / N-methylimidazolium / tetrahydrofuran solution as capping agent B (v / v / v = 10 / 16 / 74) as the capping agent, and a 0.05 M DDTT solution of pyridine / acetonitrile as the thiochemical agent (v / v = 4 / 6).

[0184] After solid-phase synthesis, the support was transferred to a 2 mL centrifuge tube, and 0.8 mL of concentrated ammonia was added. The mixture was then sealed and reacted at 55 °C or room temperature (25 °C) for 16 h. After cooling to room temperature, the solution was transferred to a 2 mL centrifuge tube and concentrated to dryness. After cooling to room temperature, the crude sequence was obtained by ethanol precipitation.

[0185] The crude product was purified by reversed-phase HPLC, and the collected fraction was lyophilized. Ethanol precipitation was performed by adding 0.3 mL of 1M sodium acetate solution and 0.9 mL of ethanol to replace the sequence with sodium salt. Then, desalting was carried out using a 3KD ultrafiltration tube to remove excess free salt.

[0186] The sense and antisense chains were prepared into an aqueous solution of a certain concentration. The sense and antisense chains were mixed at a molar ratio of 1:1.05, incubated at 95°C for 5 minutes, and then naturally cooled to room temperature. The product was then freeze-dried to obtain the target product.

[0187] For the MS or purity characterization results of the modified duplexes, please refer to Tables 3b and 3c:

[0188] 3b: MS detection results of modified double strands

[0189] 3c: HPLC purity test results of modified double strands

[0190] Example 2: In vitro activity detection - cell transfection

[0191] Cell culture and transfection

[0192] Cell culture: Huh7 cells were cultured at 37°C and 5% CO2 in MEM complete medium (Gibco, with 10% FBS added) until near confluence. Cells were then trypsinized and seeded into 96-well plates, with 6.0 × 10⁶ cells per well. 4 Cells were cultured in 90 μL of MEM complete medium at 37°C for 16-24 h before transfection.

[0193] Cell transfection: Diluted siRNA was mixed 1:1 with lipofectamine RNAiMax (Invitrogen), added to PCR tubes, and incubated at room temperature for 5 minutes. 10 μL of this siRNA mixture was then added to the cells, and the cells were cultured for another 24 hours before RNA extraction. Experiments were performed at 0.1 nM and 0.01 nM siRNA double-strand concentrations.

[0194] RNA extraction

[0195] Using 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 instructions for the RNA isolation kit. Finally, add 30 μL of RNase-free water, let stand for 2 minutes, and then centrifuge at 14000g for 2 minutes to collect RNA.

[0196] cDNA synthesis

[0197] cDNA synthesis was performed using the TransGold gDNA Removal and cDNA Synthesis Kit (TransGold 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) following the manufacturer's instructions.

[0198] Real-time quantitative PCR

[0199] Add the synthesized cDNA and the mixed stock solution (containing primers, qPCR premix and ultrapure water) to a 384-well plate (Bokcom Biosystems Cat#PC-0040-9U) to make the final real-time quantitative PCR system contain 0.25 μM each of the upstream and downstream primers of the target gene (ACVR1C) or the internal reference gene (GADPH) and 1×SYBR Green premix (Applied Biosystems Cat#A25742).

[0200] The ΔΔCt measurement method was used in ABI QuantStudio. TM 6. Perform real-time fluorescence PCR in a real-time fluorescence PCR system. Perform 3-4 independent transfection tests for each double-stranded strain, with 3-4 assays per transfection.

[0201] Table 4. Results of transfection test of modified double strands

[0202] Note:

[0203] A: Indicates an inhibition rate ≥80%.

[0204] B: Indicates 80% > Inhibition rate ≥ 60%

[0205] C: Indicates 60% > Inhibition rate ≥ 40%

[0206] D: Indicates 40% > Inhibition rate ≥ 10%

[0207] Example 3: In vivo activity test in hACVR1C single-transfer mice

[0208] Humanized ACVR1C mice aged 6–8 weeks were divided into groups of 4 mice each based on their average body weight. On Day 0, mice were administered the drug (conjugates listed in Table 3) subcutaneously at a dose of 3 mg / kg, with an equal volume of PBS injected as a control group. On Day 14, subcutaneous fat from the groin, perigonial fat, mesenteric fat, and brown fat from the scapula were collected. The hACVR1C mRNA levels in these different adipose tissues were detected using qPCR to compare the knockdown effects of different conjugates on the target gene.

[0209] Preliminary experimental results show that the conjugate prepared in this invention exhibits good knockdown effect on target genes in mice.

Claims

1. A small interfering RNA (siRNA) for inhibiting the expression of the activin A receptor type 1C (ACVR1C) gene in cells, wherein the siRNA comprises a sense strand and an antisense strand forming a double strand, wherein the antisense strand comprises at least 17 consecutive nucleotides that differ from the nucleotide sequence of any one of SEQ ID NO: 1236 to SEQ ID NO: 2470 by no more than 4 nucleotides, the antisense strand being 17 to 30 nucleotides in length; the sense strand being 17 to 30 nucleotides in length and at least partially complementary to the antisense strand.

2. The siRNA of claim 1, wherein: The antisense strand is 19 to 30 nucleotides in length; the sense strand is 19 to 30 nucleotides in length.

3. The siRNA of claim 1, wherein: The antisense strand differs from any of the nucleotide sequences shown in SEQ ID NO: 1236 to SEQ ID NO: 2470 by no more than 4 nucleotides.

4. The siRNA of claim 1, wherein: The sense strand and antisense strand have a mismatch of no more than 3 nucleotides.

5. The siRNA according to any one of claims 1 to 4, characterized in that: The siRNA sequence is selected from the double strands listed in Table 1.

6. The siRNA according to any one of claims 1 to 5, wherein: The dual siRNA contains at least one modified nucleotide.

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

8. The siRNA of claim 7, wherein: The modified nucleotide is selected from 2'-methoxynucleotides, 2'-fluoronucleotides, 2'-deoxynucleotides, 2',3'-cleaved nucleotide analogs, 2'-fluoro-arabinonucleotides, 2'-methoxyethylnucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, 3'-methoxynucleotides, 2'-allyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate groups, nucleotides containing 5'-phosphate analogs, diol-modified nucleotides, debased nucleotides, morpholinonucleotides, locked nucleotides, unlocked nucleotides, threonucleotides, or glycerol nucleotides.

9. The siRNA according to any one of claims 1 to 8, wherein: The 5' end and 3' end of the sense chain each independently contain 0, 1, 2 or 3 thiophosphate groups; and / or the 5' end and 3' end of the antisense chain each independently contain 1, 2 or 3 thiophosphate groups.

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 optionally comprises a phosphate ester or a phosphate ester analogue; Preferably, the phosphate ester analogue is 5'-vinyl phosphate.

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

12. The siRNA according to any one of claims 1 to 11, wherein: The siRNA is selected from the modified double-stranded compounds listed in Table 2.

13. The siRNA according to any one of claims 1-12, wherein the length of the double-stranded region is 17-30 nucleotide pairs, 19-25 nucleotide pairs, 19-23 nucleotide pairs, 23-27 nucleotide pairs, or 21-23 nucleotide pairs.

14. The siRNA according to any one of claims 1-13, wherein one strand has a 0-5 nucleotide overhang.

15. The siRNA according to any one of claims 1-14, wherein at least one long hydrocarbon chain is conjoined to the end or middle of any one chain of the siRNA; Preferably, the at least one long-chain hydrocarbon chain is conjugated with a nucleobase, a sugar moiety, or a phosphate ester bond between nucleosides.

16. The siRNA of claim 15, comprising the following long chain hydrocarbon chain conjugation structure:

17. The siRNA according to claim 15 or 16, selected from the siRNA conjugates listed in Table 3.

18. A pharmaceutical composition for inhibiting the expression of a target gene of activin A receptor type 1C (ACVR1C), said pharmaceutical composition comprising siRNA according to any one of claims 1-17 and a pharmaceutically acceptable carrier.

19. Use of the siRNA of any one of claims 1-17 or the pharmaceutical composition of claim 18 in the preparation of a medicament for treating and / or preventing pathological conditions or diseases associated with overexpression of the activin A receptor type 1C (ACVR1C) gene; Preferably, the pathological condition or disease is a metabolic disease and / or a cardiovascular disease; More preferably, the metabolic disease is obesity, type 2 diabetes mellitus (T2DM), insulin resistance, lipodystrophy, fatty liver disease, non-alcoholic fatty liver disease (NAFLD) or metabolic dysfunction-related fatty liver disease (MAFLD), non-alcoholic steatohepatitis (NASH) or metabolic dysfunction-related steatohepatitis (MASH); the cardiovascular disease is hyperlipidemia or dyslipidemia, atherosclerosis, cardiomyopathy, heart failure or coronary heart disease (CHD).

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