Double-stranded sirna, conjugate thereof and use thereof

Abstract

Provided are a double-stranded siRNA, a conjugate thereof and the use thereof. The siRNA comprises a sense strand and an antisense strand that form a reverse complementary double-stranded region, wherein the antisense strand comprises at least 15 consecutive nucleotides that bind to a target sequence. The siRNA or the conjugate thereof can effectively reduce ACVR1C mRNA and ALK7 protein levels in vitro and in vivo, while exhibiting low risks of off-target effects, immunogenicity and cytotoxicity. The siRNA or the conjugate thereof can be used in the treatment of ALK7 protein-related diseases, such as abdominal obesity, obesity, hyperlipidemia and T2D.

Description

A double-stranded siRNA, its conjugates and their applications

[0001] This application claims priority to Chinese Patent Application No. 202510376473.X, filed on March 27, 2025; Chinese Patent Application No. 202511113157.X, filed on August 8, 2025; Chinese Patent Application No. 202511881831.9, filed on December 12, 2025; and Chinese Patent Application No. 202610363110.7, filed on March 24, 2026, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention belongs to the field of nucleic acid drugs, specifically providing an siRNA that inhibits the expression of ALK7 (activin receptor-like kinase 7) and its conjugates, as well as pharmaceutical compositions thereof. The siRNA and its conjugates provided by this invention, as well as pharmaceutical compositions thereof, can treat and / or improve diseases related to ALK7. Background Technology

[0003] Obesity is characterized by a significant degree of overweight and an excessively thick layer of body fat, resulting from the excessive accumulation of body fat, especially triglycerides. Obesity not only affects a patient's physical health but is also closely related to the development of various chronic diseases, such as cardiovascular disease, diabetes, and certain cancers. With the increasing number of obese patients, the need for effective treatments is becoming increasingly urgent, necessitating the development of new treatment methods and technologies to address this global health challenge.

[0004] In recent years, GLP-1 agonists have been successfully proven to treat obesity, with semaglutide and telpoxetine clinically demonstrated to significantly reduce patient weight. However, GLP-1 agonists can easily cause gastrointestinal discomfort, decreased appetite, and muscle loss, and also suffer from poor tolerability and rebound weight gain after discontinuation. The market needs a novel weight-loss drug with a novel mechanism, fewer side effects, no muscle loss, and good tolerability and long-lasting effects.

[0005] ALK7 (activin receptor-like kinase 7) is a member of the TGF-β receptor superfamily, encoded by the ACVR1C gene, and is primarily expressed in cells (such as adipocytes) and tissues involved in endocrine functions. Recent studies have shown that inhibiting ACVR1C gene expression in adipocytes and reducing ALK7 protein levels can reduce body weight and fat content in DIO (Diet-Induced Obesity) mice without affecting their food intake or muscle mass. Therefore, reducing ALK7 protein in adipocytes is considered a potentially effective treatment for weight loss.

[0006] Meanwhile, the Loss-of-Function (LOF) study showed that people with ACVR1C gene defects have lower BMI-adjusted waist-to-hip ratios, healthier lipid phenotypes, lower rates of type 2 diabetes, and lower risk of cardiovascular disease.

[0007] RNA interference (RNAi) has been closely studied since its discovery in 1998. This mechanism achieves the desired pharmacological effect by downregulating the level of target mRNA, thereby controlling the function of the corresponding target. Double-stranded compounds that regulate target genes through this mechanism are called small interfering RNAs (siRNAs). In recent years, several siRNA drugs have been launched, fully demonstrating their potential for clinical treatment. Summary of the Invention

[0008] To address the technical problems existing in the prior art, this invention provides a method for preparing and applying siRNA and its conjugates that can effectively inhibit ACVR1C gene expression. The siRNA and its conjugates can effectively regulate the ACVR1C gene in adipocytes, thereby reducing the level of ALK7 protein and achieving the treatment of diseases related to ALK7 protein, such as abdominal obesity, obesity, hyperlipidemia, type 2 diabetes, and cardiovascular diseases.

[0009] 1. siRNA

[0010] In one aspect, a siRNA targeting the ACVR1C gene is provided, the siRNA comprising a sense strand and an antisense strand, the antisense strand comprising at least 15 consecutive nucleotides that are approximately 3 (e.g., 0, 1, 2, 3) nucleotides similar to a sequence or modified fragment thereof shown in any of SEQ ID NO:51-100, 222-233, 352, and 357, and the sense strand and antisense strand are at least partially complementary; the siRNA inhibits the expression level of the ACVR1C gene.

[0011] In some embodiments, the antisense strand of the siRNA comprises at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides or modified sequences thereof in any of the sequences shown in SEQ ID NO:51-100, 222-233, 352, and 357.

[0012] In some embodiments, the positive chain comprises at least 15 consecutive nucleotides having a nucleotide sequence of approximately 3 (e.g., 0, 1, 2, or 3) nucleotides, as shown in any of SEQ ID NO:1-50, 101, 210-221, and 358.

[0013] Preferably, the positive chain comprises at least 15 consecutive, at least 16, at least 17, at least 18, or at least 19 nucleotides or modified sequences thereof in any of the sequences shown in SEQ ID NO:1-50, 101, 210-221, and 358.

[0014] In some implementations, the sense and antisense strands of the siRNA each independently comprise 16-25 nucleotides in length;

[0015] Preferably, the sense and antisense strands of the siRNA each independently comprise 18-23 nucleotides (e.g., the sense strand is 18 or 21 nucleotides long, and the antisense strand is 23 nucleotides long).

[0016] In some embodiments, the siRNA comprises blunt ends and / or 1-3 (e.g., 1, 2, or 3) nucleotide overhangs;

[0017] Preferably, the siRNA contains one or two nucleotide overhangs;

[0018] Preferably, the protruding end is present at the 5' end and / or 3' end of the antisense chain and / or the justice chain;

[0019] Preferably, the 3' end of the antisense strand of the siRNA contains a 2- or 5-nucleotide overhang.

[0020] In some implementations, the sense strand of the siRNA has a mismatch of no more than 5 nucleotides (e.g., 0, 1, 2, 3, 4, or 5) with the antisense strand.

[0021] In some embodiments, the siRNA comprises a sense strand and an antisense strand forming an inverse complementary double-stranded region, the antisense strand comprising at least 15 consecutive nucleotides binding to a target sequence; the target sequence corresponds to positions 70-121, 404-427, 531-554, 597-619, 611-633, 680-702, 785-823, 853-881, 873-929, 904-936, 944-966, and 963-986 of the sequence shown in SEQ ID NO:209. Positions 985-1030, 997-1031, 1028-1090, 1072-1107, 1080-1120, 1110-1139, 1226-1253, 1300-1340, 1304-1325, 1303-1326, or 1304-1326.

[0022] In some embodiments, a siRNA targeting the ACVR1C gene is provided, comprising a sense strand and an antisense strand forming an inverse complementary double-stranded region, wherein the antisense strand is approximately equal to at least 3, 2, or 1 bases in at least 15 consecutive bases compared to a sequence or modified fragment thereof as shown in any of SEQ ID NO:51-100, 222-233, 352, and 357.

[0023] In some embodiments, the antisense strand is at least 80% complementary to the target sequence; and / or, the antisense strand suppresses the expression level of the ACVR1C gene.

[0024] In some embodiments, the antisense strand comprises at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides or modified sequences thereof in any of the sequences shown in SEQ ID NO:51-100, 222-233, 352, and 357.

[0025] In some embodiments, the positive chain comprises at least 15 consecutive, at least 17, at least 18, or at least 19 nucleotides or modified sequences thereof in any of the sequences shown in SEQ ID NO:1-50, 101, 210-221, and 358.

[0026] In some embodiments, the lengths of the sense and antisense strands are each independently comprised of 16-25 nucleotides; preferably, they are each independently comprised of 18-23 nucleotides. For example, the length of the sense strand is 18 or 21 nucleotides, and the length of the antisense strand is 23 nucleotides.

[0027] In some embodiments, the nucleotide sequence of the antisense strand is as shown in any of SEQ ID NO:51-100, 222-233, 352 and 357 or a modified sequence thereof; and / or, the nucleotide sequence of the sense strand is as shown in any of SEQ ID NO:1-50, 101, 210-221 and 358 or a modified sequence thereof.

[0028] In some embodiments, the target sequence corresponds to positions 787-823, 879-899, 999-1031, 1080-1120, or 1300-1340 of the sequence shown in SEQ ID NO:209.

[0029] In some embodiments, the siRNA comprises the sequences shown on the sense and antisense strands of any of the double strands from A1 to A66 in Table 1, or modified sequences thereof.

[0030] In some preferred embodiments of the present invention, the siRNA comprises the sequences shown in the sense and antisense strands of any double-stranded RNA selected from A4, A6, A7, A8, A16, A17, A25, A27, A33, A50, A52, A55, A64, and A65 in Table 1, or their modified sequences.

[0031] In some preferred embodiments of the present invention, the siRNA comprises the sequences shown in the sense and antisense strands of any double strand selected from A8, A51, A52, A64, and A65 in Table 1, or their modified sequences.

[0032] 2. Modification of siRNA

[0033] siRNAs can be modified in their nucleoside base structure or ribose-phosphate backbone to enhance their ability to inhibit target mRNA expression, reduce off-target effects, increase molecular biostability, increase the physical stability of the duplex formed between antisense and sense nucleic acids, and / or reduce immunogenicity. siRNA molecules containing ribonucleoside analogs or derivatives must retain the ability to form duplexes and allow or mediate specific degradation of target RNA via the RISC pathway.

[0034] In some embodiments of the invention, the modified sequences of the sense and antisense strands of the siRNA each independently contain at least one modified nucleotide. For each of the plurality of modified ribonucleotides in the siRNA, the modifications need not be identical.

[0035] 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.

[0036] In some embodiments of the present invention, the siRNA comprises 2'-modified nucleotides.

[0037] In some embodiments of the present invention, the modified sequences of the sense and antisense strands each independently comprise at least one modified nucleotide, said at least one modified nucleotide being selected from one or more of the group consisting of: deoxy-nucleotides, 3'-terminal deoxy-thymidine nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2 Nucleotides modified with '-C-alkyl-, 2'-hydroxy-, 2'-O-methoxyethyl, 2'-O-alkyl, morpholinyl, aminophosphate, nucleotides containing non-natural bases, tetrahydropyran-modified, 1,5-dehydrohexyl-modified, cyclohexenyl-modified, nucleotides containing thiophosphate, aminophosphate, methylphosphate, 5'-phosphate, 5'-phosphate mimics, and the following structures:

[0038] In some embodiments of the present invention, the first nucleotide at the 5' end of the modified sequence of the antisense strand contains a phosphate ester structure, preferably an alkenyl phosphate ester or a cyclopropyl phosphate ester structure.

[0039] In some embodiments of the present invention, the 5' and / or 3' ends of the modified sequence of the positive strand are connected to one or more inverse abase residues (invAb).

[0040] In some embodiments of the present invention, the sense strand and / or antisense strand of the siRNA comprises modified nucleoside linkages;

[0041] Preferably, the 5' end and 3' end of the sense chain each independently contain one or two thiophosphate groups; and / or the 5' end and 3' end of the antisense chain each independently contain one or two or three thiophosphate groups.

[0042] In some embodiments of the present invention, the above-described antisense strand modification mode and / or sense strand modification mode are applied to small interfering RNA (siRNA) for reducing ACVR1C mRNA and ALK7 protein levels.

[0043] In some embodiments of the present invention, the modified sequence of the antisense strand comprises at least 15 consecutive nucleotides that are approximately 3 (e.g., 0, 1, 2, or 3) similar to any of the sequences shown in SEQ ID NO:155-208, 264-304, 327-328, or 334-336.

[0044] Preferably, the modified sequence of the positive strand comprises at least 15 consecutive nucleotides that are approximately 3 (e.g., 0, 1, 2, or 3) similar to any of the sequences shown in SEQ ID NO:102-153, 234-263, 353-356, and 359.

[0045] Preferably, the sense strand is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the antisense strand. In some embodiments of the invention, the modified sequence of the antisense strand is approximately equal to at least 3, 2, or 1 bases in at least 15 consecutive bases compared to the sequence shown in any of SEQ ID NO: 155-208, 264-304, 327-328, or 334-336.

[0046] In some embodiments of the present invention, the modified sequence of the positive chain is approximately equal to at least 3, 2 or 1 bases in at least 15 consecutive bases compared to the sequence shown in any of SEQ ID NO:102-153, 234-263, 353-356 and 359.

[0047] In some embodiments of the present invention, the sequences of the sense and antisense strands of the siRNA are selected from any of the sense and antisense strand sequences of the modified siRNA duplexes listed in Table 2, namely B001-B094, B105, and B114-B142.

[0048] In some embodiments of the present invention, the sequences of the sense and antisense strands of the siRNA are selected from any of the sense and antisense strand sequences of the modified siRNA duplexes listed in Table 2, namely B004, B006-B008, B016, B017, B025, B027, B033, B050-B051, B117-B124, B141, and B142.

[0049] In some embodiments of the present invention, the sequences of the sense and antisense strands of the siRNA are selected from the sense and antisense strand sequences of any of the B118, B120, B122, B133-B139 modified siRNA duplexes as described in Table 2.

[0050] 3. siRNA conjugates

[0051] In one aspect, a conjugate (also known as a coupling compound) of siRNA is provided, comprising siRNA or a fragment thereof as described in the first or second aspect of the invention, modified siRNA or a fragment thereof, and a ligand conjugated to said siRNA or a fragment thereof, or modified siRNA or a fragment thereof.

[0052] In some implementations, one, two, or three ligands are attached to the sense chain and / or antisense chain; preferably, one ligand is attached to each end of the sense chain.

[0053] In some embodiments, the ligand is selected from: lipid groups, small molecule ligands and / or peptides, wherein the lipid group contains at least one lipophilic group containing 4-40 carbon atoms; and a ligand is attached to the 5' and / or 3' ends of the positive chain.

[0054] In some embodiments, the lipophilic group is covalently conjugated to the siRNA or a fragment thereof.

[0055] In some embodiments, the ligand is selected from one or more lipid groups shown in the following structures:

[0056] In some embodiments, the positive chain of the conjugate is connected to the ligand via a covalent bond or linker, wherein the covalent bond is selected from phosphate ester bonds, phosphothioester bonds, and thioether bonds.

[0057] In some embodiments, the positive chain of the conjugate is linked to the lipid group via a linker; the linker is, for example, C6-S, with the following structure:

[0058] In some implementations, the ligand is attached to the 5' end, 3' end, or middle position of the justice chain.

[0059] In some embodiments, the lipid group LC-207 is connected to the 5' end of the positive chain of the conjugate via a linker C6-S. Preferably, the lipid group LC-207 and the linker C6-S are connected by a covalent bond. More preferably, the covalent bond is a thioether bond.

[0060] In some embodiments, the lipid group LC-207 is connected to the 3' end of the positive chain of the conjugate via a linker C6-S. Preferably, the lipid group LC-207 and the linker C6-S are connected by a covalent bond. More preferably, the covalent bond is a thioether bond.

[0061] In some embodiments, the lipid group LC-207 is connected to the 5' end of the positive chain via linker C6-S, and the lipid group LC-044 is connected to the 3' end of the positive chain.

[0062] In some embodiments, the lipid group LC-044 is attached to the 5' end of the positive chain, and the lipid group LC-164 is attached to the 3' end of the positive chain.

[0063] In some embodiments, the lipid group LC-207 is connected to the 5' end of the positive chain via linker C6-S, and the lipid group LC-205 or LC-231 is connected to the 3' end of the positive chain.

[0064] In some embodiments, the lipid group LC-206 is connected to the 5' end of the positive chain via linker C6-S, and optionally, the lipid group LC-205 is connected to the 3' end of the positive chain;

[0065] In some embodiments, the lipid group LC-202 is attached to the 3rd nucleotide at the 5' end of the positive strand.

[0066] In some embodiments, the antisense strand of the conjugate is approximately equal to no more than 3, 2, or 1 bases in at least 15 consecutive bases compared to any of the sequences shown in SEQ ID NO:327-341.

[0067] In some embodiments, the positive chain of the conjugate has approximately 3, 2, or 1 bases in at least 15 consecutive bases compared to the sequences shown in any of SEQ ID NO:305-326 and 342-351.

[0068] In some embodiments, the conjugate comprises a sense strand as shown in SEQ ID NO:347 and an antisense strand as shown in SEQ ID NO:328;

[0069] The conjugate comprises a sense chain as shown in SEQ ID NO:343, and an antisense chain as shown in SEQ ID NO:327;

[0070] The conjugate comprises a sense chain as shown in SEQ ID NO:344, and an antisense chain as shown in SEQ ID NO:328;

[0071] The conjugate comprises a sense chain as shown in SEQ ID NO:345, and an antisense chain as shown in SEQ ID NO:329;

[0072] The conjugate comprises a sense chain as shown in SEQ ID NO:346, and an antisense chain as shown in SEQ ID NO:327;

[0073] The conjugate comprises a sense chain as shown in SEQ ID NO:348, and an antisense chain as shown in SEQ ID NO:329;

[0074] The conjugate comprises a sense chain as shown in SEQ ID NO:343, and an antisense chain as shown in SEQ ID NO:334;

[0075] The conjugate comprises a sense chain as shown in SEQ ID NO:347, and an antisense chain as shown in SEQ ID NO:335;

[0076] The conjugate comprises a sense chain as shown in SEQ ID NO:348, and an antisense chain as shown in SEQ ID NO:336;

[0077] The conjugate comprises a sense strand as shown in SEQ ID NO:350, and an antisense strand as shown in SEQ ID NO:335;

[0078] The conjugate comprises a sense chain as shown in SEQ ID NO:350, and an antisense chain as shown in SEQ ID NO:328;

[0079] The conjugate comprises a sense chain as shown in SEQ ID NO:305, and an antisense chain as shown in SEQ ID NO:327;

[0080] The conjugate comprises a sense chain as shown in SEQ ID NO:306, and an antisense chain as shown in SEQ ID NO:328;

[0081] The conjugate comprises a sense chain as shown in SEQ ID NO:307 and an antisense chain as shown in SEQ ID NO:329;

[0082] The conjugate comprises a sense chain as shown in SEQ ID NO:308, and an antisense chain as shown in SEQ ID NO:327;

[0083] The conjugate comprises a sense chain as shown in SEQ ID NO:309, and an antisense chain as shown in SEQ ID NO:328;

[0084] The conjugate comprises a sense chain as shown in SEQ ID NO:310, and an antisense chain as shown in SEQ ID NO:329;

[0085] The conjugate comprises a sense chain as shown in SEQ ID NO:311 and an antisense chain as shown in SEQ ID NO:329;

[0086] The conjugate comprises a sense chain as shown in SEQ ID NO:312, and an antisense chain as shown in SEQ ID NO:330;

[0087] The conjugate comprises a sense chain as shown in SEQ ID NO:313, and an antisense chain as shown in SEQ ID NO:331;

[0088] The conjugate comprises a sense chain as shown in SEQ ID NO:314, and an antisense chain as shown in SEQ ID NO:330;

[0089] The conjugate comprises a sense chain as shown in SEQ ID NO:315, and an antisense chain as shown in SEQ ID NO:331;

[0090] The conjugate comprises a sense chain as shown in SEQ ID NO:316, and an antisense chain as shown in SEQ ID NO:332;

[0091] The conjugate comprises a sense chain as shown in SEQ ID NO:317 and an antisense chain as shown in SEQ ID NO:333;

[0092] The conjugate comprises a sense chain as shown in SEQ ID NO:318, and an antisense chain as shown in SEQ ID NO:333;

[0093] The conjugate comprises a sense chain as shown in SEQ ID NO:319, and an antisense chain as shown in SEQ ID NO:327;

[0094] The conjugate comprises a sense chain as shown in SEQ ID NO:320, and an antisense chain as shown in SEQ ID NO:328;

[0095] The conjugate comprises a sense chain as shown in SEQ ID NO:321, and an antisense chain as shown in SEQ ID NO:329;

[0096] The conjugate comprises a sense chain as shown in SEQ ID NO:322, and an antisense chain as shown in SEQ ID NO:329;

[0097] The conjugate comprises a sense chain as shown in SEQ ID NO:323, and an antisense chain as shown in SEQ ID NO:332;

[0098] The conjugate comprises a sense chain as shown in SEQ ID NO:324, and an antisense chain as shown in SEQ ID NO:330;

[0099] The conjugate comprises a sense chain as shown in SEQ ID NO:325, and an antisense chain as shown in SEQ ID NO:331;

[0100] The conjugate comprises a sense chain as shown in SEQ ID NO:326, and an antisense chain as shown in SEQ ID NO:333;

[0101] The conjugate comprises a sense chain as shown in SEQ ID NO:305, and an antisense chain as shown in SEQ ID NO:334;

[0102] The conjugate comprises a sense strand as shown in SEQ ID NO:306, and an antisense strand as shown in SEQ ID NO:335;

[0103] The conjugate comprises a sense chain as shown in SEQ ID NO:307 and an antisense chain as shown in SEQ ID NO:336;

[0104] The conjugate comprises a sense chain as shown in SEQ ID NO:308, and an antisense chain as shown in SEQ ID NO:334;

[0105] The conjugate comprises a sense strand as shown in SEQ ID NO:309, and an antisense strand as shown in SEQ ID NO:335;

[0106] The conjugate comprises a sense chain as shown in SEQ ID NO:310, and an antisense chain as shown in SEQ ID NO:336;

[0107] The conjugate comprises a sense chain as shown in SEQ ID NO:311 and an antisense chain as shown in SEQ ID NO:336;

[0108] The conjugate comprises a sense chain as shown in SEQ ID NO:312, and an antisense chain as shown in SEQ ID NO:337;

[0109] The conjugate comprises a sense chain as shown in SEQ ID NO:313, and an antisense chain as shown in SEQ ID NO:338;

[0110] The conjugate comprises a sense chain as shown in SEQ ID NO:314, and an antisense chain as shown in SEQ ID NO:337;

[0111] The conjugate comprises a sense chain as shown in SEQ ID NO:315, and an antisense chain as shown in SEQ ID NO:338;

[0112] The conjugate comprises a sense chain as shown in SEQ ID NO:316, and an antisense chain as shown in SEQ ID NO:339;

[0113] The conjugate comprises a sense chain as shown in SEQ ID NO:317, and an antisense chain as shown in SEQ ID NO:340;

[0114] The conjugate comprises a sense chain as shown in SEQ ID NO:318, and an antisense chain as shown in SEQ ID NO:340;

[0115] The conjugate comprises a sense chain as shown in SEQ ID NO:319, and an antisense chain as shown in SEQ ID NO:334;

[0116] The conjugate comprises a sense chain as shown in SEQ ID NO:320, and an antisense chain as shown in SEQ ID NO:335;

[0117] The conjugate comprises a sense chain as shown in SEQ ID NO:321 and an antisense chain as shown in SEQ ID NO:336;

[0118] The conjugate comprises a sense chain as shown in SEQ ID NO:322, and an antisense chain as shown in SEQ ID NO:336;

[0119] The conjugate comprises a sense chain as shown in SEQ ID NO:323, and an antisense chain as shown in SEQ ID NO:341;

[0120] The conjugate comprises a sense chain as shown in SEQ ID NO:326, and an antisense chain as shown in SEQ ID NO:340;

[0121] The conjugate comprises a sense chain as shown in SEQ ID NO:342, and an antisense chain as shown in SEQ ID NO:328;

[0122] The conjugate comprises a sense chain as shown in SEQ ID NO:344, and an antisense chain as shown in SEQ ID NO:335;

[0123] The conjugate comprises a sense chain as shown in SEQ ID NO:345, and an antisense chain as shown in SEQ ID NO:336;

[0124] The conjugate comprises a sense chain as shown in SEQ ID NO:346, and an antisense chain as shown in SEQ ID NO:334;

[0125] The conjugate comprises a sense chain as shown in SEQ ID NO:349, and an antisense chain as shown in SEQ ID NO:328;

[0126] The conjugate comprises a sense strand as shown in SEQ ID NO:349, and an antisense strand as shown in SEQ ID NO:335;

[0127] The conjugate comprises a sense chain as shown in SEQ ID NO:350, and an antisense chain as shown in SEQ ID NO:328;

[0128] The conjugate comprises a sense chain as shown in SEQ ID NO:351 and an antisense chain as shown in SEQ ID NO:327;

[0129] The conjugate comprises a sense chain as shown in SEQ ID NO:351 and an antisense chain as shown in SEQ ID NO:334.

[0130] 4. Delivery of the composition

[0131] The siRNA of the present invention may be delivered or introduced by any means known in the art (e.g., in vitro delivery or introduction into cells, or in vivo delivery or introduction into a patient). For example, for in vivo delivery, the siRNA may be injected into a tissue site or administered systemically. In vivo delivery may also be performed via a β-glucan delivery system. In vitro introduction into cells includes methods known in the art, such as electroporation, lipid transfection, and receptor-mediated endocytosis.

[0132] In some embodiments, the delivery composition comprises the siRNA or siRNA conjugate described in this invention, and optionally, further comprises a delivery vector.

[0133] In some embodiments, the delivery vector is selected from: liposomes, lipid nanoparticles, polymer nanoparticles, exosomes, viral vectors, inorganic nanoparticles, bioconjugates; and / or, the siRNA molecule is linked to the delivery vector.

[0134] In some embodiments, the delivery vector is selected from lipid carriers, lipid-like carriers, coupling ligands, polymers, and exosome carriers.

[0135] In some embodiments, the coupling ligand is selected from carbohydrates, peptides, antibodies, aptamers, and small molecules; the lipid carrier is selected from LNP, cholesterol, Dlin-DMA / MC3-DMA; the lipid carrier is selected from nanoparticles (e.g., endoplasmic reticulum membrane-modified nanoparticles, polymer nanoparticles (PNP), lipid polypolymers (LPP)), PEI particles, PLGA particles, preferably inorganic nanoparticles; the polymer is a dendritic molecule, such as PBAVE polymer, DPC1.0, or DPC2.0; the reverse complementary double-stranded region is connected to the delivery carrier via a linker with monovalent, divalent, or trivalent branches; preferably, the 3' end of the positive strand of the reverse complementary double-stranded region is connected to the delivery carrier via a linker.

[0136] In some embodiments, the carbohydrate is selected from sugars, fatty acid oligosaccharides, and polysaccharides, wherein the oligosaccharides preferably include monosaccharides, disaccharides, trisaccharides, and tetrasaccharides; the monosaccharides are preferably GalNAc, such as L96; and the polypeptide is an RGD polypeptide.

[0137] In some embodiments, the delivery methods include, but are not limited to, viral delivery (retrovirus, adenovirus, lentivirus, baculovirus, AAV); liposomes (Lipofectamine, cationic DOTAP, neutral DOPC); nanoparticles (cationic polymers, PEI); bacterial delivery (tkRNAi); lipid nanoparticles (LNP); neutral liposomes (NL); polymer nanoparticles (low molecular weight polymers or high molecular weight polymers); double-stranded RNA binding motifs (dsRBMs); and other delivery systems known in the art that are suitable for nucleic acid or oligonucleotide delivery.

[0138] 5. Composition

[0139] This invention provides a nucleic acid protein composition comprising siRNA, modified siRNA, siRNA conjugate, or the delivery composition described herein, and a nuclease; the nuclease is preferably an AGO protein.

[0140] Alternatively, the nucleic acid protein composition comprises an antisense strand of siRNA as described in the first or second aspect of the present invention, and a nuclease; the nuclease is preferably an AGO protein.

[0141] 6. Recombinant vector

[0142] The present invention provides a recombinant vector comprising the coding sequence of siRNA or modified siRNA as described in the first or second aspect of the present invention.

[0143] In some implementations, the starting vector of the recombinant vector is a plasmid or a viral vector.

[0144] 7. Transformation

[0145] The present invention provides a transformant comprising siRNA, modified siRNA, or recombinant vector as described in the first or second aspect of the present invention, or as described in the sixth aspect of the present invention; the host cell of the transformant is a eukaryotic cell or a prokaryotic cell.

[0146] In some implementations, the transformant is neither a plant variety nor an animal variety.

[0147] 8. Preparation method

[0148] The present invention provides a method for preparing siRNA as described in the first aspect of the present invention, the method comprising culturing a transformant as described in the seventh aspect of the present invention, or directly obtaining the nucleic acid molecule by means of chemical synthesis and mixing.

[0149] 9. Pharmaceutical Composition

[0150] The present invention provides a pharmaceutical composition comprising siRNA as described in the first or second aspect of the present invention, modified siRNA, conjugate as described in the third aspect of the present invention, delivery composition as described in the fourth aspect of the present invention, recombinant vector as described in the sixth aspect of the present invention, or transformant as described in the seventh aspect of the present invention, and pharmaceutically acceptable carriers and / or excipients.

[0151] In some embodiments, the pharmaceutical composition contains at least one of the siRNAs, modified siRNAs, or siRNA conjugates described above.

[0152] In some embodiments, the pharmaceutical composition contains at least two of the siRNAs, modified siRNAs, or siRNA conjugates (e.g., but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) as active ingredients. Each of the at least two siRNAs targets a different target sequence in the ACVR1C gene, thereby potentially resulting in a synergistic effect by acting simultaneously on different target sequences. Here, "different target sequences" refers to target sequences that do not overlap, or where the number of overlapping consecutive nucleotides between target sequences is less than 5 (e.g., 4, 3, 2, 1, or 0 overlapping consecutive nucleotides). In this case, the at least two of the above-described siRNAs can be present in any different proportions.

[0153] In some embodiments, the pharmaceutical composition comprises an effective amount of siRNA. "Effective amount" refers to the amount of siRNA that is effective in producing the desired pharmacological, therapeutic, or preventative outcome. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or condition is reduced by at least 10%, then the therapeutically effective amount of a drug used to treat that disease or condition is the amount required to achieve that at least 10% reduction in the parameter. For example, a therapeutically effective amount of siRNA targeting ACVR1C can reduce ACVR1C mRNA levels by at least 10%.

[0154] In some implementations, the carrier is water, saline solution, or a buffer solution.

[0155] In some embodiments, the buffer solution contains a buffering agent including acetate, citrate, alcohol-soluble gluten, carbonate or phosphate, Tris-hydrochloric acid or any combination thereof; for example, phosphate.

[0156] In some embodiments, the pharmaceutical compositions of the present invention are formulated into dosage forms compatible with their intended route of administration, such as local administration (e.g., direct injection or implantation), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral administration, including intracranial (e.g., intraventricular, intradural, or intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.

[0157] In some embodiments, the pharmaceutical composition is administered by inhalation, intranasal administration, intratracheal administration, or oropharyngeal inhalation. Formulations suitable for inhalation can be prepared by incorporating the desired amount of the active ingredient into a suitable solvent, followed by sterile filtration. Typically, formulations for inhalation are sterile solutions at physiological pH and have low viscosity. Salts may be added to the formulation to balance surface tension. In some cases, surfactants or co-solvents may be added to increase the solubility of the active ingredient and improve aerosol properties. In some cases, excipients may be added to control viscosity to ensure the size and distribution of atomized droplets.

[0158] In some embodiments, the pharmaceutical composition can be administered by injection, such as intravenous, intramuscular, subcutaneous, intradermal, intra-articular, intraocular, intraperitoneal, or local administration. Suitable pharmaceutical compositions for injectable use include sterile aqueous solutions or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, or phosphate-buffered saline (PBS). The pharmaceutical composition should remain stable under manufacturing and storage conditions and should be protected against contamination by microorganisms such as bacteria and fungi. For example, a sterile injectable solution can be prepared by incorporating the required dose of the active ingredient into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives, or any combination thereof), followed by sterile filtration. Alternatively, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze-drying) for easy storage and use.

[0159] The siRNA, modified siRNA, and siRNA conjugates of the present invention can be formulated in dosage units for easy administration. Dosage unit form refers to physically discrete units suitable for use as a single dose in a subject to be treated; each unit contains a predetermined amount of the active ingredient, calculated to produce the desired therapeutic effect when combined with a desired drug carrier.

[0160] In this invention, the dosing regimen can be adjusted to obtain the optimal target response (e.g., treatment or prevention). For example, it can be administered as a single dose, multiple times over a period of time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.

[0161] 10. Pill Set

[0162] The present invention provides a kit comprising a kit A containing a reagent kit, the kit comprising one or more of the following: siRNA as described in the first or second aspect of the present invention, modified siRNA, conjugate as described in the third aspect of the present invention, delivery composition as described in the fourth aspect of the present invention, recombinant vector as described in the sixth aspect of the present invention, transformant as described in the seventh aspect of the present invention, or pharmaceutical composition as described in the ninth aspect of the present invention.

[0163] In some embodiments, the pillbox further includes a pillbox B, which contains one or more of the following:

[0164] (1) Other drugs that inhibit ACVR1C gene expression or compositions containing said drugs that inhibit ACVR1C gene expression; and

[0165] (2) One or more of the following groups: hormone preparations, targeted small molecule preparations, proteasome inhibitors, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0166] 11. Application

[0167] 11.1 Treatment of ACVR1C gene expression-related diseases

[0168] The present invention provides the use of siRNA, modified siRNA, conjugate as described in the first or second aspect of the present invention, delivery composition as described in the fourth aspect of the present invention, recombinant vector as described in the sixth aspect of the present invention, transformant as described in the seventh aspect of the present invention, or pharmaceutical composition as described in the ninth aspect of the present invention in the preparation of a medicament for diseases related to ACVR1C gene expression.

[0169] In some embodiments, the diseases associated with ACVR1C gene expression are selected from one or more of the following: metabolic diseases, cardiovascular diseases, liver diseases, kidney diseases, endocrine diseases, and tumor diseases. The metabolic diseases are selected from obesity (e.g., abdominal obesity), overweight, insulin resistance, type 2 diabetes (T2D), metabolic syndrome, lipid disorders (such as hyperlipidemia, hypertriglyceridemia, or dyslipidemia), lipoprotein disorders, and impaired glucose tolerance (IGT); the cardiovascular diseases are selected from heart diseases (such as heart disease including coronary heart disease, congestive heart failure), stroke, peripheral vascular disease, atherosclerosis, arteriosclerosis, and hypertension; the liver diseases are selected from inflammation (e.g., liver inflammation and / or adipose tissue inflammation) and fatty liver disease (including non-alcoholic fatty liver disease); the kidney diseases are selected from nephropathy (e.g., nephropathy); the endocrine diseases are selected from pancreatitis; and the tumor diseases are selected from cancers (e.g., myeloma (e.g., multiple myeloma, plasmacytoma, localized myeloma, or extramedullary myeloma), melanoma, ovarian cancer, breast cancer, colon cancer, endometrial cancer, liver cancer, kidney cancer, pancreatic cancer, gastric cancer, uterine cancer, or colon cancer).

[0170] 11.2 Suppressing ACVR1C gene expression

[0171] The present invention provides a method for inhibiting the expression level of the ACVR1C gene in vitro or in vivo, the method comprising administering one or more of the following to an in vivo or in vitro sample: siRNA as described in the first or second aspect of the present invention, modified siRNA conjugates as described in the third aspect of the present invention, delivery compositions as described in the fourth aspect of the present invention, pharmaceutical compositions as described in the ninth aspect of the present invention, and kits as described in the tenth aspect of the present invention, to reduce the expression level of the ACVR1C gene.

[0172] In some implementations, the method is for non-therapeutic purposes.

[0173] 12. Methods of treating diseases

[0174] The present invention provides a method for inhibiting the gene expression level of ACVR1C in a subject or sample or for treating ACVR1C gene expression-related diseases, the method comprising: step (a) contacting the cells of the subject or sample with an effective amount of one or more of the following: siRNA as described in the first or second aspect of the present invention, conjugates as described in the third aspect of the present invention, delivery compositions as described in the fourth aspect of the present invention, pharmaceutical compositions as described in the ninth aspect of the present invention, and kits as described in the tenth aspect of the present invention.

[0175] In some embodiments, the method further includes maintaining the contact described in step (a) for a period of time to allow for the degradation of the ACVR1C gene mRNA transcript, thereby inhibiting the expression level of the ACVR1C gene in the subject or sample.

[0176] In some embodiments, the diseases associated with ACVR1C gene expression are selected from one or more of the following: metabolic diseases, cardiovascular diseases, liver diseases, kidney diseases, endocrine diseases, and tumor diseases. The metabolic diseases are selected from obesity (e.g., abdominal obesity), overweight, insulin resistance, type 2 diabetes (T2D), metabolic syndrome, lipid disorders (such as hyperlipidemia, hypertriglyceridemia, or dyslipidemia), lipoprotein disorders, and impaired glucose tolerance (IGT); the cardiovascular diseases are selected from heart diseases (such as heart disease including coronary heart disease, congestive heart failure), stroke, peripheral vascular disease, atherosclerosis, arteriosclerosis, and hypertension; the liver diseases are selected from inflammation (e.g., liver inflammation and / or adipose tissue inflammation) and fatty liver disease (including non-alcoholic fatty liver disease); the kidney diseases are selected from nephropathy (e.g., nephropathy); the endocrine diseases are selected from pancreatitis; and the tumor diseases are selected from cancers (e.g., myeloma (e.g., multiple myeloma, plasmacytoma, localized myeloma, or extramedullary myeloma), melanoma, ovarian cancer, breast cancer, colon cancer, endometrial cancer, liver cancer, kidney cancer, pancreatic cancer, gastric cancer, uterine cancer, or colon cancer).

[0177] The present invention provides one or more of the following: siRNA as described in the first or second aspect of the present invention, conjugates as described in the third aspect of the present invention, delivery compositions as described in the fourth aspect of the present invention, pharmaceutical compositions as described in the ninth aspect of the present invention, and kits as described in the tenth aspect of the present invention, for inhibiting the gene expression level of ACVR1C or treating ACVR1C gene expression-related diseases.

[0178] In some embodiments, the diseases associated with ACVR1C gene expression are selected from one or more of the following: metabolic diseases, cardiovascular diseases, liver diseases, kidney diseases, endocrine diseases, and tumor diseases. The metabolic diseases are selected from obesity (e.g., abdominal obesity), overweight, insulin resistance, type 2 diabetes (T2D), metabolic syndrome, lipid disorders (such as hyperlipidemia, hypertriglyceridemia, or dyslipidemia), lipoprotein disorders, and impaired glucose tolerance (IGT); the cardiovascular diseases are selected from heart diseases (such as heart disease including coronary heart disease, congestive heart failure), stroke, peripheral vascular disease, atherosclerosis, arteriosclerosis, and hypertension; the liver diseases are selected from inflammation (e.g., liver inflammation and / or adipose tissue inflammation) and fatty liver disease (including non-alcoholic fatty liver disease); the kidney diseases are selected from nephropathy (e.g., nephropathy); the endocrine diseases are selected from pancreatitis; and the tumor diseases are selected from cancers (e.g., myeloma (e.g., multiple myeloma, plasmacytoma, localized myeloma, or extramedullary myeloma), melanoma, ovarian cancer, breast cancer, colon cancer, endometrial cancer, liver cancer, kidney cancer, pancreatic cancer, gastric cancer, uterine cancer, or colon cancer).

[0179] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0180] The reagents and raw materials used in this invention are all commercially available.

[0181] The positive and progressive effects of this invention are as follows:

[0182] The compounds provided by this invention can effectively reduce ACVR1C mRNA levels in vitro and in vivo. In humanized mouse models, the compounds of this invention can effectively reduce the expression of the hALK7 gene in various adipose tissues, exhibiting stable and excellent inhibitory effects. They have low off-target risk, immunogenicity risk, and cytotoxicity risk, while also exhibiting excellent pharmacokinetic performance. They can be applied to treat diseases related to ALK7 protein, such as abdominal obesity, obesity, hyperlipidemia, and type 2 diabetes.

[0183] Terminology Definitions and Explanations

[0184] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below:

[0185] In this invention, unless otherwise specified, “G,” “C,” “A,” “T,” and “U” refer to guanine ribonucleotide, cytosine ribonucleotide, adenine ribonucleotide, thymine ribonucleotide, and uracil ribonucleotide, respectively. However, it should be understood that the terms “ribonucleotide,” “nucleotide,” or “deoxyribonucleotide” can also refer to a modified nucleotide (as further detailed below) or an alternative substitution. Those skilled in the art will readily recognize that guanine, cytosine, adenine, and uracil can be substituted with other portions without substantially altering the base-pairing properties of an oligonucleotide (including a nucleotide having such a substitution). For example, and not limited to, a nucleotide containing inosine as its base can base-pair with a nucleotide containing adenine, cytosine, or uracil. Therefore, a nucleotide containing uracil, guanine, or adenine can be substituted in the nucleotide sequence of this disclosure by a nucleotide containing, for example, inosine. Sequences containing such substitution portions are embodiments of this disclosure.

[0186] In this invention, the "d" mark before a nucleotide (A, U, C, G, and T, etc.) indicates that the nucleotide is 2'-deoxygenated.

[0187] In this invention, the label “f” after a nucleotide (A, U, C, G, and T, etc.) indicates that the nucleotide is modified by 2'-fluorine (2'-F modification).

[0188] In this invention, the prefix "GNA-" before nucleotides (A, U, C, G, and T, etc.) indicates that the nucleotide has been modified with ethylene glycol nucleic acid (GNA modification). In this invention, Tgn is the code for GNA-T, and the meanings are equivalent.

[0189] In this invention, the "m" mark after the nucleotide (A, U, C, G, and T, etc.) indicates that the nucleotide is modified by 2'-methoxyribose (2'-O-methyl).

[0190] In this invention, the "s" marking between nucleotides (A, U, C, G, and T, etc.) indicates that the two nucleotides are linked by a phosphate thioester bond (i.e., a phosphate thioester bond), meaning they are modified with phosphate thioester (PS). In this invention, the absence of the "s" marking between nucleotides (A, U, C, G, and T, etc.) indicates that the two nucleotides are linked by a phosphate ester bond (i.e., a phosphate diester bond).

[0191] The terms "double-stranded RNAi agent," "small interfering RNA," or "siRNA" refer to small interfering RNA (siRNA) molecules. It is a class of double-stranded RNA molecules, also known in the art as short interfering RNA or silent RNA. siRNA typically comprises a sense strand (also called a guest strand) and an antisense strand (also called a leader strand), each strand being 15 to 30 nucleotides in length, typically 18 to 25 nucleotides in length. The antisense strand is complementary to the target nucleic acid (suitably a mature mRNA sequence) (e.g., at least 80%, 85%, 90%, or 95% complementarity, such as 100% complete complementarity), and the sense strand is complementary to the antisense strand, such that the sense and antisense strands form a double-stranded structure or a double-stranded region. The siRNA strands may form blunt-ended double-stranded structures, or preferably, the 3′ ends of the sense and antisense strands may form 3′ overhangs, such as one, two, or three nucleotides, similar to the products produced by Dicer, which can form RISC substrates in vivo. In some embodiments, both the sense and antisense strands have a 2nt 3′ overhang. Therefore, the length of the double-stranded region can be, for example, 17 to 25 nucleotides, such as 21 to 23 nucleotides in length.

[0192] The term "antisense strand" refers to the strand of siRNA that includes a region substantially complementary to the target sequence. As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., the target sequence). When the complementary region is not perfectly complementary to the target sequence, mismatches can occur within the molecule or in terminal regions. Typically, the most permissible mismatches are in terminal regions, for example, within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends.

[0193] When used herein, the term "sense chain" refers to the chain of siRNA that includes regions substantially complementary to the regions of the antisense chain (as defined herein).

[0194] As used herein, the term "ACVR1C-encoded protein" specifically refers to ALK7 (activin receptor-like kinase 7), and the term refers to the corresponding gene, encoding mRNA, encoding protein / peptide, and functional fragment thereof. The human ACVR1C gene is preferred. The term "ACVR1C gene / sequence" refers not only to the wild-type sequence but also to mutations and alterations that may be included in said gene / sequence. Therefore, the invention is not limited to the specific siRNA molecules provided herein. The invention also relates to siRNA molecules comprising an antisense strand that is at least 85% complementary to a corresponding nucleotide segment of an RNA transcript comprising a mutated / altered ACVR1C gene.

[0195] As used herein, “target sequence” refers to a continuous portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the ACVR1C gene, including the mRNA processed from the primary transcription product.

[0196] As used herein and unless otherwise stated, the term "complementary" when used to describe a first nucleotide sequence associated with a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide including the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide including the second nucleotide sequence under certain conditions and form a double-stranded structure. As used herein, a "complementary" sequence may also include non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, or formed entirely from them, provided that the conditions for its hybridization ability described above are met.

[0197] A “fully complementary” sequence includes an oligonucleotide or polynucleotide containing a first nucleotide sequence and an oligonucleotide or polynucleotide containing a second nucleotide sequence, with base pairing along the full length of the first and second nucleotide sequences.

[0198] However, when the first sequence is referred to as “substantially complementary” with respect to the second sequence in this document, the two sequences may be completely complementary, or they may form one or more, but preferably no more than 13 mismatched base pairs after hybridization.

[0199] The terms “complementary,” “fully complementary,” and “substantially complementary” can be used herein to refer to base pairing between the sense and antisense strands of the siRNA, or between the antisense strand of the siRNA and the target sequence, as can be understood from the context in which they are used. In some embodiments herein, a first nucleotide sequence can be considered complementary to a second nucleotide sequence if the first nucleotide sequence exhibits sequence complementarity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. In one exemplary embodiment, 18 out of 20 nucleobases of the first nucleotide sequence pair with the corresponding region of the second nucleotide sequence, achieving 90% complementarity.

[0200] The nucleic acid molecules of the present invention can also be chemically modified and can be synthesized and / or modified by methods well known in the art, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. Modifications include, but are not limited to: terminal modifications, such as 5′-terminal modifications (phosphorylation, coupling, reverse linkage, etc.) or 3′-terminal modifications (coupling, DNA nucleotides, reverse linkage, etc.); base modifications, such as substitution with a stable base, an unstable base, or a base that pairs with more partners, base removal (base-free nucleotides), or coupled bases; sugar modifications (e.g., at the 2′ or 4′ position) or sugar substitution; or backbone modifications, including modification or substitution of phosphodiester bonds. Specific examples of siRNAs described in this invention include, but are not limited to, RNAs containing a modified backbone or without natural nucleoside linkages. RNAs containing a modified backbone include, but are not limited to, RNAs whose backbone does not contain phosphorus atoms. For the purposes of this specification, and as sometimes mentioned in the art, modified RNA without phosphorus atoms in its internucleotide backbone may also be considered an oligonucleotide. In some embodiments, the modified siRNA reagent will contain phosphorus atoms in its internucleotide backbone.

[0201] Modified RNA backbones include, but are not limited to: thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkyl phosphates (including 3′-alkyl phosphates and chiral phosphates), phosphoimine esters, phosphatases (including 3′-aminophosphatases and aminoalkyl phosphatases), thiophosphatases, thioalkyl phosphates, thioalkyl phosphate triesters, boran phosphates, etc., which have normal 3′-5′ linkages, 2′-5′ linkages, analogs, and those with reverse polarity, wherein adjacent nucleoside units are linked by 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts are also included, such as sodium salts, mixed salts, and free acid forms.

[0202] Modified RNA backbones that do not contain phosphorus atoms are formed by linkages between short-chain alkyl or cycloalkyl nucleosides, between mixed heteroatoms and alkyl or cycloalkyl nucleosides, or between one or more short-chain heteroatoms or heterocyclic nucleosides. These include morpholino linkages partially formed from the sugar moieties of nucleosides; siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formyl acetate and thioformyl acetate backbones; methylformyl acetate and thioformyl acetate backbones; olefin-containing backbones; sulfonamide backbones; methylimine and methylhydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones containing mixed N, O, S, and CH2 components.

[0203] In other embodiments, the present invention also considers the use of RNA mimics as RNAi reagents, wherein the sugar and nucleoside links (i.e., backbones) of the nucleotide units are replaced with new groups, while the base units are retained for hybridization with suitable nucleic acid targets. One RNA mimic exhibiting excellent hybridization properties is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The bases are retained and are directly or indirectly bound to the nitrogen atom of the amide moiety. Representative U.S. patents teaching methods for preparing PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of which are incorporated herein by reference. Other PNA compounds suitable for the RNAi reagents of the present invention are described in Nielsen et al. (Science, 1991, 254, 1497-1500).

[0204] Some embodiments of the present invention include RNA with a phosphate thioester backbone and oligonucleotides with a heteroatom backbone, particularly -CH2-NH-CH2-, -CH2-N(CH3)-O-CH- [referred to as the methylimine (MMI) backbone], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- (US Patent No. 5,489,677), and the amide backbone of US Patent No. 5,602,240. In some embodiments, the RNA involved in the present invention has the morpholine backbone structure of US Patent No. 5,034,506. The native phosphate diester backbone can be represented as OP(O)(OH)-OCH2-.

[0205] The modified RNA may also contain one or more substituted sugar groups. The RNAi reagents involved in this invention (e.g., oligonucleotides, dsRNA, siRNA) may contain the following groups at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-ynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and ynyl groups may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and ynyl groups. Exemplary suitable modifications include O[(CH2)] n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, wherein n and m are 1 to about 10. In other embodiments, the dsRNA contains the following groups at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkylaryl, aralkyl, O-alkylaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl, heterocyclic aralkyl, aminoalkylamino, polyalkylamino, substituted silyl, RNA restriction enzyme group, reporter group, group that improves the pharmacokinetic properties of the RNAi reagent or the pharmacodynamic properties of the RNAi reagent, and other substituents with similar properties. In some embodiments, the modification includes 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxyalkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (also known as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O-CH2-O-CH2-N(CH2)2. Further exemplary modifications include: 5′-Me-2′-F nucleotide, 5′-Me-2′-OMe nucleotide, 5′-Me-2′-deoxynucleotide (R and S enantiomers of these three families); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

[0206] Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2), 2′-O-hexadecyl, and 2′-fluoro (2′-F). Similar modifications can also be made at other positions of the RNA in the RNAi reagent, particularly at the 3′ position of the sugar of the 3′-terminal nucleotide or in 2′-5′ linked dsRNAs, and at the 5′ position of the 5′-terminal nucleotide. RNAi reagents can also contain sugar mimics, such as replacing the pentofuranose with a cyclobutyl group. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. patents 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, some of which are co-owned with this application. The entire contents of each of the foregoing patents are incorporated herein by reference.

[0207] The RNAi reagent of the present invention may also contain nucleoside base modifications or substitutions (generally referred to in the art simply as "bases"). As used herein, "unmodified" or "natural" nucleoside bases include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleoside bases include other synthetic and natural nucleoside bases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl adenine and guanine derivatives, 2-propyl and other alkyl adenine and guanine derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halogenated uracil and cytosine, 5-propynyluracil and cytosine, 6-azauracil, and cytosine. And thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated, especially 5-bromine, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deadenine and 7-deadenine, and 3-deadenine and 3-deadenine. Further nucleoside bases include those disclosed in the following literature: U.S. Patent No. 3,687,808; *Modified Nucleosides in Biochemistry, Biotechnology and Medicine* (Herdewijn, P., ed., Wiley-VCH, 2008); *The Concise Encyclopedia Of Polymer Science and Engineering* (pp. 858-859, Kroschwitz, JL, ed., John Wiley & Sons, 1990); disclosed by Englisch et al. (1991) in *Angewandte Chemie, International Edition*, 30:613; and disclosed by Sanghvi, YS in *dsRNA Research and Applications* (Chapter 15, pp. 289-302, Crooke, ST. and Lebleu, B., eds., CRC Press, 1993). Certain of these nucleoside bases are particularly suitable for increasing the binding affinity of the oligomeric compounds involved in this invention. These include 5-substituted pyrimidines, 6-azapurines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-Methylcytosine substitution has been shown to increase the stability of nucleic acid double strands by 0.6–1.2 °C (Sanghvi, YS; Crooke, ST; and Lebleu, B., eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276–278), and is an exemplary base substitution, particularly when combined with 2′-O-methoxyethyl sugar modification.

[0208] The RNAi reagent of the present invention can also be modified to include one or more bicyclic sugar groups. "Bicyclic sugar" refers to a ring formed by a furanose ring connecting two carbon atoms (whether adjacent or not) via a bridging ring. "Bicyclic nucleoside" (BNA) refers to a bicyclic system formed by a sugar moiety connecting two carbon atoms (whether adjacent or not) via a bridging ring. In some embodiments, the bridging ring connects the 4′-carbon and 2′-carbon of the sugar ring, optionally via a 2′-acyl oxygen atom. Therefore, in some embodiments, the reagent of the present invention may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide whose modified ribose moiety includes an additional bridging ring connecting the 2′ and 4′ carbon atoms. In other words, an LNA is a nucleotide containing a bicyclic sugar group comprising a 4′-CH2-O-2′ bridging ring. This structure effectively "locks" the 3′-inward conformation of the ribose. Adding locking nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Exemplary bicyclic nucleotides of the oligonucleotides involved in this invention include, but are not limited to, nucleotides containing a 4′ to 2′ sugar ring bridge. In some embodiments, the antisense oligonucleotide reagent of this invention comprises one or more bicyclic nucleotides containing a 4′ to 2′ bridge.

[0209] In this invention, tautomerism refers to the phenomenon where a functional group in a compound changes its structure to become an isomer of another functional group, and these two isomers can rapidly interconvert. This rapid and reversible conversion process allows the two isomers to coexist in a certain proportion under given conditions, forming a dynamic equilibrium state, such as enol-ketone tautomers. Therefore, those skilled in the art will understand that tautomers that can rapidly interconvert to reach a certain equilibrium essentially represent the same compound. In other words, for a compound existing in different tautomer forms, the compound is not limited to any specific tautomer, but rather aims to encompass all tautomer forms.

[0210] The RNAi reagent of the present invention can also be modified to include one or more restricted ethyl nucleosides. As used herein, "restricted ethyl nucleoside" or "cEt" is a locked nucleic acid containing a bicyclic sugar group that includes a 4′-CH(CH3)—O-2′ bridge (i.e., L in the above structure). In one embodiment, the restricted ethyl nucleoside is in the S conformation, referred to herein as "S-cEt".

[0211] The RNAi reagent of the present invention may also contain one or more "conformation-restricted nucleosides" (CRNs). CRNs are nucleoside analogs that increase hybridization affinity with mRNA by locking the ribose ring in a stable conformation through a linker chain connecting the C2′ and C4′ carbon atoms or the C3 and C5′ carbon atoms of the ribose. The length of the linker chain is sufficient to place the oxygen atom in the optimal position for both stability and affinity, thereby reducing ribose ring shrinkage.

[0212] In some embodiments, the RNAi reagent of the present invention comprises one or more UNA (unlocked nucleic acid) nucleosides. An UNA is an unlocked open-ring nucleic acid in which any bonds of the sugar are removed, forming an "unlocked" sugar residue. In one example, the UNA also includes a monomer with the bond between C1′ and C4′ removed (i.e., the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbon atoms). In another example, the C2′-C3′ bond of the sugar (i.e., the covalent carbon-carbon bond between the C2′ and C3′ carbon atoms) is removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, both cited herein by reference).

[0213] Possible stabilizing modifications to the ends of RNA molecules include N-(acetaminohexanoyl)-4-hydroxyproline (Hyp-C6-NHAc), N-(hexanoyl-4-hydroxyproline)(Hyp-C6), N-(acetyl-4-hydroxyproline)(Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminohexanoyl)-4-hydroxyproline (Hyp-C6-amino), 2-icosicoyl-uridine-3′-phosphate, and ribonucleotides with reverse 2′-deoxy modifications, such as reverse dT (idT), reverse dA (idA), and reverse abase-free 2′-deoxyribonucleotides (iAb). Disclosures of these modifications can be found in WO 2011 / 005861.

[0214] In one example, the 3′ or 5′ end of the oligonucleotide is linked to a reverse 2′-deoxy-modified ribonucleotide, such as reverse dT (idT), reverse dA (idA), or reverse abase-free 2′-deoxyribonucleotide (iAb). In a particular example, the reverse 2′-deoxy-modified ribonucleotide is linked to the 3′ end of the oligonucleotide, such as the 3′ end of the positive strand described herein, via a 3′-3′ phosphodiester bond or a 3′-3′ thiophosphate bond.

[0215] In another example, the 3′ end of the positive chain is linked to the inverse abasic ribonucleotide (iAb) via a 3′-3′ thiophosphate bond. In yet another example, the 3′ end of the positive chain is linked to the inverse dA (idA) via a 3′-3′ thiophosphate bond.

[0216] In one particular example, the reverse 2′-deoxy modified ribonucleoside is linked to the 3′ end of the oligonucleotide, such as the 3′ end of the positive strand described herein, via a 3′-3′ phosphodiester bond or a 3′-3′ thiophosphate bond.

[0217] In another example, the 3′ terminal nucleotide of the positive strand is the reverse dA (idA) and is linked to the preceding nucleotide via a 3′-3′ linker (e.g., a 3′-3′ phosphate thioester bond).

[0218] Other modifications to the RNAi reagent of the present invention include 5′ phosphate or 5′ phosphate mimicry, such as the 5′ terminal phosphate or phosphate mimicry of the antisense strand of the RNAi reagent. Suitable phosphate mimicry are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0219] In some embodiments, the modified nucleotide is selected from deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-deoxy-2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformation-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, and 2'-methoxyethyl modified nucleotides. 2'-O-alkyl modified nucleotides, morpholinonucleotides, aminophosphates, non-natural bases including nucleotides, tetrahydropyran modified nucleotides, 1,5-dehydrohexyl modified nucleotides, cyclohexenyl modified nucleotides, nucleotides including thiophosphate groups, nucleotides including methylphosphonate groups, nucleotides including 5'-phosphates, nucleotides including 5'-phosphate mimics, heat-labile nucleotides, ethylene glycol modified nucleotides (GNA), nucleotides including 2'-phosphates, and nucleotides modified with 2-O-(N-methylacetamide).

[0220] In this invention, "siRNA-conjugate" refers to a complex formed by covalently linking small interfering RNA (siRNA) or modified siRNA with a specific molecule (such as a lipid group, a small molecule ligand, and / or a polypeptide) through a chemical method. The lipid group comprises at least one lipid group and contains 4-40 carbon atoms.

[0221] In this invention, the delivery composition comprises the nucleic acid molecule of this invention and an RNA vector. The RNA vector may be selected from lipid vectors, lipid-like vectors, coupling ligands, polymers, and exosome vectors.

[0222] In this invention, the RNA vector may also be selected from liposomes, lipid nanoparticles, polymer nanoparticles, exosomes, viral vectors, inorganic nanoparticles, and bioconjugates.

[0223] In this invention, a liposome refers to a closed vesicle composed of a phospholipid bilayer that can encapsulate a hydrophilic drug in its aqueous interior region, while a hydrophobic drug can be embedded in the hydrocarbon chain region of the lipid bilayer.

[0224] In this invention, lipid nanoparticles (LNPs) refer to stable nanoparticles composed of functional lipids, polyethylene glycol-modified lipids, saturated phospholipids, and cholesterol.

[0225] In this invention, polymer nanoparticles refer to nanoscale particles made of natural polymers (such as chitosan and gelatin) or synthetic polymers (such as polyvinyl alcohol and polylactic acid).

[0226] In this invention, exosomes refer to nanoscale vesicles secreted by cells, exhibiting good biocompatibility and low immunogenicity. The surface of exosomes can be engineered, for example, by introducing specific ligands or markers through genetic engineering or chemical modification, to achieve targeting of specific cells or tissues. Exosomes have significant applications in RNA delivery, effectively protecting RNA from degradation and promoting its uptake within cells. In this invention, viral vectors refer to tools that utilize the natural infection mechanisms of viruses to deliver genetic material. Common viral vectors include adeno-associated virus (AAV), adenovirus (AdV), lentivirus (LV), and retrovirus (RV).

[0227] In this invention, inorganic nanoparticles refer to nanoscale particles made of inorganic materials (such as gold, silver, silicon dioxide, etc.), such as metal nanoparticles (e.g., gold nanoparticles, silver nanoparticles) and oxide nanoparticles (e.g., silicon dioxide nanoparticles, iron oxide nanoparticles). The surface of inorganic nanoparticles can be polyethylene glycol-modified or ligand-modified to achieve targeting and biocompatibility.

[0228] In this invention, a bioconjugate refers to a complex formed by chemically binding a drug (such as the siRNA or modified siRNA of this invention), a gene or other bioactive molecule to a carrier (such as an antibody or protein).

[0229] In some embodiments, the coupling ligand is selected from carbohydrates, peptides, antibodies, aptamers, and small molecules; the lipid carrier is selected from LNP, cholesterol, Dlin-DMA / MC3-DMA; the lipid carrier is selected from nanoparticles (e.g., endoplasmic reticulum membrane-modified nanoparticles, polymer nanoparticles (PNP), lipid polypolymer complexes (LPP)), PEI particles, PLGA particles, preferably inorganic nanoparticles; the polymer is a dendritic molecule, such as PBAVE polymer, DPC1.0, or DPC2.0.

[0230] In some embodiments, the carbohydrate includes monosaccharides (e.g., GalNAc (N-acetylgalactosamine molecule)), disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides. The polypeptide is an RGD polypeptide.

[0231] In some embodiments, nucleic acid molecules are attached to carbohydrates and their derivatives via linkers that are monovalent, divalent, or trivalent. In some embodiments, the double-stranded RNAi reagent of the present invention comprises GalNAc or GalNAc derivatives attached to the iRNA reagent. In another embodiment, the double-stranded RNAi reagent of the present invention comprises a plurality of (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double-stranded RNAi reagent via a plurality of monovalent linkers. In one embodiment, the iRNA reagent of the present invention is conjugated to a carbohydrate via linkers. Non-limiting examples of linker-containing iRNA carbohydrate conjugates of these compositions and methods of the present invention include, but are not limited to, L96; L96 can be prepared according to methods known to those skilled in the art, such as those described in PCT disclosure WO 2009 / 073809, which is incorporated herein by reference.

[0232] In this invention, the term "nucleic acid protein composition" refers to the RNA-induced silencing complex (RISC) formed by the binding of siRNA to Argonaute protein (AGO). The siRNA is then unwound into a sense strand and an antisense strand. The sense strand is degraded, and the antisense strand (guide strand) RISC binds to a target mRNA homologous to the siRNA via base pairing. The RISC functions as a nuclease; the siRNA guides the RISC to cleave the homologous single-stranded mRNA, causing the mRNA to lose its function, i.e., it cannot be translated to produce protein, thus "silencing" the gene.

[0233] As used herein, a “recombinant vector” is preferably a vector comprising a regulatory sequence operatively linked to a nucleotide sequence encoding the positive strand contained in the nucleic acid molecule of the present invention. A “transformation” is a cell in which at least one recombinant vector capable of expressing a nucleic acid molecule or at least one strand of such nucleic acid molecule has been introduced.

[0234] The nucleic acid molecules of this invention can be introduced into cells by promoting uptake or absorption into cells. The absorption or uptake of nucleic acid molecules can occur through passive diffusion or active cellular processes, or through auxiliary reagents or devices. The term "introduced into cells" is not limited to cells in vitro; cells can also be parts of a living organism. In such cases, intracellular introduction will include delivery to the organism. For example, for in vivo delivery, nucleic acid molecules can be injected into tissue sites or administered systemically. For example, the administration of the nucleic acid molecules of this invention to a subject requiring medical intervention can be considered. Such administration can include injecting the nucleic acid molecules, recombinant vectors, or transformants of this invention into the diseased site of the subject, or injection adjacent to the diseased tissue. In vitro intracellular introduction includes methods known in the art, such as electroporation and lipid transfection.

[0235] The term “inhibition of ACVR1C gene expression” in this document refers to at least partial inhibition of ACVR1C gene expression, which can be manifested in the following way: the amount of mRNA transcribed from the ACVR1C gene isolated from a first cell or cell population that has been transcribed from the ACVR1C gene and has been treated to inhibit ACVR1C gene expression is reduced compared to a second cell or cell population (control cell) that is substantially the same as the first cell or cell population except for not having been treated.

[0236] As illustrated in the examples and tables provided herein, the siRNA molecules of the present invention are capable of inhibiting the expression of human ACVR1C by at least about 50%, at least about 60%, preferably at least 70%, preferably at least 80%, and most preferably at least 90% in in vitro assays. As used herein, the term "in vitro" includes, but is not limited to, cell culture assays. In particular, given the assays provided herein, those skilled in the art can readily determine the inhibition rate and associated effects.

[0237] As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of siRNA and a pharmaceutically acceptable vector. However, such a “pharmaceutical composition” may also comprise a single strand of this siRNA molecule or a vector as described herein, said vector comprising a regulatory sequence operatively linked to a nucleotide sequence encoding at least one of the sense and antisense strands contained in the siRNA of the present invention. It is also conceivable that cells, tissues, or isolated organs expressing or comprising siRNA as defined herein may be used as “pharmaceutical compositions.” As used herein, “pharmacologically effective amount,” “therapeuticly effective amount,” or simply “effective amount” refers to the amount of RNA that effectively produces the desired pharmacological, therapeutic, or preventative outcome.

[0238] The term "pharmaceutically acceptable carrier" refers to a carrier used for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients, such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavorings, colorants, and preservatives, as known to those skilled in the art.

[0239] In particular, pharmaceutically acceptable carriers are considered to allow for systemic administration of the nucleic acid molecules, recombinant vectors, or transformants of the present invention. However, enteric administration, parenteral administration, and transdermal or transmucosal (e.g., inhalation, buccal, vaginal, anal) administration, as well as drug inhalation, are also feasible methods for administering the compounds of the present invention to patients requiring medical intervention. When using parenteral administration, this may include direct injection of the compounds of the present invention into or at least immediately adjacent to the diseased tissue. However, intravenous, intra-arterial, subcutaneous, intramuscular, intraperitoneal, intradermal, intrathecal, and other administrations of the compounds of the present invention are also within the scope of the skill of a skilled person, such as an attending physician.

[0240] For intramuscular, subcutaneous, and intravenous use, the pharmaceutical compositions of the present invention are generally provided in a sterile aqueous solution or suspension buffered to a suitable pH and isotonic. In a preferred embodiment, the carrier consists only of an aqueous buffer. In this case, "only" means the absence of any adjuvants or encapsulating substances that could affect or mediate the uptake of siRNA in cells expressing the ACVR1C gene. The aqueous suspension according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone, and tragali gum, and wetting agents such as lecithin. Suitable preservatives for the aqueous suspension include ethylparaben and n-propylparaben. The pharmaceutical compositions useful according to the invention also include encapsulated formulations to protect the siRNA from rapid clearance from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as those described in PCT Publication WO 91 / 06309, which is incorporated herein by reference.

[0241] The term "treatment" in this invention refers to the relief or reduction of a disease mediated by ACVR1C gene expression, the disease being selected from one or more of the following: metabolic diseases, cardiovascular diseases, liver diseases, kidney diseases, endocrine diseases, and tumor diseases. The metabolic diseases are selected from obesity (e.g., abdominal obesity), overweight, insulin resistance, type 2 diabetes (T2D), metabolic syndrome, lipid disorders (such as hyperlipidemia, hypertriglyceridemia, or dyslipidemia), lipoprotein disorders, and impaired glucose tolerance (IGT); the cardiovascular diseases are selected from heart diseases (such as heart disease including coronary heart disease, congestive heart failure), stroke, peripheral vascular disease, atherosclerosis, arteriosclerosis, and hypertension; the liver diseases are selected from inflammation (e.g., liver inflammation and / or adipose tissue inflammation) and fatty liver disease (including non-alcoholic fatty liver disease); the kidney diseases are selected from nephropathy (e.g., nephropathy); the endocrine diseases are selected from pancreatitis; and the tumor diseases are selected from cancers (e.g., myeloma (e.g., multiple myeloma, plasmacytoma, localized myeloma, or extramedullary myeloma), melanoma, ovarian cancer, breast cancer, colon cancer, endometrial cancer, liver cancer, kidney cancer, pancreatic cancer, gastric cancer, uterine cancer, or colon cancer).

[0242] In this invention, "non-diagnostic or therapeutic purpose" refers to reducing the expression of the ACVR1C gene for research purposes, such as in a laboratory setting. Detailed Implementation

[0243] The invention will now be described in the following non-limiting embodiments.

[0244] Those skilled in the art will understand that the embodiments are described by way of example only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0245] 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.

[0246] Example 1: Synthesis of LC-044-phosphamide monomer LC-044-P

[0247] Step 1: Compound LC-044-1 (6 g, 16 mmol), compound LC-044-2 (13.09 g, 32 mmol), and N,N-diisopropylethylamine (5.16 g, 40 mmol) were dissolved in N,N-dimethylformamide (20 mL) and stirred at room temperature for 12 hours. After TLC showed that the reaction was complete, the reaction solution was poured into water (100 mL), extracted with ethyl acetate, and the organic phase was collected by separation. The organic phase was washed twice with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by normal-phase silica gel column chromatography (EA:PE 0%-30%) to obtain compound LC-044-3 (2.2 g, yield: 22%).

[0248] Step 2: Compound LC-044-3 (2.2 g, 3.6 mmol) was dissolved in dichloromethane (30 mL), and trifluoroacetic acid (5 mL) was added. The reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to give compound LC-044-4 (1.8 g, yield: 97%).

[0249] Step 3: Compound LC-044-4 (1.8 g, 3.5 mmol) and potassium carbonate (1.4 g, 10.5 mmol) were dissolved in a mixed solvent of dichloromethane (30 mL) and water (30 mL). Then, compound LC-044-5 was added, and the mixture was stirred at room temperature for 12 hours. After TLC showed that the reaction was complete, the reaction solution was extracted with dichloromethane, the organic phase was collected by separation, washed twice with saturated brine (30 mL), dried over anhydrous sodium sulfate, and the crude product was concentrated and purified by normal phase silica gel column chromatography (EA:PE 0%-30%) to obtain compound LC-044-6 (2.4 g, yield: 93%).

[0250] Step 4: Dissolve compound LC-044-6 (2.4 g, 51 mmol) in methanol (50 mL) and add palladium on carbon (100 mg, 10%). Stir at room temperature for 4 hours under a hydrogen atmosphere. After the reaction is complete, filter and concentrate the filtrate to obtain compound LC-044-7 (1.8 g, 85%).

[0251] Step 5: Dissolve compound LC-044-7 (1.8 g, 2.86 mmol) in dichloromethane (30 mL), add thionyl chloride (2 mL), and heat the reaction solution to 40 °C with stirring for 1 hour. Concentrate the reaction solution directly to obtain crude compound LC-044-8 (1.8 g), which is used directly in the next step.

[0252] Step 6: Compound LC-044-9 (780 mg, 2.8 mmol) and potassium carbonate (1.1 g, 8.4 mmol) were dissolved in dichloromethane (30 mL) and water (30 mL), followed by the addition of compound LC-044-8 (1.8 g, 2.8 mmol). The mixture was stirred at room temperature for 12 hours. After TLC showed that the reaction was complete, the reaction solution was extracted with dichloromethane, and the organic phase was collected by separation. The organic phase was washed twice with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by normal-phase silica gel column chromatography (MeOH:DCM 0%-8%) to obtain compound LC-044-10 (1.3 g, yield: 51%).

[0253] Step 7: Compound LC-044-10 (1.3 g, 1.4 mmol), DMTr-Cl (597 mg, 1.7 mmol), and 4-dimethylaminopyridine (90 mg, 0.7 mmol) were dissolved in pyridine (30 mL). The reaction mixture was stirred at 40 °C for 12 hours under nitrogen protection. The reaction mixture was washed with saturated ammonium chloride aqueous solution (100 mL), extracted twice with ethyl acetate (30 mL), dried over anhydrous sodium sulfate, and the crude product was concentrated and purified by normal-phase silica gel column chromatography (MeOH / DCM 0%-5%) to obtain compound LC-044-11 (580 mg, yield: 33%).

[0254] Step 8: Compound LC-044-11 (580 mg, 0.49 mmol) and 4,5-dicyanimidazole (87 mg, 0.74 mmol) were dissolved in anhydrous dichloromethane (5 mL). Under nitrogen protection, compound bis(diisopropylamino)(2-cyanoethoxy)phosphine (223 mg, 0.74 mmol) was added. The reaction was stirred at 25 °C for 12 hours under nitrogen protection. The reaction was monitored by LCMS under alkaline conditions. After the reaction was complete, the solvent was evaporated to obtain the crude product. The crude product was purified by silica gel column chromatography using a wet loading method to obtain LC-044-P (140.05 mg, yield: 20%).

[0255] 1H NMR(400MHz, DMSO-d6)δ7.76(d,J=4.4Hz,1H),7.39–7.18(m,9H),7.03–7.01(m,2H),6.87(d,J=7.6Hz,4H),4.03 (dd,J=14.0,7.0Hz,2H),3.91–3.82(m,6H),3.73(s,6H),3.62–3.53(m,3H),3.43(d,J=6.0Hz,2H),3.17(dd,J=26 .4,10.6Hz,4H),3.03–2.90(m,5H),2.71–2.64(m,2H),2.25(t,J=7.2Hz,2H),2.18–1.97(m,3H),1.53–1.45(m,8 H),1.36–1.34(m,5H),1.23(s,37H),1.19–1.13(m,6H),1.09–1.08(m,6H),0.99–0.95(m,9H),0.86–0.83(m,3H).

[0256] Example 2: Synthesis of LC-044-succinic acid monomer LC-044-SA

[0257] Compound LC-044-11 (930 mg, 0.78 mmol), succinic anhydride (235 mg, 2.35 mmol), and 4-dimethylaminopyridine (287 mg, 2.35 mmol) were dissolved in dichloromethane (20 mL). The reaction was carried out under nitrogen protection and stirred at 25 °C for 12 hours. The reaction was monitored by LCMS. After the reaction was completed, the solvent was evaporated to obtain the crude product. The crude product was purified by silica gel column chromatography (MeOH:DCM 0%-10%) using a wet loading method to obtain LC-044-SA (649 mg, yield: 64%).

[0258] 1H NMR (400MHz, DMSO) δ7.77(dd,J=10.0,5.0Hz,1H),7.32(dt,J=22.6,7.2Hz,4H),7.22(dt,J=4.4,3.8Hz,5H),7.06 –6.96(m,2H),6.87(d,J=8.4Hz,4H),4.03(q,J=7.0Hz,2H),3.93–3.82(m,5H),3.73(s,6H),3.25–3.18(m,2H),3.0 4–2.85(m,10H),2.42–2.35(m,4H),2.25(t,J=7.4Hz,2H),2.17–2.06(m,2H),1.53–1.45(m,8H),1.39–1.34(m,4H) ,1.23(s,32H),1.15(dd,J=15.4,7.4Hz,9H),1.08(d,J=10.0Hz,3H),0.97(d,J=3.8Hz,3H),0.85(t,J=6.6Hz,3H).

[0259] Example 3: Synthesis of C-161-phosphamide monomer LC-161-P

[0260] Step 1: Compound LC-161-0 (2 g, 7.05 mmol) and potassium carbonate (1.93 g, 14.1 mmol) were dissolved in dichloromethane (30 mL) and water (30 mL). Then, compound LC-161-1 (1.1 g, 7.05 mmol) was added, and the mixture was stirred at room temperature for 2 hours. After TLC showed that the reaction was complete, the reaction solution was extracted with dichloromethane, and the organic phase was collected by separation. The organic phase was washed twice with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by normal-phase silica gel column chromatography (MeOH:DCM 0%-8%) to obtain compound LC-161-2 (1.5 g, yield: 52%).

[0261] Step 2: Compound LC-161-2 (11.1 g, 27.34 mmol) was dissolved in dichloromethane, and triethylamine (7.7 mL, 41 mmol) and DMAP (309 mg, 2.73 mmol) were added. Then, DMTrCl (9.34 g, 27.34 mmol) was slowly added under ice bath conditions. The reaction was stirred overnight at room temperature. After TLC showed that the reaction had ceased, the reaction mixture was extracted with dichloromethane, and the organic phase was collected separately. The organic phase was washed twice with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by normal-phase silica gel column chromatography (MeOH:DCM (+1% TEA) 0%-8%) to obtain compound LC-161-3 (9.8 g, yield: 51%).

[0262] Step 3: Compound LC-161-3 (5 g, 7.06 mmol) and palladium on carbon (20 g) were dissolved in methanol (500 mL) and stirred at room temperature for 3 hours under a hydrogen atmosphere. After LCMS showed that the reaction was complete, the reaction solution was filtered, and the filtrate was collected and concentrated to obtain compound LC-161-4 (4 g, yield: 99%).

[0263] Step 4: Dissolve LC-161-4 (0.7 g, 1.22 mmol) in dichloromethane (10 mL), then add triethylamine (247 mg, 2.44 mmol) and 2,5-dioxopyrrolidine-1-yl palmitate LC-161-5 (474 ​​mg, 1.34 mmol). Stir the reaction at room temperature for 3 hours. After TLC showed that the reaction was complete, the solvent was evaporated to obtain the crude product. The crude product was then sent to pre-HPLC for further separation (under the following conditions: column specifications: Waters XBridge C8 5μm 19*150mm; mobile phase A: water (+0.1% NH4HCO3), mobile phase B: acetonitrile; flow rate: 15mL / min; elution gradient: 80-95 (20 min); detection wavelength: UV 214nm; retention time (min): 11.00; solvent: acetonitrile; single injection volume: 400μL). After purification, compound LC-161-6 (650mg, yield: 66%) was obtained.

[0264] Step 5: Dissolve compound LC-161-6 (250 mg, 0.31 mmol) in a dry dichloromethane solution (5 mL) and add dicyanimidazole (48 mg, 0.37 mmol) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (139 mg, 0.37 mmol). Stir the reaction solution at room temperature for 16 hours. No concentration of the reaction solution is required. The crude product is purified by wet loading and silica gel column chromatography (PE / EA 20%-60%, 1% TEA) to obtain compound LC-161-P (mg, yield: 48%).

[0265] 1H NMR (400MHz, DMSO-d6) δ7.73–7.65(m,1H),7.39–7.27(m,4H),7.26–7.17(m,5H),6.87(d,J=7.6Hz, 4H),3.73(s,6H),3.66–3.50(m,3H),3.49–3.37(m,3H),3.26–3.12(m,4H),3.05–2.92(m,4H),2.92 –2.85(m,1H),2.71–2.64(m,2H),2.13(s,1H),2.04–1.98(m,2H),1.45(s,4H),1.40–1.32(m,2H),1 .29–1.20(m,26H),1.20–1.17(m,3H),1.15–1.06(m,9H),1.01–0.96(m,6H),0.85(t,J=6.4Hz,3H).

[0266] Example 4: Synthesis of LC-164-phosphamide monomer LC-164-P

[0267] Step 1: Compound LC-164-1 (700 mg, 2.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (564 mg, 2.9 mmol), and 1-hydroxybenzotriazole (398 mg, 2.9 mmol) were dissolved in tetrahydrofuran (30 mL) and stirred at room temperature for 0.5 hours. Then, compound LC-161-4 (1.1 g, 2.0 mmol) was added, and the mixture was stirred at room temperature for 12 hours. After TLC showed complete reaction, the reaction mixture was poured into water (100 mL), extracted three times with ethyl acetate (30 mL), and the organic phase was collected separately. The organic phase was washed twice with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by normal-phase silica gel column chromatography (MeOH:DCM 0%-8%) to obtain compound LC-164-2 (712 mg, yield: 35%).

[0268] Step 2: Compound LC-164-2 (380 mg, 0.42 mmol) and 4,5-dicyanimidazole (119 mg, 0.62 mmol) were dissolved in anhydrous dichloromethane (5 mL), and compound LC-009-12 (188 mg, 0.62 mmol) was added under nitrogen protection. The reaction was stirred at 25 °C for 12 hours under nitrogen protection. The reaction was monitored by LCMS under alkaline conditions. After the reaction was completed, the solvent was evaporated to obtain the crude product. The crude product was purified by silica gel column chromatography (EA:PE 0%-40%) using a wet loading method to obtain LC-164-P (210 mg, yield: 43%).

[0269] 1H NMR(400MHz, DMSO-d6)δ7.70–7.66(m,1H),7.32(dt,J=22.4,7.0Hz,4H),7.25–7.15(m,5H),6.87(d,J=8 .6Hz,4H),3.73(s,2H),3.68–3.58(m,2H),3.57(s,3H),3.50–3.36(m,3H),3.24–3.11(m,4H),3.08–2.85 (m,5H),2.70–2.65(m,2H),2.27(t,J=7.4Hz,1H),2.21–2.08(m,2H),2.04–1.98(m,2H),1.51–1.42(m,6 H),1.36(dd,J=13.6,6.4Hz,2H),1.22(s,31H),1.16–1.11(m,4H),1.10–1.07(m,6H),1.01–0.96(m,7H).

[0270] Example 5: Synthesis of LC-164-SA, a monomer of LC-164-succinic acid

[0271] Step 1: Dissolve compound LC-164-2 (530 mg, 0.58 mmol), succinic anhydride (174 mg, 1.74 mmol), triethylamine (176 mg, 1.74 mmol), and 4-dimethylaminopyridine (212 mg, 1.74 mmol) in dichloromethane (10 mL). The reaction was carried out under nitrogen protection and stirred at 25 °C for 12 hours. The reaction was monitored by LCMS. After the reaction was completed, the solvent was evaporated to obtain the crude product. The crude product was then purified by silica gel column chromatography (MeOH:DCM 0%-10%) using a wet loading method, followed by pre-HPLC separation (conditions as follows: column specifications: Waters XBridge C8 5μm 19*150mm; mobile phase A: water (+0.1% NH4OH), mobile phase B: acetonitrile; flow rate: 15mL / min; elution gradient: 50-90 (10.5 min); detection wavelength: UV 214nm; retention time (min): 10.00; solvent: acetonitrile; single injection volume: 500μL) to obtain LC-164-SA (258mg, yield: 41%).

[0272] 1H NMR(400MHz,DMSO-d6)δ12.03(s,1H),7.74–7.68(m,1H),7.38–7.27(m,4H),7.24–7.20(m,5H) ,6.90–6.85(m,4H),3.73(s,6H),3.57(s,3H),3.34–3.32(m,5H),3.26–3.16(m,2H),3.01–2.8 4(m,4H),2.42–2.34(m,4H),2.27(t,J=7.4Hz,2H),2.17–2.09(m,2H),2.03–1.99(m,2H),1.54 –1.41(m,6H),1.41–1.32(m,2H),1.22(s,30H),1.08(d,J=10.4Hz,3H),0.97(d,J=4.2Hz,3H).

[0273] Example 6: Synthesis of LC-206-P

[0274] Step 1: The reaction temperature was controlled at -55℃. Compounds LC-206-1 (0.620 g, 1.56 mmol, 1.00 eq) and LC-206-2 (521.9 mg, 1.88 mmol, 1.20 eq) were dissolved in dichloromethane (12.4 mL), and N,N-diisopropylethylamine (151.9 mg, 1.17 mmol, 194.8 μL, 0.75 eq) was slowly added dropwise. The reaction was stirred at this temperature for 1 hour, and the reaction was confirmed to be complete by LCMS. The reaction solution was quenched in ice-cold citric acid aqueous solution (50 mL), followed by extraction with dichloromethane (50 mL). The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound LC-206-3 (0.87 g, crude product, yellow oil).

[0275] Step 2: Compound LC-206-3 (0.870 g, 1.56 mmol, 1.00 eq) was dissolved in dichloromethane (4.35 mL), and trifluoroacetic acid (6.69 g, 58.7 mmol, 4.35 mL, 37.6 eq) was added at room temperature. The reaction was carried out at 25 °C for 1 hour, and the reaction was confirmed to be complete by LCMS. The reaction solution was concentrated under reduced pressure to obtain compound LC-206-4 (0.780 g, crude product, yellow oil).

[0276] Step 3: Dissolve compound LC-206-4 (0.330 g, 657.9 μmol, 1.00 eq) in dichloromethane (5.00 mL), and add N,N-diisopropylethylamine (510.2 mg, 3.94 mmol, 654.1 μL, 6.00 eq), compound LC-206-5 (214.2 mg, 657.9 μmol, 1.00 eq), and TBTU (253.5 mg, 789.5 μmol, 1.20 eq) at room temperature. The reaction was carried out at 25 °C for 1 hour, and the reaction was confirmed by LCMS. The reaction solution was quenched in 50 mL of citric acid aqueous solution and extracted with 100 mL of dichloromethane. The organic phase was washed with an aqueous sodium bicarbonate solution (50 mL) and saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound LC-206-P (354 mg, 96.5% yield).

[0277] LCMS:[M+H] + =809.5.

[0278] 1 H NMR: 400MHz CDCl3, δ8.55(s,1H),6.26-6.21(m,2H),4.15(s,3H),3.72(t,J=6.0Hz,2H),3. 62-3.61(m,8H),3.56-3.54(m,2H),3.47-3.43(m,2H),3.32(s,3H),3.23-3.18 (m,2H),2.57(t,J=7.0Hz,2H),2.43(t,J=6.0Hz,2H),2.36(t,J=7.4Hz,2H),2. 01-1.94(m,2H),1.51-1.43(m,2H),1.27-1.24(m,38H),0.87(t,J=6.8Hz,3H).

[0279] Example 7: Synthesis of LC-207-P

[0280] Step 1: Compound LC-206-4 (0.450 g, 897.2 μmol, 1.00 eq) was dissolved in dichloromethane (7.00 mL). N,N-diisopropylethylamine (695.7 mg, 5.38 mmol, 891.9 μL, 6.00 eq), compound LC-207-1 (216.6 mg, 897.2 μmol, 1.00 eq), and TBTU (345.6 mg, 1.07 mmol, 1.20 eq) were added at room temperature. The reaction was carried out at 25 °C for 1 hour, and the reaction was confirmed by LCMS. The reaction solution was quenched in 50 mL of citric acid aqueous solution and extracted with 100 mL of dichloromethane. The organic phase was washed with sodium bicarbonate aqueous solution (50 mL) and saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound LC-207-P (0.540 g, 744.8 μmol, 83.0% yield).

[0281] LCMS:[M+H] + =725.5.

[0282] 1 H NMR: 400MHz CDCl3, δ8.54(s,1H),6.27-6.21(m,2H),4.15(s,3H),3.72(t,J=5.8Hz,2H),3. 62-3.58(m,8H),3.56-3.54(m,2H),3.47-3.43(m,2H),3.32(s,3H),3.23-3.18 (m,2H),2.57(t,J=7.0Hz,2H),2.43(t,J=5.8Hz,2H),2.36(t,J=7.4Hz,2H),2. 01-1.94(m,2H),1.49-1.43(m,2H),1.27-1.24(m,28H),0.87(t,J=6.8Hz,3H).

[0283] Example 8: Synthesis of LC-205-SA

[0284] Step 1: Compound LC-205-1 (10 g, 32.6 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (9.3 g, 48.9 mmol), and 1-hydroxybenzotriazole (6.6 g, 48.9 mmol) were dissolved in dichloromethane (100 mL) and stirred at room temperature for 0.5 hours. Subsequently, compound LC-205-2 (2.0 g, 35.9 mmol) and triethylamine (9.0 mL, 65.2 mmol) were added, and the mixture was stirred at room temperature for 16 hours. After TLC showed that the reaction was complete, the reaction solution was poured into water (200 mL), extracted twice with dichloromethane (200 mL), the organic phase was collected by separation, washed twice with saturated brine (200 mL), dried over anhydrous sodium sulfate, and the crude product obtained after concentration was purified by normal phase silica gel column chromatography (MeOH:DCM = 0%-5%) to obtain compound LC-205-3 (8.7 g, yield: 78%).

[0285] Step 2: Dissolve compound LC-205-3 (7.7 g, 22.4 mmol) in HCl / dioxane (70 mL) and stir at room temperature for 1 hour. After the reaction is complete as detected by TLC, concentrate to obtain compound LC-205-4 (6.8 g, crude product). Repeat the above operation to prepare the raw materials. The crude product obtained is directly used for the next step.

[0286] Step 3: Compound LC-205-4 (7.3 g, 25.4 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (7.3 g, 38.1 mmol), and 1-hydroxybenzotriazole (5.1 g, 38.1 mmol) were dissolved in dichloromethane (80 mL) and stirred at room temperature for 0.5 hours. Then, compound LC-161-4 (14.6 g, 25.4 mmol) and triethylamine (7.0 mL, 50.4 mmol) were added, and the mixture was stirred at room temperature for 16 hours. After TLC showed that the reaction was complete, the reaction solution was poured into water (100 mL), extracted twice with dichloromethane (100 mL), the organic phase was collected by separation, washed twice with saturated brine (100 mL), dried over anhydrous sodium sulfate, and the crude product obtained after concentration was purified by normal phase silica gel column chromatography (MeOH:DCM = 0%-7%) to obtain compound LC-205-6 (16 g, yield: 75%).

[0287] Step 4: Compound LC-205-6 (2.0 g, 2.37 mmol), compound LC-205-7 (505 mg, 2.37 mmol), copper sulfate pentahydrate (1.8 g, 7.11 mmol), and sodium L-ascorbate (2.3 g, 11.85 mmol) were dissolved in tert-butanol / water (24 mL, 3 / 1) and stirred at room temperature for 2 hours. After TLC showed that the reaction was complete, the reaction solution was poured into water (50 mL), extracted three times with dichloromethane (50 mL), and the organic phase was collected by separation. The solution was washed once with saturated sodium bicarbonate (50 mL) and twice with saturated brine (50 mL). After drying with anhydrous sodium sulfate, the crude product was concentrated and purified by normal-phase silica gel column chromatography (MeOH:DCM 0%-8%) to obtain compound LC-205-8 (2.1 g, yield: 77%).

[0288] Step 5: Dissolve compound LC-205-8 (1.5 g, 1.4 mmol), succinic anhydride (426 mg, 4.3 mmol), triethylamine (429 mg, 4.3 mmol), and 4-dimethylaminopyridine (342 mg, 2.8 mmol) in dichloromethane (10 mL). The reaction was carried out under nitrogen protection and stirred at 25 °C for 16 hours. The reaction was monitored using LCMS. After the reaction was completed, the solvent was evaporated to obtain the crude product. The crude product was then purified by silica gel column chromatography (MeOH:DCM 0%-10%) using a wet loading method, followed by pre-HPLC separation (under the following conditions: column specifications: Waters XBridge C8 5μm 19*150mm; mobile phase A: water (+0.1% NH4OH), mobile phase B: acetonitrile; flow rate: 15mL / min; elution gradient: 40-90 (125 min); detection wavelength: UV 214nm; retention time (min): 10.00; solvent: acetonitrile; single injection volume: 150μL) to obtain LC-205-SA (610mg, yield: 38%).

[0289] MS m / z (ESI): 1157.1 (M+1).

[0290] 1H NMR(400MHz,MeOD)δ7.82(d,J=2.8Hz,1H),7.40(d,J=8.4Hz,2H),7.32–7.24(m,6H),7.23–7.17(m,1H),6.89–6.82 (m,4H),4.43(s,2H),4.37–4.31(m,2H),3.78(s,6H),3.77–3.75(m,1H),3.74–3.66(m,5H),3.64–3.62(m,3H),3.5 9–3.52(m,8H),3.34(s,1H),3.25–3.12(m,3H),3.11–2.99(m,2H),2.50–2.38(m,8H),2.32–2.20(m,4H),1.91–1.8 3(m,2H),1.64–1.55(m,4H),1.53–1.46(m,2H),1.40–1.24(m,12H),1.17(d,J=17.6Hz,3H),1.09(d,J=4.0Hz,3H).

[0291] Example 9: Synthesis of LC-202-P, a phosphorus amide monomer

[0292] Step 1: Dissolve compound LC-202-1 (1 g, 1.5 mmol) and sodium hydroxide (300 mg, 7.5 mmol) in dimethyl sulfoxide (50 mL) and stir at room temperature for 0.5 hours. Then add compound LC-202-2 (522 mg, 3 mmol) and stir at 45 degrees Celsius for 12 hours. After TLC showed the reaction was complete, the reaction was quenched with ammonium chloride, and the mixture was extracted three times with ethyl acetate (30 mL). The organic phase was collected by separation, washed twice with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by normal-phase silica gel column (MeOH:DCM 0%-8%), and then separated by pre-HPLC (conditions as follows: column specifications: XBridge Prep C18 19*150mm 5μm; mobile phase A: water (+0.1% NH4HCO3), mobile phase B: acetonitrile; flow rate: 15 mL / min; elution gradient: 50-80 (19 min); detection wavelength: UV 214nm; retention time (min): 11.1; solvent: acetonitrile; single injection volume: 300 μL) to obtain compound LC-202-3 (300 mg, yield: 26%).

[0293] Step 2: Dissolve compound LC-202-3 (1g, 1.3mmol), copper sulfate pentahydrate (227mg, 3.9mmol), sodium L-ascorbate (1.3g, 6.5mmol) and compound N3-21 (501.8mg, 1.3mmol) in 30mL of tert-butanol and 10mL of water, and stir at room temperature for 4 hours. After TLC showed that the reaction was complete, the reaction was quenched with water, extracted three times with dichloromethane (30 mL), and the organic phase was collected by separation. The organic phase was washed twice with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by normal-phase silica gel column (MeOH:DCM 0%-8%), and then separated by pre-HPLC (under the following conditions: column specifications: XBridge Prep C18 19*150mm 5μm; mobile phase A: water (+0.1% NH4HCO3), mobile phase B: acetonitrile; flow rate: 15 mL / min; elution gradient: 70-90 (27 min); detection wavelength: UV 214nm; retention time (min): 9.1; solvent: acetonitrile; single injection volume: 300 μL) to obtain compound LC-202-4 (300 mg, yield: 26%).

[0294] Step 3: Compound LC-202-4 (300 mg, 0.26 mmol) and 4,5-dicyanimidazole (46 mg, 0.39 mmol) were dissolved in anhydrous dichloromethane (10 mL), and compound LC-009-12 (118 mg, 0.39 mmol) was added under nitrogen protection. The reaction was stirred at 25 °C for 12 hours under nitrogen protection. The reaction was monitored by LCMS under alkaline conditions. After the reaction was completed, the solvent was evaporated to obtain the crude product. The crude product was purified by silica gel column chromatography (EA:PE 0%-50%) using a wet loading method to obtain LC-202-P (100 mg, yield: 29%).

[0295] 1H NMR(400MHz,DMSO-d6)δ12.11(s,1H),11.59(s,1H),8.15(s,1H),7.73(s,1H),7.35(s,2H),7.29–7.19(m,7H),6 .96(t,J=5.6Hz,1H),6.88–6.80(m,4H),5.93–5.83(m,1H),4.63(t,J=5.9Hz,1H),4.44–4.35(m,1H),4.30–4.10 (m,3H),3.72(d,J=2.1Hz,6H),3.70–3.40(m,6H),3.27–3.18(m,1H),2.97–2.90(m,2H),2.81–2.70(m,2H),2.61 –2.57(m,2H),1.81–1.69(m,2H),1.60–1.38(m,7H),1.28–1.19(m,26H),1.16–1.10(m,16H),1.01–0.86(m,8H).

[0296] Example 10: Synthesis of N3-21

[0297] Step 1: Compound N3-21-1 (10 g, 82.51 mmol) and di-tert-butyl dicarbonate (21.56 g, 99.01 mmol) were dissolved in dichloromethane. Triethylamine (17.3 mL, 123.76 mmol) and 4-dimethylaminopyridine (500 mg, 4.13 mmol) were added under ice bath conditions. The mixture was stirred at room temperature for 12 hours. After TLC showed that the reaction was complete, the reaction solution was evaporated to dryness, water (100 mL) was added, and the mixture was extracted three times with ethyl acetate (100 mL). The organic phase was collected by separation. The organic phase was washed twice with 1 M hydrochloric acid aqueous solution (100 mL), then twice with saturated brine (100 mL), and dried over anhydrous sodium sulfate to give compound N3-21-2 (15 g, yield: 83%).

[0298] Step 2: Compound N3-21-2 (3 g, 13.60 mmol) and triphenylphosphine (5.35 g, 20.30 mmol) were dissolved in anhydrous tetrahydrofuran (40 mL). Compound N3-21-3 (3.85 g, 13.60 mmol) and diisopropyl azodicarbonate (4.13 g, 20.30 mmol) were added under nitrogen protection. The reaction was stirred at 25 °C for 2 hours under nitrogen protection. The reaction was monitored by TLC (PE:EA = 10:1). After the reaction was complete, the crude product was concentrated and purified by silica gel column chromatography (EA:PE 0%-10%) to obtain compound N3-21-4 (3.5 g, yield: 54%).

[0299] Step 3: Compound N3-21-4 (3.5 g, 7.20 mmol) was dissolved in dichloromethane (40 mL), and trifluoroacetic acid (12 mL) was added. The reaction was stirred overnight at 25 °C. After the reaction was confirmed to be complete by TLC (PE:EA = 3:1), the reaction solution was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (EA:PE 0%-30%) to obtain compound N3-21 (2.1 g, yield: 76%).

[0300] 1 H NMR (400MHz, DMSO) δ6.97(t,J=6.0Hz,1H),3.31(t,J=3.6Hz,2H),2.94(q,J=6.8Hz,2H),1.58–1.41(m,4H),1.35–1.19(m,25H),0.97–0.83(m,4H).

[0301] Example 11: Synthesis of LC-231-SA, a monomer of succinic acid.

[0302] Step 1: Compound LC-231-1 (10 g, 82.51 mmol) and di-tert-butyl dicarbonate (21.56 g, 99.01 mmol) were dissolved in dichloromethane. Triethylamine (17.3 mL, 123.76 mmol) and 4-dimethylaminopyridine (500 mg, 4.13 mmol) were added under ice bath conditions. The mixture was stirred at room temperature for 12 hours. After TLC showed that the reaction was complete, the reaction solution was evaporated to dryness, water (100 mL) was added, and the mixture was extracted three times with ethyl acetate (100 mL). The organic phase was collected by separation. The organic phase was washed twice with 1 M hydrochloric acid aqueous solution (100 mL), then washed twice with saturated brine (100 mL), and dried over anhydrous sodium sulfate to give compound LC-231-2 (15 g, yield: 83%).

[0303] Step 2: Compound LC-231-2 (3 g, 13.60 mmol) and triphenylphosphine (5.35 g, 20.30 mmol) were dissolved in anhydrous tetrahydrofuran (40 mL). Compound LC-231-3 (3.85 g, 13.60 mmol) and diisopropyl azodicarbonate (4.13 g, 20.30 mmol) were added under nitrogen protection. The reaction was stirred at 25 °C for 2 hours under nitrogen protection. The reaction was monitored by TLC (PE:EA = 10:1). After the reaction was complete, the crude product was concentrated and purified by silica gel column chromatography (EA:PE 0%-10%) to obtain compound LC-231-4 (3.5 g, yield: 54%).

[0304] Step 3: Compound LC-231-4 (3.5 g, 7.20 mmol) was dissolved in dichloromethane (40 mL), and trifluoroacetic acid (12 mL) was added. The reaction was stirred overnight at 25 °C. After the reaction was confirmed to be complete by TLC (PE:EA = 3:1), the reaction solution was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (EA:PE 0%-30%) to obtain compound LC-231-5 (2.1 g, yield: 76%).

[0305] 1 H NMR (400MHz, DMSO-d6): δ6.97(t,J=6.0Hz,1H),3.31(t,J=3.6Hz,2H),2.94 (q,J=6.8Hz,2H),1.58–1.41(m,4H),1.35–1.19(m,25H),0.97–0.83(m,4H).

[0306] Step 4: Dissolve compound LC-231-5 (1.2 g, 3.1 mmol) in methanol (20 mL), add Pd / C (200 mg), replace with hydrogen balloons three times, and stir at room temperature for 2 hours. After TLC showed that the reaction was complete, filter and concentrate the filtrate to obtain crude compound LC-231-6 (1 g). The crude product was used directly in the next step of the reaction.

[0307] Step 5: Compound LC-231-6 (1 g, 2.78 mmol), compound LC-205-1 (850 mg, 2.78 mmol), 1-hydroxybenzotriazole (450 mg, 3.34 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (637 mg, 3.34 mmol) were dissolved in dichloromethane (20 mL). The reaction mixture was stirred at room temperature for 12 hours. After TLC showed that the reaction was complete, the crude product was concentrated and then purified by normal-phase silica gel column chromatography (MeOH:DCM 0%-10%) to obtain compound LC-231-7 (1.1 g, yield: 61%).

[0308] Step 6: Dissolve compound LC-231-7 (1.1 g, 1.69 mmol) in dichloromethane (3 mL), followed by the addition of trifluoroacetic acid (15 mL). Stir the reaction mixture at room temperature for 2 hours. After TLC showed that the reaction was complete, concentrate the solution directly to obtain crude compound LC-231-8 (1 g), which was used directly in the next step.

[0309] Step 7: Compounds LC-231-8 (1 g, 1.69 mmol) and LC-161-4 (970 mg, 1.69 mmol) were dissolved in dichloromethane (20 mL), and 1-hydroxybenzotriazole (274 mg, 2.03 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (388 mg, 2.03 mmol) were added. The reaction was stirred at room temperature for 12 hours. After TLC showed that the reaction was complete, the mixture was concentrated and then purified by normal-phase silica gel column chromatography (MeOH:DCM (+1% TEA)) to give crude compound LC-231-9 (1.1 g, yield: 52%). The crude product (800 mg) was sent for pre-HPLC preparation and separation under the following conditions: column specifications: Waters Xbridge C8μm 19*150mm; mobile phase A: water, mobile phase B: acetonitrile; flow rate: 15 mL / min; elution gradient: 5-95 (10 min); detection wavelength: UV 214 nm; retention time (min): 10.00; solvent: acetonitrile; the target compound LC-231-9 (300 mg) was obtained.

[0310] Step 8: Compound LC-231-9 (300 mg, 0.22 mmol) and succinic anhydride (66 mg, 0.66 mmol) were dissolved in dichloromethane (8 mL), and 4-dimethylaminopyridine (80 mg, 0.66 mmol) was added. The reaction mixture was stirred at room temperature for 12 hours. After TLC showed that the reaction was complete, the solution was directly concentrated and purified by normal-phase silica gel column chromatography (MeOH:DCM 0%-10%) to obtain the crude compound LC-231-SA (500 mg). The crude product was sent to pre-HPLC for separation (under the following conditions: column specifications: Waters Xbridge C8 5μm 19*150mm; mobile phase A: water, mobile phase B: acetonitrile; flow rate: 15mL / min; elution gradient: 5-95 (10 min); detection wavelength: UV 214nm; retention time (min): 10.00; solvent: acetonitrile), yielding the target compound LC-231-SA (173mg).

[0311] 1H NMR (400MHz, DMSO-d6) δ7.82(t,J=20.8Hz,2H),7.33(dd,J=14.1,6.2Hz,4H),7.27–7.17(m,5H),6.98(t,J=5.8Hz,1H),6.87( dd,J=8.8,2.5Hz,4H),3.73(s,6H),3.67(s,1H),3.57(t,J=6.4Hz,5H),3.46(s,9H),3.26–3.18(m,2H),3.15–3.07(m,1H),3. 00(dd,J=12.5,6.6Hz,4H),2.97–2.90(m,3H),2.85(d,J=9.1Hz,1H),2.36(d,J=5.0Hz,4H),2.28(dd,J=8.1,4.7Hz,4H),2.20 –2.02(m,2H),1.44(s,4H),1.40–1.30(m,5H),1.23(s,26H),1.08(d,J=10.7Hz,3H),0.97(d,J=4.1Hz,3H),0.94–0.83(m,4H).

[0312] Example 12: Synthesis of siRNA or its conjugates

[0313] The synthesis of siRNA compounds is based on a mature solid-phase synthesis method using phosphoramidite chemistry. The general procedure is described below:

[0314] 1. Single fixed load

[0315] Taking LC-205-SA as an example, the general method for immobilization is described as follows: At room temperature, DCC (98 mg, 5 eq), HOBT (65 mg, 5 eq), DIEA (83 μL, 5 eq), and lcaa-CPG ( ) are added to a solution of LC-205-SA (100 mg, 0.9 eq) in ACN (1.7 mL). 164 μmol / g, 580 mg, 1 eq). Shake for 16 hours. After the reaction is complete, wash CPG with ACN, add CAP A (acetic anhydride:tetrahydrofuran = 1:9, v / v, 10.0 mL) and CAP B (N-methylimidazolium:pyridine:acetonitrile = 15:10:75, v / v / v, 10.0 mL), and shake the mixture at room temperature for 1 hour. Then filter the mixture and wash three times with ACN (20 mL). After freeze-drying, LC-205-CPG is given as a white powder (600 mg).

[0316] The monomers containing succinic acid groups in this invention can all be immobilized using the above method.

[0317] 2. Solid-phase synthesis and ammonolysis

[0318] Using an Oligo 48 solid-phase synthesizer, phosphoramide monomers were synthesized on a general-purpose or self-made synthetic support. Nucleotides and / or ligands were sequentially linked from the 3'-5' direction. The phosphoramide monomers were dissolved in anhydrous acetonitrile (100 mM) and molecular sieves were added. Each phosphoramide monomer linkage involved four steps: deprotection, coupling, capping, and oxidation (or thiolation), yielding a sequence with a solid support and protecting groups. The conditions for each reaction step were as follows:

[0319] (1) Deprotection: Add 3% TCA decapping agent to remove the protecting group of 5'-OH of nucleotides attached to the solid support. The reaction time is 60s.

[0320] (2) Coupling reaction: With the addition of an activator, the nucleoside phosphoramide monomer reacts with the exposed 5'-OH to form a phosphate triester bond, and the coupling time is 300s.

[0321] (3) Oxidation reaction: Add 0.05M iodine solution and oxidize for 25 seconds.

[0322] (4) Capping reaction: Add CAPA / CAPB (1 / 1, v / v) solution to block the unreacted 5'-OH group. The capping time is 20s.

[0323] When synthesizing thiophosphate bonds, the oxidation reaction is replaced with a thioreaction step, and the reaction conditions are as follows:

[0324] Thioation reaction: Add 0.2M hydroflavin pyridine solution, and the thioation time is 200s.

[0325] After solid-phase synthesis, the dried solid support was treated with ammonia solution at 55°C for 16 hours, then purified, desalted, and freeze-dried.

[0326] 3. Reagents for synthesis and purification

[0327] The crude product was purified by reversed-phase HPLC using a Waters XBridge C18 column and an Autotide 100 system. Buffer A was 100 mM TEAA, pH 7.5, containing 5% acetonitrile, and buffer B was 100% acetonitrile. UV traces were recorded at 260 nm, and appropriate fractions were collected.

[0328] 4. Reagents

[0329] 5. Oxidation reaction

[0330] Some compounds of this invention contain specific nucleosides with a 5' terminal phosphate group (such as NT-100 and NT-177). In addition to the conventional solid-phase synthesis method described above, an oxidation reaction step is also required, as described below:

[0331] (1) Deprotection: Add 3% TCA decapping agent to remove the protecting group of 5'-OH of nucleotides attached to the solid support. The reaction time is 60s.

[0332] (2) Coupling reaction: An activator is added, and the phosphate monomer reacts with the exposed 5'-OH to form a phosphate triester bond. The coupling time is 900s.

[0333] (3) Oxidation reaction: Add the corresponding oxidizing agent, and react for 500 seconds * 3 times. The oxidizing agent corresponding to NT-100 is... The oxidizing agent corresponding to the specific nucleoside NT-177 is

[0334] (4) Capping reaction: Add CAP A / CAP B (1 / 1, v / v) solution to block the unreacted 5'-OH group. The capping time is 20s.

[0335] 6. Coupling reaction

[0336] Some compounds of this invention (such as LC-207) need to be conjugated with siRNA via a liquid-phase coupling reaction. The coupling reaction is described as follows: the sense or antisense strand of the oligonucleotide is dissolved in 0.1M sodium bicarbonate buffer solution, 10 equivalents of LC-207-P (dissolved in DMSO) are added, the mixture is stirred and vortexed, and then reacted at 25°C for 1 h to obtain the conjugate of the target compound.

[0337] 7. Annealing

[0338] The obtained sense and antisense strands were mixed in a 1:1 ratio, heated to 90°C and held for 5 minutes, then slowly cooled to room temperature to form siRNA or its conjugates. After annealing, the purity was determined by HPLC, and after passing the test, the siRNA was lyophilized.

[0339] Example 13: siRNA sequence design targeting the ACVR1C gene

[0340] The CDS sequence (SEQ ID NO: 209) in the human ACVR1C gene (NCBI Reference Sequence: NM_001111031.2) was used as the target gene to extract siRNA sequences containing 21 nucleotides in the sense strand and 23 nucleotides in the antisense strand.

[0341] SEQ ID NO:209:

[0342] After off-target risk assessment and activity assessment, siRNA sequences for testing were selected. Unmodified siRNA sequences are shown in Table 1, modified siRNA sequences are shown in Table 2, and sequences of conjugates of modified siRNAs are shown in Table 3.

[0343] The compounds in Tables 1-3 can be synthesized by the method described in Example 12.

[0344] Table 1. Sequences of unmodified siRNAs

[0345] The uppercase letters represent natural ribonucleotides, and I represents inosine.

[0346] Table 2. Sequences of modified siRNA compounds

[0347] m represents 2'OMe-modified nucleoside, f represents 2'F-modified nucleoside, s represents a phosphothioester bond, d represents a 2'deoxynucleoside, and the cPrpu structure is... The vpU structure is The Im structure is The NT-100 structure is The NT-177 structure is as follows: invAb at the 5' end represents invAb at the 3' end represents Chd represents Uda represents

[0348] Table 3. Sequences of modified siRNA conjugates

[0349] The structures m, f, s, d, cPrpu, vpU, Im, NT-177, and invAb at the 5' end represent the structure, while invAb at the 3' end represents the modified structure, which is consistent with the structures defined in Table 2.

[0350] Chd represents Uda represents C6-S represents In this design, the -S terminus is attached to a lipid group (such as LC-207, LC206, etc.), and the phosphate ester terminus is attached to the 3rd or 5th position of the invAb. For example, LC-206 C6-S invAbs represents...

[0351] LC-044 represents The structure represented by LC-202 is LC-205 represents LC-206 represents LC-207 represents LC-231 represents

[0352] Example 14: Evaluation of the inhibitory activity of compounds on ACVR1C mRNA in Huh7 cells at different concentrations

[0353] 1. Experimental Objective: The purpose of this study is to evaluate the effect of the compounds described in this invention (shown in Tables 1 and 2) on inhibiting ACVR1C mRNA at different concentrations (10 nM, 1 nM, 0.1 nM) using Huh7 cells, so as to assess the in vitro activity of the compounds described in this invention.

[0354] 2. Experimental Materials

[0355] 2.1 Cell lines

[0356] Huh7 cells were purchased from JCRB (catalog number: JCRB0403). The Huh7 cells were revived and cultured in appropriate culture medium.

[0357] 2.2 Reagents

[0358] Lipofectamine TM RNAiMAX transfection reagent (13778030), FlysisAmp Cells-to-cDNA Kit (CL111-00), TaqMan TM Fast Advanced premix (4444963) and Opti-MEM medium (Gibco, catalog number 31985-070).

[0359] 3. Experimental Procedures and Methods

[0360] 3.1 Transfection

[0361] 1) On the first day, take Lipofectamine. TM RNAiMAX transfection reagent was prepared using Opti-MEM medium at a volume ratio of 0.3:9.7, and incubated at room temperature for 15 minutes.

[0362] 2) Add 10 μL of RNAiMAX mixture and 10 μL of compound dilution (10 nM, 1 nM, 0.1 nM) to each well, and add 10 μL of LOPTI-MEM medium to the control well. Mix well and incubate at room temperature for 15 min.

[0363] 3) Remove the cells to be transfected from the incubator, discard the culture medium, and rinse with PBS;

[0364] 4) Add 2 mL of trypsin and digest at 37°C until cells detach. Then add complete culture medium to stop digestion and mix thoroughly by pipetting.

[0365] 5) Centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in 3 mL of culture medium;

[0366] 6) Add 80 μL of diluted cell suspension (i.e., 30,000 cells per well) to the experimental wells, and then place the cell plate in an incubator and incubate at 37°C and 5% CO2 for 24 h.

[0367] 3.2 RNA extraction and reverse transcription

[0368] The next day, cells were collected, and the RNA from the lysed cells was reverse transcribed into cDNA according to the instructions of the FlysisAmp Cells-to-cDNA Kit (CL111-00).

[0369] 3.3 qPCR detection

[0370] The target gene cDNA was detected by qPCR, with GAPDH as an internal reference gene. qPCR was performed in a 384-well plate. The qPCR reaction program was as follows: heat at 95°C for 10 minutes, then enter cycling mode, heat at 95°C for 15 seconds, followed by 60°C for 1 minute, for a total of 40 cycles.

[0371] 3.4 Data Analysis:

[0372] This experiment used GAPDH as an internal reference gene and employed the ΔΔCt method (Ct difference comparison method) to calculate the relative expression level of the target gene. After qRT-PCR, the Ct value of the internal reference was recorded and referred to as Ct(GAPDH), the Ct value of the sample was referred to as Ct(sample), and the baseline blank group was referred to as Ct(control). The 2^-ΔΔCt method was used to calculate the relative expression level of the target gene. Using GAPDH as the internal reference gene, the following formulas were used for calculation: ΔCt(sample) = Ct(sample) - Ct(GAPDH) ΔCt(control) = Ct(control) - Ct(GAPDH) ΔΔCt = ΔCt(sample) - ΔCt(control) Relative expression level of the gene = 2^-ΔΔCt Target gene inhibition rate (%) = (1 – average relative expression level of the target gene in the sample well / average relative expression level of the target gene in the control well) * 100 (%)

[0373] 4. Experimental Results

[0374] The degree of inhibition of the ALK7 gene by some of the compounds of this invention in the Huh7 cell line is shown in Table 4. The numbers in the table are percentages, representing the degree of inhibition of ACVR1C mRNA.

[0375] Table 4. Inhibition rate (%) of the compounds of this invention on the ALK7 gene in the Huh7 line.

[0376] 5. Experimental conclusion: The compound of this invention exhibits an inhibitory effect on ALK7.

[0377] Example 15: Compound inhibits IC50 of ACVR1C mRNA in Huh7 cells 50 Measurement and evaluation of inhibitory activity

[0378] 1. Experimental Objective: The purpose of this study is to evaluate the knockdown effect (inhibition rate) and half-maximal inhibitory concentration (IC50) of the compound described in this invention on ACVR1C mRNA in the Huh7 cell line, so as to evaluate the in vitro activity of the compound described in this invention.

[0379] 2. Experimental Materials

[0380] 2.1 Cell lines

[0381] Same as Example 14.

[0382] 2.2 Reagents

[0383] Lipofectamine TMRNAiMAX transfection reagent (13778030), RNeasy Kits for RNA Purification (74182), HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (R312-01), HelyxQuest Elite qPCR premix (HB-D01018).

[0384] 3. Experimental Procedures and Methods

[0385] 3.1 Transfection, 3.2 RNA extraction and reverse transcription, 3.3 qPCR detection are the same as in Example 14. Only the RNA extraction and reverse transcription steps are changed to "On the second day, cells are collected, RNA is extracted according to the RNeasy Kits for RNA Purification (74182) instructions, and the RNA is reverse transcribed into cDNA according to the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (R312-01) instructions".

[0386] 3.4 Data Analysis:

[0387] 3.4.1 Calculation of maximum inhibition rate

[0388] Same as section 3.4 of Example 14.

[0389] 3.4.2 Absolute IC 50 calculate

[0390] Dose-response curves were plotted and IC was calculated using GraphPad Prism software. 50 value.

[0391] 4. Experimental results.

[0392] The degree of inhibition of the ALK7 gene and absolute IC50 of some compounds of this invention in the Huh7 cell line 50 The values ​​are shown in Table 5-8. The maximum inhibition rate figures in the table are percentages, representing the degree to which ACVR1C mRNA is inhibited; the absolute IC50 values ​​are... 50 This indicates the molar concentration of the compound required to reduce ACVR1C mRNA expression by 50%.

[0393] Table 5. IC50 of the compounds of this invention in inhibiting ACVR1C mRNA in the Huh7 cell line 50 and inhibition rate results

[0394] Table 6. IC50 of the compounds of the present invention in inhibiting ACVR1C mRNA in the Huh7 cell line 50 and inhibition rate results

[0395] Table 7. IC50 of the compounds of the present invention in inhibiting ACVR1C mRNA in the Huh7 cell line 50 and inhibition rate results

[0396] Table 8. IC50 of the compounds of the present invention in inhibiting ACVR1C mRNA in the Huh7 cell line 50 and inhibition rate results

[0397] 5. Experimental Conclusions

[0398] The compounds of this invention exhibit good inhibitory activity against ACVR1C mRNA in vitro.

[0399] Example 16: Compound inhibits the IC50 of ACVR1C mRNA in SK-MEL-28 cells 50 Determination and activity evaluation

[0400] 1. Experimental Objective: The purpose of this study is to evaluate the knockdown effect of the compound described in this invention on ACVR1C mRNA in the SK-MEL-28 cell line, so as to evaluate the in vitro activity of the compound described in this invention.

[0401] 2. Experimental Materials

[0402] 2.1 Cell lines

[0403] SK-MEL-28 cells were purchased from ATCC (catalog number: HTB-72). SK-MEL-28 cells were revived and cultured in appropriate culture medium.

[0404] 2.2 Reagents

[0405] Lipofectamine TM RNAiMAX transfection reagent (Invitrogen, catalog number 13778150), Opti-MEM medium (Gibco, catalog number 31985-070), RNA extraction kit (Zymo, catalog number R2056), PrimerScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, catalog number RR047A), and HelyxQuest Elite qPCR premix (HelyxQuest, catalog number HB-D01018).

[0406] 3. Experimental Procedures and Methods

[0407] 3.1 Transfection

[0408] 1) On the first day, take Lipofectamine. TM RNAiMAX transfection reagent was prepared using Opti-MEM medium at a volume ratio of 0.3:9.7 and incubated at room temperature for 15 minutes.

[0409] 2) Add 10 μL of RNAiMAX mixture and 10 μL of compound dilution buffer to each well (start with a final concentration of 10 nM and dilute 10 times, or start with 10 nM and dilute 5 times). Add 10 μL of Opti-MEM to the control well, mix well, and incubate at room temperature for 15 min.

[0410] 3) Remove the cells to be transfected from the incubator, discard the culture medium, and rinse with PBS;

[0411] 4) Add 2 mL of trypsin and digest at 37°C until cells detach. Then add complete culture medium to stop digestion and mix thoroughly by pipetting.

[0412] 5) Centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in 5 mL of culture medium;

[0413] 6) Add 80 μL of diluted cell suspension (i.e., 20,000 cells per well) to the experimental wells, and then place the cell plate in an incubator and incubate at 37°C and 5% CO2 for 24 h.

[0414] 3.2 RNA extraction and reverse transcription

[0415] On the second day, cells were collected, and RNA was extracted according to the instructions of the RNA extraction kit. The RNA was then reverse transcribed into cDNA according to the instructions of the PrimerScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, catalog number RR047A).

[0416] 3.3 qPCR detection

[0417] Same as Example 14.

[0418] 3.4 Data Analysis:

[0419] 3.4.1 Calculation of maximum inhibition rate

[0420] Same as section 3.4 of Example 14.

[0421] 3.4.2 Absolute IC 50 calculate

[0422] The dose-response curve was plotted and the IC50 value was calculated using GraphPad Prism software.

[0423] 4. Experimental results.

[0424] The absolute IC50 values ​​of some compounds of the present invention against the ACVR1C gene in the SK-MEL-28 cell line are shown in Tables 9-14.

[0425] Table 9 shows the IC50 values ​​of the compounds of this invention in inhibiting ACVR1C mRNA in the SK-MEL-28 cell line. 50 result

[0426] Table 10. IC50 of the compounds of the present invention in inhibiting ACVR1C mRNA in the SK-MEL-28 cell line 50 result

[0427] Table 11. IC50 of the compounds of the present invention in inhibiting ACVR1C mRNA in the SK-MEL-28 cell line 50 result

[0428] Table 12. IC50 of the compounds of the present invention in inhibiting ACVR1C mRNA in the SK-MEL-28 cell line 50 result

[0429] Table 13. IC50 of the compounds of the present invention in inhibiting ACVR1C mRNA in the SK-MEL-28 cell line 50 result

[0430] Table 14. IC50 of the compounds of the present invention in inhibiting ACVR1C mRNA in the SK-MEL-28 cell line 50 result

[0431] 5. Experimental Conclusions

[0432] The compound of this invention exhibits good inhibitory activity against ACVR1C mRNA in SK-MEL-28 cells, meeting experimental expectations.

[0433] Example 17: Study on the effect of a compound on inhibiting ACVR1C mRNA in SK-MEL-28 cells using the free uptake method

[0434] 1. Experimental Objective: The purpose of this study is to evaluate the inhibitory effect of the compound described in this invention on ACVR1C mRNA in the SK-MEL-28 cell line, so as to evaluate the in vitro activity of the compound described in this invention.

[0435] 2. Experimental Materials

[0436] 2.1 Cell lines

[0437] Same as Example 16.

[0438] 2.2 Reagents

[0439] Same as Example 16.

[0440] 3. Experimental Procedures and Methods

[0441] 3.1 Cell-based drug delivery

[0442] 1) Add 20 μL of compound dilution buffer (starting at a final concentration of 2 μM, diluted 2 times) to each well, and add 20 μL of FBS-free medium to each control well;

[0443] 3) Remove the cells from the incubator, discard the culture medium, and rinse with PBS;

[0444] 4) Add 2 mL of trypsin and digest at 37°C until cells detach. Then add complete culture medium to stop digestion and mix thoroughly by pipetting.

[0445] 5) Centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in 5 mL of FBS-free medium;

[0446] 6) Add 180 μL of diluted cell suspension (i.e., 20,000 cells per well) to the experimental wells, and then place the cell plate in an incubator and incubate at 37°C and 5% CO2 for 72 h.

[0447] 3.2 RNA extraction and reverse transcription

[0448] Cells were collected after 72 hours. RNA was extracted according to the instructions of the Zymo R2056 RNA extraction kit. RNA was reverse transcribed into cDNA according to the instructions of the PrimerScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, catalog number RR047A).

[0449] 3.3 qPCR detection

[0450] The target gene cDNA was detected using qPCR, with GAPDH as an internal control gene. qPCR was performed in 384-well plates. The IDT mix qPCR program was: 95°C for 3 minutes, followed by cycling at 95°C for 5 seconds, then at 60°C for 30 seconds, for a total of 40 cycles. The Hybosys mix qPCR program was: 37°C for 2 minutes, 95°C for 20 seconds, followed by cycling at 95°C for 1 second, then at 60°C for 20 seconds, for a total of 40 cycles.

[0451] 3.4 Data Analysis:

[0452] 3.4.1 Calculation of maximum inhibition rate

[0453] Same as section 3.4 of Example 14.

[0454] 3.4.2 Absolute IC 50 calculate

[0455] The dose-response curve was plotted and the IC50 value was calculated using GraphPad Prism software.

[0456] 4. Experimental Results

[0457] The degree of inhibition of the ACVR1C gene by some of the compounds of this invention in the SK-MEL-28 cell line is shown in Table 15. The numbers in the table are percentages, representing the degree of inhibition of ACVR1C mRNA.

[0458] Table 15. IC50 of the compounds of the present invention in inhibiting ACVR1C mRNA in the SK-MEL-28 cell line 50 and inhibition rate results

[0459] 5. Experimental Conclusions

[0460] The compounds of this invention exhibit good inhibitory activity against ACVR1C mRNA in vitro.

[0461] Example 18: Study on the effect of different concentrations of compounds on inhibiting ACVR1C mRNA in SK-MEL-28 cells using the free uptake method

[0462] 1. Experimental Objective: The purpose of this study is to evaluate the inhibitory effect of different concentrations of the compound described in this invention on ACVR1C mRNA in the SK-MEL-28 cell line, so as to evaluate the in vitro activity of the compound described in this invention.

[0463] 2. Experimental Materials

[0464] 2.1 Cell lines

[0465] Same as Example 16.

[0466] 2.2 Reagents

[0467] Same as Example 16.

[0468] 3. Experimental Procedures and Methods

[0469] 3.1 The cell drug delivery method is the same as section 3.1 of Example 17.

[0470] Set up group PC, where PC compound is compound AC006189 in patent WO2025137390, and the synthesis method is referred to patent WO2025137390);

[0471] 3) Remove the cells from the incubator, discard the culture medium, and rinse with PBS;

[0472] 4) Add 2 mL of trypsin and digest at 37°C until cells detach. Then add complete culture medium to stop digestion and mix thoroughly by pipetting.

[0473] 5) Centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in 5 mL of FBS-free medium;

[0474] 6) Add 180 μL of diluted cell suspension (i.e., 20,000 cells per well) to the experimental wells, and then place the cell plate in an incubator and incubate at 37°C and 5% CO2 for 72 h.

[0475] 3.2 RNA extraction and reverse transcription, same as section 3.2 of Example 17.

[0476] 3.3 qPCR detection, same as section 3.2 of Example 17.

[0477] 3.4 Calculation of maximum inhibition rate: Same as section 3.4 of Example 14.

[0478] 4. Experimental Results

[0479] The degree of inhibition of the ACVR1C gene by some of the compounds of this invention in the SK-MEL-28 cell line is shown in Table 16. The numbers in the table are percentages, representing the degree of inhibition of ACVR1C mRNA.

[0480] Table 16. Inhibition rate (%) of the compound of the present invention against ACVR1C mRNA in SK-MEL-28 cell line at different concentrations

[0481] 5. Experimental Conclusions

[0482] Experimental results show that the compounds of this invention exhibit concentration-dependent inhibitory effects, and each compound has excellent inhibitory activity against ACVR1C mRNA in SK-MEL-28 cells via free uptake, demonstrating good in vitro inhibitory activity against ACVR1C mRNA.

[0483] Example 19: Knockdown of human ACVR1C mRNA by the compound in humanized mice

[0484] 1. Experimental Objective

[0485] The modified siRNA and modified siRNA conjugates of the present invention were injected subcutaneously, and the in vivo knockdown activity of the compounds of the present invention was evaluated by detecting the expression of human ALK7 mRNA in mouse adipose tissue.

[0486] 2. Experimental Materials

[0487] hALK7 humanized female mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0488] 3. Experimental Methods

[0489] Mice were placed under quarantine for one week after arriving at the animal facility. On day 1, mice were randomly divided into groups of three according to their body weight. After grouping, all mice were subcutaneously injected with the compounds and conjugates described in Tables 2 and 3 of this invention, as a single dose at a volume of 10 mL / kg. The first group of mice was injected with DPBS, and the second group was injected with the control compound PC (compound PC was the same as in Example 18). The remaining groups of mice were injected with a DPBS solution of the compounds of this invention at a dose of 3 MPa.

[0490] On day 15, all mice were euthanized by CO2 inhalation. Different types of adipose tissue were collected from each mouse (inguinal white adipose tissue iWAT, perirenal adipose tissue piWAT, perigonial white adipose tissue pgWAT, interscapular adipose tissue BAT, and parametrial adipose tissue), approximately 30-100 mg per tissue. Liver, pituitary gland, muscle, pancreas, kidney, heart, and testes were also collected. Samples were treated with RNAlater overnight at 4°C, then the RNAlater was removed, and the samples were stored at -80°C for the detection of hALK7 gene expression levels.

[0491] The knockdown efficiency of hALK7 mRNA in adipose tissue was analyzed using qPCR. mArl1 was used as the internal reference gene. Data from each treatment group were normalized to the control group (DPBS) and the hALK7 gene inhibition rate in each mouse tissue was calculated.

[0492] 4. Experimental Results

[0493] On day 15, the degree of inhibition of the hALK7 gene in various tissues of mice by the compound of the present invention is shown in Tables 17-19. The results show that the compound of the present invention has good inhibitory activity on the hALK7 gene in the parauterine fat, inguinal fat and perirenal fat tissue of mice.

[0494] Table 17. Inhibition rate (%) of the compounds of this invention on hALK7 gene in parauterine and inguinal fat of mice.

[0495] Table 18. Inhibition rate (%) of the compounds of this invention on hALK7 gene in interscapular and inguinal fat of mice.

[0496] Table 19. Results of the inhibition rate (%) of the compounds of the present invention on the hALK7 gene in parauterine fat, inguinal fat, and perirenal fat of mice.

[0497] Example 20: Studying off-target effects of compounds in human primary adipocytes using RNA-Seq technology

[0498] 1. Experimental Objective: The purpose of this study is to evaluate the effects of the compounds described in this invention on gene expression in human primary adipocyte lines, in order to assess the potential off-target risks of the compounds of this invention.

[0499] 2. Experimental Materials

[0500] 2.1 Cell lines

[0501] Primary adipocytes (Brand: Pronosai, Product No.: CP-H112)

[0502] 2.2 Reagents

[0503] Same as Example 15, except that Trizol (R0016) is added.

[0504] 3. Experimental Procedures and Methods

[0505] 3.1 Cell Plating

[0506] 1) Add 500 μL of gelatin to a 12-well plate, incubate at 37°C for 30 min, then discard the supernatant. Dry in a clean bench.

[0507] 2) Remove the cells to be transfected from the incubator, discard the culture medium, and rinse with PBS; add 2 mL of trypsin, digest at 37°C until the cells detach, add complete culture medium to stop digestion, and mix by pipetting.

[0508] 3) Centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cells in 5 ml of culture medium;

[0509] 4) Add 1000 μL of diluted cell suspension (i.e., 250,000 cells per well) to 24 wells and incubate at 37°C and 5% CO2 for 3 days.

[0510] 3.2 Cell transfection and drug administration

[0511] 1) Change the medium, discard the culture medium from the previous step, and add 800 μL of culture medium.

[0512] 2) siRNA dilution: Add 4 μL of the stock solution to 396 μL of PTI-MEM, mix well, and the concentration after dilution is 200 nM.

[0513] 3) Prepare RNAiMAX mix: Add 60 μL of RNAiMAX transfection reagent to 1940 μL of OPTI-MEM, mix well, and incubate at room temperature for 15 min.

[0514] 4) Take 350 μl of each diluted molecule and add it to 350 μl of RNAiMAX mix, and incubate at room temperature for 15 min.

[0515] 5) Add 200 μL of the mix from the previous step to each well, and repeat the process 3 times per molecule.

[0516] 3.3 Cell lysis

[0517] After incubation for 24 hours, the cells were lysed and frozen at -80°C according to the Trizol (R0016) reagent instructions.

[0518] 3.4 Database Construction and Analysis

[0519] Sample lysis buffers were sent to Beijing Novogene Technology Co., Ltd., where library construction and high-throughput sequencing were performed according to the company's standard operating procedures (SOP). Sequencing data were analyzed for bioinformatics using the company's cloud platform. Differentially expressed genes were identified using a corrected p-value (padj) ≤ 0.05 and |log2FoldChange| ≥ 1 as screening thresholds.

[0520] 4. Experimental Results

[0521] The number of genes regulated by the compounds of the present invention in human primary adipocytes under the current testing conditions is shown in Table 20 below. The results show that the compounds of the present invention have a lower off-target risk compared with the control group.

[0522] Table 20. Number of genes regulated by the compounds of the present invention in human primary adipocytes

[0523] Example 21: Studying off-target effects of compounds in SK-MEL-28 cells using RNA-Seq technology

[0524] 1. Experimental Objective: The purpose of this study is to evaluate the effect of the compounds described in this invention on gene expression in the SK-MEL-28 cell line, in order to assess the potential off-target risk of the compounds of this invention.

[0525] 2. Experimental Materials

[0526] 2.1 Cell line: Same as in Example 16.

[0527] 2.2 Reagents: Same as in Example 16, except Trizol (R0016) is added.

[0528] 3. Experimental Procedures and Methods

[0529] 3.1 Transfection: Same as section 3.1 in Example 16.

[0530] 3.2 Cell lysis

[0531] After incubation for 24 hours, the cells were lysed and frozen at -80°C according to the Trizol (R0016) reagent instructions.

[0532] 3.3 Database Construction and Analysis

[0533] Sample lysis buffers were sent to Beijing Novogene Technology Co., Ltd., where library construction and high-throughput sequencing were performed according to the company's standard operating procedures (SOP). Sequencing data were analyzed for bioinformatics using the company's cloud platform. Differentially expressed genes were identified using a corrected p-value (padj) ≤ 0.05 and |log2FoldChange| ≥ 1 as screening thresholds.

[0534] 4. Experimental Results

[0535] The number of genes regulated by the compounds of the present invention in the SK-MEL-28 cell line under the current testing conditions is shown in Tables 21-22 below. The results indicate that the compounds of the present invention have a low off-target risk.

[0536] Table 21. Number of regulatory genes in the SK-MEL-28 cell line

[0537] Table 22. Number of regulatory genes in the SK-MEL-28 cell line

[0538] Example 22: Study on the stability of fat homogenate

[0539] 1. Experimental objective: The objective of this study is to evaluate the stability of the compounds described in this invention in fat homogenates, thereby assessing the stability of the compounds of this invention.

[0540] 2. Experimental Materials

[0541] Mouse fat homogenate

[0542] 3. Experimental Procedures and Methods

[0543] The compound of this invention was co-incubated with mouse fat homogenate for 48 hours at a temperature of 37 degrees Celsius. The remaining amount of antisense strand was analyzed by high-resolution mass spectrometry.

[0544] 4. Experimental Results

[0545] The remaining amount of antisense chain after incubation at different time points of the present invention is shown in the table below.

[0546] Table 23. Results of Antisense Chain Residue Measurement

[0547] 5. Experimental Conclusions

[0548] The compounds of this invention exhibit good stability.

[0549] Example 23: In vivo efficacy experiment in hALK7 DIO mice

[0550] 1. Experimental Objective

[0551] The modified siRNAs described in Tables 2 and 3 of this invention were injected subcutaneously, and the in vivo activity of the compounds of this invention was evaluated by detecting changes in the body weight of mice.

[0552] 2. Experimental Materials

[0553] hALK7 humanized DIO mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0554] 3. Experimental Methods

[0555] On day 1, mice were randomly divided into groups of 7 mice each according to their body weight. After grouping, the first group of mice was injected with saline, and the second group of mice was injected with PC (the same PC as in Example 18). All mice were subcutaneously injected with the modified siRNA or modified siRNA conjugates described in Tables 2 and 3 of this invention, at a volume of 10 mL / kg. The control group mice were injected with saline, and the experimental group mice were injected with a saline solution containing the compounds described in this invention, at a dose of 3 MPa; administration was performed once a week. Changes in mouse body weight and food intake were recorded every 3 days.

[0556] 4. Experimental Results

[0557] The rate of change in body weight of hALK7 DIO mice after injection of the compound of the present invention is shown in Table 24.

[0558] Table 24. Results of body weight changes in the hALK7 DIO mouse model

[0559] 5. Experimental Conclusions

[0560] After injection of the compounds of this invention, in the hALK7 DIO mouse model, each compound showed varying degrees of weight-reducing effect, with a reduction of more than 9% compared to the saline group, and the weight loss effect was better than that of the control group at 81 days.

[0561] Example 24: ACVR1C mRNA knockdown experiment in cynomolgus monkeys

[0562] 1. Experimental Principle

[0563] The modified siRNA and modified siRNA conjugates of the present invention were injected subcutaneously, and the in vivo activity of the compounds of the present invention was evaluated by detecting the expression of ALK7 mRNA in the abdominal adipose tissue of cynomolgus monkeys.

[0564] 2. Experimental Materials

[0565] The crab-eating macaques were purchased from Hainan Jingang Biotechnology Co., Ltd.

[0566] 3. Experimental Methods

[0567] Abdominal fat was collected from each group of animals on day 2 or 7 before the start of the experiment.

[0568] On day 1, the cynomolgus monkeys were randomly divided into groups of 3 according to their body weight. After grouping, all cynomolgus monkeys were subcutaneously injected with the modified siRNA and modified siRNA conjugates described in Tables 2 and 3 of this invention, as a single dose at a volume of 0.5 mL / kg. The first group of cynomolgus monkeys was injected with DPBS, and the second group was injected with the control compound PC, which was the same as in Example 18. The remaining groups of cynomolgus monkeys were injected with a DPBS solution of the compounds described in this invention at a dose of 3 MPa.

[0569] On day 28 after drug administration, subcutaneous fat was collected from the abdomen of all cynomolgus monkeys via biopsy. The samples were treated with RNAlater overnight at 4°C, then the RNAlater was removed and the samples were stored at -80°C for the detection of hALK7 gene expression levels.

[0570] The knockdown efficiency of ALK7 mRNA in adipose tissue was analyzed using qPCR. mArl1 was used as the internal reference gene. Data from each treatment group were normalized to the control group (DPBS).

[0571] 4. Experimental Results

[0572] It can significantly reduce the expression of the ALK7 gene in the abdominal fat of cynomolgus monkeys, indicating that the compound of the present invention has good in vivo activity.

[0573] Example 25: Comparison Experiment of Plasma and Fat in Crab-Eating Monkeys

[0574] 1. Experimental Objective

[0575] Subcutaneous injection of the modified siRNA and its conjugates as described in Tables 2 and 3 of this invention was performed, and the pharmacokinetic (PK) properties of the compounds were analyzed by detecting the drug concentrations in the plasma and adipose tissue of cynomolgus monkeys.

[0576] 2. Experimental Materials

[0577] Same as Example 24.

[0578] 3. Experimental Methods

[0579] On day 1, the cynomolgus monkeys were randomly divided into groups of 3 according to their body weight. After grouping, all cynomolgus monkeys were subcutaneously injected with the modified siRNA or modified siRNA conjugates described in Tables 2 and 3 of this invention, as a single dose at a volume of 0.5 mL / kg. The first group of cynomolgus monkeys was injected with DPBS; the second group was injected with the control compound PC, which was the same as in Example 18; the remaining groups were injected with a DPBS solution of the compounds described in this invention at a dose of 3 MPa.

[0580] Plasma samples were collected from cynomolgus monkeys at 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, and 48 h post-drug administration. Abdominal adipose tissue samples were collected on days 14, 28, and 56 post-drug administration. The concentrations of the sense and antisense chains of the compounds in the plasma and abdominal adipose tissue of the cynomolgus monkeys were analyzed, and p-q parameters were fitted.

[0581] 4. Experimental Results

[0582] The composition of this invention exhibits good pharmacokinetic properties in cynomolgus monkeys, with systemic exposure and half-life superior to the PC group. It also shows good early accumulation ability in abdominal adipose tissue, providing pharmacokinetic evidence for the sustained pharmacological effect of the compound of this invention at the adipose tissue target.

[0583] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A siRNA targeting the ACVR1C gene, said siRNA comprising a sense strand and an antisense strand, characterized in that, The antisense strand comprises at least 15 consecutive nucleotides that are approximately 3 (e.g., 0, 1, 2, 3) nucleotides similar to the sequences or modified fragments thereof shown in any of SEQ ID NO:51-100, 222-233, 352, and 357, and the sense and antisense strands are at least partially complementary; the siRNA inhibits the expression level of the ACVR1C gene.

2. The siRNA as described in claim 1, characterized in that, The antisense strand comprises at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides or modified sequences thereof in any of the sequences shown in SEQ ID NO:51-100, 222-233, 352, and 357.

3. The siRNA as described in claim 1 or 2, characterized in that, The positive strand comprises at least 15 consecutive nucleotides having a nucleotide sequence of approximately 3 (e.g., 0, 1, 2, or 3) nucleotides, as shown in any of SEQ ID NO:1-50, 101, 210-221, and 358. Preferably, the positive chain comprises at least 15 consecutive, at least 16, at least 17, at least 18, or at least 19 nucleotides or modified sequences thereof in any of the sequences shown in SEQ ID NO:1-50, 101, 210-221, and 358.

4. The siRNA according to any one of claims 1-3, characterized in that, The sense and antisense strands of the siRNA each independently comprise 16-25 nucleotides in length; Preferably, the sense and antisense strands of the siRNA each independently comprise 18-23 nucleotides (e.g., the sense strand is 18 or 21 nucleotides long, and the antisense strand is 23 nucleotides long).

5. The siRNA according to any one of claims 1-4, characterized in that, The siRNA comprises blunt ends and / or 1-3 (e.g., 1, 2, or 3) nucleotide overhangs; Preferably, the siRNA contains one or two nucleotide overhangs; Preferably, the protruding end is present at the 5' end and / or 3' end of the antisense chain and / or the justice chain; Preferably, the 3' end of the antisense strand of the siRNA contains a 2- or 5-nucleotide overhang.

6. The siRNA according to any one of claims 1-5, characterized in that, The sense strand and the antisense strand have a mismatch of no more than 5 nucleotides (e.g., 0, 1, 2, 3, 4 or 5).

7. The siRNA according to any one of claims 1-6, characterized in that, The nucleotide sequence of the antisense strand is as shown in any of SEQ ID NO:51-100, 222-233, 352 and 357 or a modified sequence thereof; and / or, the nucleotide sequence of the sense strand is as shown in any of SEQ ID NO:1-50, 101, 210-221 and 358 or a modified sequence thereof.

8. The siRNA according to any one of claims 1-7, characterized in that, The siRNA comprises the sense and antisense strand sequences selected from any of the A1-A66 siRNAs described in Table 1.

9. The siRNA as described in claim 8, characterized in that, The modified sequences of the sense strand and the antisense strand each independently contain at least one modified nucleotide; Preferably, all nucleotides in the sense and / or antisense strands of the siRNA are modified nucleotides or nucleotide analogs.

10. The siRNA as described in claim 9, characterized in that, The modified nucleotide is selected from one or more of the group consisting of: deoxy-nucleotides, 3'-terminal deoxy-thymidine nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinyl nucleotides, aminophosphates, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl alcohol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing aminophosphate groups, nucleotides containing methylphosphate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimics, and nucleotides with the following structures: And / or, the first nucleotide at the 5' position of the modified sequence of the antisense strand, starting from the 5' end, contains a phosphate ester structure; And / or, the modified sequence of the positive strand is connected to one or more inverse abase residues (invAb) at the 5' and / or 3' ends.

11. The siRNA as described in claim 10, characterized in that, The sense and / or antisense strands of the siRNA contain modified internucleotide links; Preferably, the 5' end and 3' end of the sense chain each independently contain one or two thiophosphate groups; and / or the 5' end and 3' end of the antisense chain each independently contain one or two or three thiophosphate groups.

12. The siRNA as described in claim 11, characterized in that, The modified sequence of the antisense strand comprises at least 15 consecutive nucleotides that are approximately 3 (e.g., 0, 1, 2, or 3) similar to any of the sequences shown in SEQ ID NO:155-208, 264-304, 327-328, or 334-336. Preferably, the modified sequence of the positive strand comprises at least 15 consecutive nucleotides that are approximately 3 (e.g., 0, 1, 2, or 3) similar to any of the sequences shown in SEQ ID NO:102-153, 234-263, 353-356, and 359; Preferably, the justice chain and the antisense chain are complementary by at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

13. The siRNA as described in claim 12, characterized in that, The sequences of the sense and antisense strands of the siRNA are selected from any of the sense and antisense strand sequences of the modified siRNAs listed in Table 2, namely B001-B094, B105, and B114-B142.

14. A conjugate of siRNA, characterized in that, It includes siRNA or a fragment thereof as described in any one of claims 1-13, and a ligand conjugated to said siRNA or a fragment thereof; Preferably, one, two, or three ligands are attached to the sense chain and / or antisense chain.

15. The siRNA conjugate according to claim 14, characterized in that, The ligand is selected from: lipid groups, small molecule ligands and / or peptides; the lipid group contains at least one lipophilic group, and the lipophilic group contains 4-40 carbon atoms; a ligand is attached to the 5' and / or 3' ends of the positive chain; Optionally, the lipophilic group is covalently conjugated to the siRNA or a fragment thereof.

16. The siRNA conjugate according to claim 14 or 15, characterized in that, The ligand is selected from one or more lipid groups shown in the following structures: Preferably, the positive chain is connected to the lipid group via a covalent bond or a linker, wherein the covalent bond is selected from phosphate ester bonds, phosphothioester bonds, and thioether bonds; the linker is, for example, C6-S, with the following structure:

17. The siRNA conjugate according to any one of claims 14-16, characterized in that, The lipid group is attached to the 5' end, 3' end, or middle position of the positive chain; Preferably, any of the following positions 1)-5): 1) The lipid group LC-207 is connected to the 5' end of the positive chain via linker C6-S, and the lipid group LC-044 is connected to the 3' end of the positive chain; 2) The lipid group LC-044 is attached to the 5' end of the positive chain, and the lipid group LC-164 is attached to the 3' end of the positive chain; 3) The lipid group LC-207 is connected to the 5' end of the positive chain via the linker C6-S, and the lipid group LC-205 or LC-231 is connected to the 3' end of the positive chain; 4) The lipid group LC-206 is connected to the 5' end of the positive chain via the linker C6-S. Optionally, the lipid group LC-205 is connected to the 3' end of the positive chain. 5) The lipid group LC-202 is attached to the 3rd nucleotide at the 5' end of the positive strand.

18. The conjugate as claimed in claim 17, characterized in that, The antisense strand of the conjugate is approximately equal to no more than 3, 2 or 1 bases in at least 15 consecutive bases compared to any of the sequences shown in SEQ ID NO:327-341; Preferably, the positive chain of the conjugate has approximately 3, 2, or 1 bases in at least 15 consecutive bases compared to the sequences shown in any of SEQ ID NO:305-326 and 342-351.

19. The conjugate according to any one of claims 14-18, characterized in that, The conjugate is selected from any of the following: The conjugate comprises a sense chain as shown in SEQ ID NO:347, and an antisense chain as shown in SEQ ID NO:328; The conjugate comprises a sense chain as shown in SEQ ID NO:343, and an antisense chain as shown in SEQ ID NO:327; The conjugate comprises a sense chain as shown in SEQ ID NO:344, and an antisense chain as shown in SEQ ID NO:319; The conjugate comprises a sense chain as shown in SEQ ID NO:345, and an antisense chain as shown in SEQ ID NO:329; The conjugate comprises a sense chain as shown in SEQ ID NO:346, and an antisense chain as shown in SEQ ID NO:327; The conjugate comprises a sense chain as shown in SEQ ID NO:348, and an antisense chain as shown in SEQ ID NO:329; The conjugate comprises a sense chain as shown in SEQ ID NO:343, and an antisense chain as shown in SEQ ID NO:334; The conjugate comprises a sense chain as shown in SEQ ID NO:347, and an antisense chain as shown in SEQ ID NO:335; The conjugate comprises a sense chain as shown in SEQ ID NO:348, and an antisense chain as shown in SEQ ID NO:336; The conjugate comprises a sense strand as shown in SEQ ID NO:350, and an antisense strand as shown in SEQ ID NO:335; The conjugate comprises a sense chain as shown in SEQ ID NO:350, and an antisense chain as shown in SEQ ID NO:328; The conjugate comprises a sense chain as shown in SEQ ID NO:305, and an antisense chain as shown in SEQ ID NO:327; The conjugate comprises a sense chain as shown in SEQ ID NO:306, and an antisense chain as shown in SEQ ID NO:328; The conjugate comprises a sense chain as shown in SEQ ID NO:307 and an antisense chain as shown in SEQ ID NO:329; The conjugate comprises a sense chain as shown in SEQ ID NO:308, and an antisense chain as shown in SEQ ID NO:327; The conjugate comprises a sense chain as shown in SEQ ID NO:309, and an antisense chain as shown in SEQ ID NO:328; The conjugate comprises a sense chain as shown in SEQ ID NO:310, and an antisense chain as shown in SEQ ID NO:329; The conjugate comprises a sense chain as shown in SEQ ID NO:311 and an antisense chain as shown in SEQ ID NO:329; The conjugate comprises a sense chain as shown in SEQ ID NO:312, and an antisense chain as shown in SEQ ID NO:330; The conjugate comprises a sense chain as shown in SEQ ID NO:313, and an antisense chain as shown in SEQ ID NO:331; The conjugate comprises a sense chain as shown in SEQ ID NO:314, and an antisense chain as shown in SEQ ID NO:330; The conjugate comprises a sense chain as shown in SEQ ID NO:315, and an antisense chain as shown in SEQ ID NO:331; The conjugate comprises a sense chain as shown in SEQ ID NO:316, and an antisense chain as shown in SEQ ID NO:332; The conjugate comprises a sense chain as shown in SEQ ID NO:317 and an antisense chain as shown in SEQ ID NO:333; The conjugate comprises a sense chain as shown in SEQ ID NO:318, and an antisense chain as shown in SEQ ID NO:333; The conjugate comprises a sense chain as shown in SEQ ID NO:319, and an antisense chain as shown in SEQ ID NO:327; The conjugate comprises a sense chain as shown in SEQ ID NO:320, and an antisense chain as shown in SEQ ID NO:328; The conjugate comprises a sense chain as shown in SEQ ID NO:321, and an antisense chain as shown in SEQ ID NO:329; The conjugate comprises a sense chain as shown in SEQ ID NO:322, and an antisense chain as shown in SEQ ID NO:329; The conjugate comprises a sense chain as shown in SEQ ID NO:323, and an antisense chain as shown in SEQ ID NO:332; The conjugate comprises a sense chain as shown in SEQ ID NO:324, and an antisense chain as shown in SEQ ID NO:330; The conjugate comprises a sense chain as shown in SEQ ID NO:325, and an antisense chain as shown in SEQ ID NO:331; The conjugate comprises a sense chain as shown in SEQ ID NO:326, and an antisense chain as shown in SEQ ID NO:333; The conjugate comprises a sense chain as shown in SEQ ID NO:305, and an antisense chain as shown in SEQ ID NO:334; The conjugate comprises a sense strand as shown in SEQ ID NO:306, and an antisense strand as shown in SEQ ID NO:335; The conjugate comprises a sense chain as shown in SEQ ID NO:307 and an antisense chain as shown in SEQ ID NO:336; The conjugate comprises a sense chain as shown in SEQ ID NO:308, and an antisense chain as shown in SEQ ID NO:334; The conjugate comprises a sense strand as shown in SEQ ID NO:309, and an antisense strand as shown in SEQ ID NO:335; The conjugate comprises a sense chain as shown in SEQ ID NO:310, and an antisense chain as shown in SEQ ID NO:336; The conjugate comprises a sense chain as shown in SEQ ID NO:311 and an antisense chain as shown in SEQ ID NO:336; The conjugate comprises a sense chain as shown in SEQ ID NO:312, and an antisense chain as shown in SEQ ID NO:337; The conjugate comprises a sense chain as shown in SEQ ID NO:313, and an antisense chain as shown in SEQ ID NO:338; The conjugate comprises a sense chain as shown in SEQ ID NO:314, and an antisense chain as shown in SEQ ID NO:337; The conjugate comprises a sense chain as shown in SEQ ID NO:315, and an antisense chain as shown in SEQ ID NO:338; The conjugate comprises a sense chain as shown in SEQ ID NO:316, and an antisense chain as shown in SEQ ID NO:339; The conjugate comprises a sense chain as shown in SEQ ID NO:317, and an antisense chain as shown in SEQ ID NO:340; The conjugate comprises a sense chain as shown in SEQ ID NO:318, and an antisense chain as shown in SEQ ID NO:340; The conjugate comprises a sense chain as shown in SEQ ID NO:328, and an antisense chain as shown in SEQ ID NO:334; The conjugate comprises a sense chain as shown in SEQ ID NO:320, and an antisense chain as shown in SEQ ID NO:335; The conjugate comprises a sense chain as shown in SEQ ID NO:321 and an antisense chain as shown in SEQ ID NO:336; The conjugate comprises a sense chain as shown in SEQ ID NO:322, and an antisense chain as shown in SEQ ID NO:336; The conjugate comprises a sense chain as shown in SEQ ID NO:323, and an antisense chain as shown in SEQ ID NO:341; The conjugate comprises a sense chain as shown in SEQ ID NO:326, and an antisense chain as shown in SEQ ID NO:340; The conjugate comprises a sense chain as shown in SEQ ID NO:342, and an antisense chain as shown in SEQ ID NO:328; The conjugate comprises a sense chain as shown in SEQ ID NO:344, and an antisense chain as shown in SEQ ID NO:335; The conjugate comprises a sense chain as shown in SEQ ID NO:345, and an antisense chain as shown in SEQ ID NO:336; The conjugate comprises a sense chain as shown in SEQ ID NO:346, and an antisense chain as shown in SEQ ID NO:334; The conjugate comprises a sense chain as shown in SEQ ID NO:349, and an antisense chain as shown in SEQ ID NO:328; The conjugate comprises a sense strand as shown in SEQ ID NO:349, and an antisense strand as shown in SEQ ID NO:335; The conjugate comprises a sense chain as shown in SEQ ID NO:350, and an antisense chain as shown in SEQ ID NO:328; The conjugate comprises a sense chain as shown in SEQ ID NO:351 and an antisense chain as shown in SEQ ID NO:327; The conjugate comprises a sense chain as shown in SEQ ID NO:351 and an antisense chain as shown in SEQ ID NO:

334.

20. A delivery composition, characterized in that, The delivery composition comprises siRNA as described in any one of claims 1-13 or a conjugate as described in any one of claims 14-19, and optionally further comprises a delivery vector; Preferably, the delivery vector is selected from: liposomes, lipid nanoparticles, polymer nanoparticles, exosomes, viral vectors, inorganic nanoparticles, bioconjugates; and / or, the siRNA molecule is linked to the delivery vector.

21. A nucleic acid protein composition, characterized in that, The nucleic acid protein composition comprises siRNA as described in any one of claims 1-13, a conjugate as described in any one of claims 14-19, or a delivery composition as described in claim 20, and a nuclease; preferably, the nuclease is an AGO protein; Alternatively, the nucleic acid protein composition comprises siRNA as described in any one of claims 1-13, a conjugate as described in any one of claims 14-19, or an antisense strand in the delivery composition as described in claim 20, and a nuclease; preferably, the nuclease is an AGO protein.

22. A recombinant vector, characterized in that, The recombinant vector comprises the coding sequence of siRNA as described in any one of claims 1-8; preferably, the starting vector of the recombinant vector is a plasmid or a viral vector.

23. A transformant, characterized in that, The transformant comprises the siRNA as described in any one of claims 1-8 or the recombinant vector as described in claim 22; the host cell of the transformant is a eukaryotic cell or a prokaryotic cell; Preferably, the transformant is neither a plant variety nor an animal variety.

24. A method for preparing siRNA as described in any one of claims 1-8, characterized in that, The method includes culturing the transformant as described in claim 23, or directly obtaining the nucleic acid molecule using chemical synthesis and mixing.

25. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises siRNA as described in any one of claims 1-13, conjugates as described in any one of claims 14-19, delivery compositions as described in claim 20, recombinant vectors as described in claim 22, or transformants as described in claim 23, as well as pharmaceutically acceptable carriers and / or excipients.

26. A medicine box set, characterized in that, The kit includes kit A, which contains a reagent kit, the reagent kit comprising one or more of the following: siRNA as described in any one of claims 1-13, conjugates as described in any one of claims 14-19, delivery composition as described in claim 20, recombinant vector as described in claim 22, transformant as described in claim 23, or pharmaceutical composition as described in claim 25; Preferably, the pillbox further includes a pillbox B, which contains one or more of the following: (1) Other drugs that inhibit ACVR1C gene expression or compositions containing said drugs that inhibit ACVR1C gene expression; and (2) One or more of the following groups: hormone preparations, targeted small molecule preparations, proteasome inhibitors, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

27. The use of the siRNA as described in any one of claims 1-13, the conjugate as described in any one of claims 14-19, the delivery composition as described in claim 20, the recombinant vector as described in claim 22, the transformant as described in claim 23, or the pharmaceutical composition as described in claim 25 in the preparation of a medicament for treating and / or preventing pathological conditions or diseases related to ACVR1C gene expression; Preferably, the ACVR1C gene expression-related diseases are selected from one or more of the following: metabolic diseases, cardiovascular diseases, liver diseases, kidney diseases, endocrine diseases, and tumor diseases; More preferably, the metabolic disease is selected from obesity (e.g., abdominal obesity), overweight, insulin resistance, type 2 diabetes, metabolic syndrome, lipid disorders (such as hyperlipidemia, hypertriglyceridemia or dyslipidemia), lipoprotein abnormalities and impaired glucose tolerance (IGT). The cardiovascular diseases mentioned are selected from heart disease (such as heart disease including coronary heart disease, congestive heart failure), stroke, peripheral vascular disease, atherosclerosis, arteriosclerosis and hypertension; The liver diseases mentioned are selected from inflammation (e.g., liver inflammation and / or adipose tissue inflammation) and fatty liver disease (including non-alcoholic fatty liver disease); The kidney disease mentioned is selected from kidney lesions (e.g., nephropathy); The endocrine disease mentioned is selected from pancreatitis; The tumor diseases mentioned are selected from cancers (such as myeloma (e.g., multiple myeloma, plasmacytoma, localized myeloma or extramedullary myeloma), melanoma, ovarian cancer, breast cancer, colon cancer, endometrial cancer, liver cancer, kidney cancer, pancreatic cancer, stomach cancer, uterine cancer).

28. A method for inhibiting the expression level of the ACVR1C gene in vitro or in vivo, characterized in that, The method comprises administering one or more of the following to an in vivo or in vitro sample: siRNA as described in any one of claims 1-13, conjugates as described in any one of claims 14-19, delivery compositions as described in claim 20, pharmaceutical compositions as described in claim 25, and kits as described in claim 26, to reduce the expression level of the ACVR1C gene. Preferably, the method is for non-therapeutic purposes.

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