Nucleic acid for inhibiting the expression of activin receptor type iib gene and use thereof

Abstract

Disclosed in the present invention are an antisense oligonucleotide or RNAi agent for inhibiting the expression of the ACVR2B gene in cells and a pharmaceutical composition thereof. The present invention also relates to the therapeutic use of the antisense oligonucleotide and RNAi agent.

Description

Nucleic acids for inhibiting activin type IIB receptor gene expression and uses thereof Technical Field

[0001] The present invention relates to nucleic acids that inhibit the expression of activin type IIB receptor (ACVR2b) genes in cells, including antisense oligonucleotides (ASOs) and RNAi agents (such as siRNAs) and pharmaceutical compositions thereof. The present invention also relates to the medical uses of these nucleic acids. Background Art

[0002] Activin type IIB receptor (ACVR2B) is a type II serine / threonine kinase receptor of the transforming growth factor-β (TGF-β) superfamily. The TGF-β superfamily includes secreted polypeptide signaling molecules such as TGF-β, activins, inhibins (INH), bone morphogenetic proteins (BMPs), growth differentiation factor (GDP), myostatin (MSTN), follistatin (FST), and the Smad protein family. The mechanism of action of the TGF-β signaling pathway is that TGF-β superfamily members bind to ACVR2B on the cell surface, which then recruits activin type I receptor (ActRI). The resulting ligand-receptor complex activates ActRI kinases (ALK4 / 5), leading to phosphorylation of the Smad family of transcription factors (primarily Smad2 and Smad3). These then bind to Smad4 to form a complex that enters the cell nucleus and regulates the target genes of multiple transcription factors, thereby controlling the proliferation, differentiation, migration, and apoptosis of various cells. The TGF-β signaling pathway plays a crucial regulatory role in embryonic development, skeletal formation, and muscle growth and development. Research has found that genetic deletion of the key gene Dystrophin in mice modeling Duchenne muscular dystrophy (DMD), a clinically intractable disease, is accompanied by a significant increase in TGF-β expression. Similar findings have been found in mice modeling Marfan syndrome (MFS), where genetic deletion of the key gene FBN1 is also associated with a significant increase in TGF-β expression. Skeletal muscle in both animal models lacks the ability to repair and regenerate after injury, but blocking the TGF-β pathway allows skeletal muscle to regain its repair and regenerative function.

[0003] Muscle atrophy is a debilitating and life-threatening condition associated with a variety of chronic, neurological, genetic, inflammatory, fibrotic, or infectious conditions, including muscular dystrophy, amyotrophic lateral sclerosis (ALS), myositis, cancer, rheumatoid arthritis, osteoarthritis, diabetes, sarcopenic obesity, androgen deficiency, corticosteroid myopathy, inflammatory bowel disease, cirrhosis, chronic obstructive pulmonary disease, pulmonary fibrosis, chronic kidney disease, trauma, cardiomyopathy, chronic heart failure, and HIV infection. The SMAD pathway, downstream of ACVR2B, regulates numerous muscle-building genes, such as myogenic determinant (MyoD), myogenin, and myogenic factor 5 (Myf5), which are involved in cell hypertrophy, proliferation, and differentiation. Studies have shown that inhibition of ACVR2B leads to rapid and robust muscle growth, making it a promising treatment for conditions such as muscle atrophy and muscular dystrophy.

[0004] Studies have shown that inhibiting MSTN pathway signaling by inhibiting ACVR2B can reduce overall fat content in mice, improve glucose metabolism, increase insulin sensitivity and energy expenditure, and even promote the conversion of white fat to brown fat, thereby exerting systemic anti-diabetic and anti-obesity functions. Furthermore, increasing evidence suggests that the MSTN / activin pathway not only affects the heart and blood vessels but also influences vascular remodeling, cellular aging, and fibrosis. Therefore, inhibition of the MSTN pathway may play an important role in the weight loss, inflammation, vascular damage, and potentially progressive renal dysfunction seen in patients with chronic kidney disease (CKD).

[0005] Among the many ligands of ACVR2B, activin A promotes the synthesis and secretion of gonadotropins by the pituitary gland to regulate reproductive physiology. Besides being expressed in reproductive organs, it is also present in other organs and systems, such as the brain, liver, lungs, kidneys, bones, and intestines. Activin A is expressed during renal development and plays a crucial role in kidney development and repair. It delays ureteric bud branching in mice and reduces the size of the mouse metanephros in vitro. It is a negative regulator of kidney development and is involved in renal pathologies such as renal fibrosis. Elevated activin A levels in chronic kidney disease can lead to anemia and promote the development of renal fibrosis, exacerbating disease progression. Furthermore, activin A can exacerbate renal damage caused by diseases such as systemic lupus erythematosus, vasculitis, and multiple myeloma (MM). Reducing activin A levels by inhibiting ACVR2A and ACVR2B can prevent acute kidney injury, alleviate renal anemia, improve mineral and bone abnormalities, and inhibit the development of renal fibrosis.

[0006] Currently, there are no small nucleic acid drugs targeting this target on the market, and there is still a need to develop such drugs with better efficacy, long-term effectiveness, specific targeting and / or safety. Summary of the Invention

[0007] The present invention is dedicated to targeting the ACVR2B gene through nucleic acid drugs (such as siRNA or ASO), specifically and efficiently inhibiting target genes and proteins in target organs, and can block a variety of diseases caused by abnormal TGF-β and its downstream signaling pathways, such as muscle atrophy, muscular dystrophy, obesity, diabetes, chronic kidney disease, acute kidney injury, postoperative healing of trauma, maxillofacial tumors, eclampsia during pregnancy, liver fibrosis, atherosclerosis, ischemic stroke, Alzheimer's disease, etc.

[0008] One aspect of the invention provides an antisense oligonucleotide comprising an oligonucleotide consisting of 12 to 30 linked nucleotides, wherein the oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 nucleotides that are at least 80%, 85%, 90%, 95% or 100% complementary to an equal length of consecutive nucleotides of a target region of an mRNA encoding an activin type IIB receptor (ACVR2B).

[0009] In some embodiments, the target region is 504-554, 588-617, 648-686, 837-866, 1234-1268, 1360-1403, 1503-1532 or 1545-1574 of the nucleotide sequence shown in SEQ ID NO:1.

[0010] In some embodiments, the target region is 504-533, 510-539, 519-548, 525-554, 588-617, 648-677, 653-682, 657-686, 837-866, 1234-1263, 1239-1268, 1360-1389, 1374-1403, 1503-1532 or 1545-1574 of the nucleotide sequence shown in SEQ ID NO: 1. In some embodiments, the target region is 509-528, 515-534, 524-543, 530-549, 593-612, 653-672, 658-677, 662-681, 842-861, 1239-1258, 1244-1263, 1365-1384, 1379-1398, 1508-1527 or 1550-1569 of the nucleotide sequence shown in SEQ ID NO: 1.

[0011] In some embodiments, the oligonucleotide comprises a nucleotide sequence of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleotides of any one of SEQ ID NOs: 3 to 17.

[0012] In some embodiments, the antisense oligonucleotide has a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to the nucleotide sequence shown in SEQ ID NO.1 and / or SEQ ID NO.2 when measured over the entire nucleotide sequence of the antisense oligonucleotide.

[0013] In some embodiments, the antisense-oligonucleotide comprises at least one modification selected from a modified sugar moiety, a modified nucleobase moiety, and a modified internucleotide linkage, preferably, the antisense-oligonucleotide comprises one or more of the following modifications: (i) at least one bicyclic sugar moiety, wherein the bicyclic sugar moiety preferably has a 4'-2' bridge, and the 4'-2' bridge is preferably cEt or LNA; (ii) at least one non-bicyclic modified sugar moiety, preferably selected from 2'-deoxy, 2'-F, 2'-MOE, and 2'-OMe sugar moieties; (iii) a modified nucleoside, preferably comprising 5-methylcytosine (m5C); and (iv) at least one sugar surrogate, preferably selected from POM, PNA, THP, and F-HNA.

[0014] In some embodiments, the antisense oligonucleotide comprises a gapmer. In some embodiments, the gapmer comprises: (a) a 5' region consisting of 1-6 linked 5' region nucleotides; (b) a central region consisting of 6-10 linked central region nucleotides; and (c) a 3' region consisting of 1-6 linked 3' region nucleotides; wherein each of the nucleotides in the 5' region and the 3' region comprises a sugar moiety selected from 2'-MOE, LNA, and cEt modifications, and at least 6 of the nucleotides in the central region comprise a 2'-deoxy sugar moiety.

[0015] In some embodiments, the antisense oligonucleotide has a sugar motif (5' to 3') selected from the following: eekddddddddddkke, ekkddddddddddkke, kkkdyddddddddkkk, kkkddydddddddkkk, kkkdddyddddddkkk, kkkddddddddddddkkk or eeeeeddddddddddeeeee; wherein e represents a 2'-MOE sugar moiety, k represents a cEt sugar moiety, d represents a 2'-deoxy sugar moiety, and y represents a 2'-OMe sugar moiety.

[0016] In some embodiments, the antisense-oligonucleotide comprises at least one modified internucleotide linkage; preferably, each internucleotide linkage of the antisense-oligonucleotide is a modified internucleotide linkage; preferably, the modified internucleotide linkage is preferably selected from phosphorothioate (PS) and phosphoramidate (PN) (e.g., methylsulfonyl phosphoramidate (MsPA)), preferably PS.

[0017] In some embodiments, the antisense oligonucleotide comprises an oligonucleotide consisting of 12-30, 14-22, 14-20, 14-18, 14-20, 15-17, 15-25 or 16-20 linked nucleotides, preferably, the oligonucleotide consists of 20 linked nucleotides; preferably, the antisense oligonucleotide comprises a nucleotide sequence as shown in any one of SEQ ID NO. 18 to 32 or a nucleotide sequence having 1 to 3 nucleotide differences therefrom, and consists of any one thereof.

[0018] In some embodiments, the antisense oligonucleotide comprises a ligand; preferably, the ligand comprises a ligand targeting hepatocytes, preferably, the ligand comprises a galactose moiety, a galactosamine moiety or an N-acetylgalactosamine moiety, further preferably, the ligand is a trivalent or tetravalent N-acetylgalactosamine moiety, further preferably, the ligand targeting hepatocytes is L96, NAG25 or NAG37; or the ligand comprises a ligand targeting non-hepatocytes or an integrin ligand, a transferrin receptor 1 ligand, preferably, the ligand is a lipophilic group, and the lipophilic group is preferably selected from: lipids, vitamins, steroids, C5-C 30 Saturated or unsaturated fatty acids, C5-C 30 alkyl, and a polypeptide comprising at least one positively charged amino acid residue; the lipophilic group is more preferably selected from cholesterol, C 16 Saturated or unsaturated fatty acids, C 16 Alkyl, C 22 Saturated or unsaturated fatty acids or C 22 alkyl.

[0019] Another aspect of the present invention provides an RNAi agent for inhibiting the expression of the activin type IIB receptor (ACVR2B) gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21 consecutive nucleotides of equal length that are at least 80%, 85%, 90%, 95% or 100% complementary to a target region of an mRNA encoding ACVR2B, or differs from it by no more than 3 nucleotides.

[0020] In some embodiments, the target region is 508-534, 634-660, 655-695, 733-774, 756-782, 929-955, 1049-1089, 1141-1180, 1189-1215, 1243-1269, 1276-1321, 1377-1403, 1547-1596, 1642-1690, 1744-1773, 1916-1942 or 1949-1975 of the nucleotide sequence shown in SEQ ID NO: 1.

[0021] In some embodiments, the target region is 508-534, 634-660, 655-681, 658-684, 669-695, 733-759, 748-774, 756-782, 929-955, 1049-1075, 1052-1078, 1063-1089, 1141-1167, 1154-1180, 1189-1215, 1243-12 69, 1276-1302, 1280-1306, 1283-1309, 1291-1317, 1295-1321, 1377-1403, 1547-1573, 1552-1578, 1570-1596, 1642-1668, 1664-1690, 1744-1770, 1747-1773, 1916-1942, or 1949-1975. In some embodiments, the target region is SEQ ID 511-531, 637-657, 658-678, 661-681, 672-692, 736-756, 751-771, 759-779, 932-952, 1052-1072, 1055-1075, 1066-1086, 1144-1164, 1157-1177, 1192-1212, 1246-12 66, 1279-1299, 1283-1303, 1286-1306, 1294-1314, 1298-1318, 1380-1400, 1550-1570, 1555-1575, 1573-1593, 1645-1665, 1667-1687, 1747-1767, 1750-1770, 1919-1939 or 1952-1972.

[0022] In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides selected from SEQ ID NOs: 64 to 94 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

[0023] In some embodiments, the length of the double-stranded region is 17 to 23 base pairs, preferably 18 to 21 base pairs, and more preferably 19 base pairs.

[0024] In some embodiments, the sense strand and the antisense strand are each 17 to 23 nucleotides in length, preferably 19 to 21 nucleotides in length.

[0025] In some embodiments, the RNAi agent comprises one or two blunt ends, preferably one blunt end.

[0026] In some embodiments, the RNAi agent comprises one or two overhangs, preferably one overhang, each overhang having 1 to 4 unpaired nucleotides, preferably 2 unpaired nucleotides.

[0027] In some embodiments, the overhang is located at the 3' end of the sense strand, the 3' end of the antisense strand, or at both the 3' end of the sense strand and the 3' end of the antisense strand; preferably, the overhang is located at the 3' end of the antisense strand, and further preferably, the RNAi agent has a blunt end.

[0028] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides selected from any one nucleotide sequence of SEQ ID NOs: 33 to 63 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

[0029] In some embodiments, the antisense strand has no more than 23 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 64 to 94; the sense strand has no more than 21 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 33 to 63.

[0030] In some embodiments, in the RNAi agent:

[0031] The sense strand comprises or is the sequence set forth in SEQ ID NO:33, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:64, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0032] The sense strand comprises or is the sequence set forth in SEQ ID NO:34, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:65, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0033] The sense strand comprises or is the sequence set forth in SEQ ID NO:35, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:66, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0034] The sense strand comprises or is the sequence set forth in SEQ ID NO:36, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:67, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0035] The sense strand comprises or is the sequence set forth in SEQ ID NO:37, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:68, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0036] The sense strand comprises or is the sequence set forth in SEQ ID NO:38, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:69, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0037] The sense strand comprises or is the sequence set forth in SEQ ID NO:39, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:70, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0038] The sense strand comprises or is the sequence set forth in SEQ ID NO:40, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:71, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0039] The sense strand comprises or is the sequence set forth in SEQ ID NO:41, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:72, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0040] The sense strand comprises or is the sequence set forth in SEQ ID NO:42, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:73, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0041] The sense strand comprises or is the sequence set forth in SEQ ID NO:43, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:74, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0042] The sense strand comprises or is the sequence set forth in SEQ ID NO:44, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:75, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0043] The sense strand comprises or is the sequence set forth in SEQ ID NO:45, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:76, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0044] The sense strand comprises or is the sequence set forth in SEQ ID NO:46, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:77, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0045] The sense strand comprises or is the sequence set forth in SEQ ID NO:47, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:78, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0046] The sense strand comprises or is the sequence set forth in SEQ ID NO:48, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:79, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0047] The sense strand comprises or is the sequence set forth in SEQ ID NO:49, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:80, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0048] The sense strand comprises or is the sequence set forth in SEQ ID NO:50, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:81, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0049] The sense strand comprises or is the sequence set forth in SEQ ID NO:51, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:82, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0050] The sense strand comprises or is the sequence set forth in SEQ ID NO:52, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:83, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0051] The sense strand comprises or is the sequence set forth in SEQ ID NO:53, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:84, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0052] The sense strand comprises or is the sequence set forth in SEQ ID NO:54, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:85, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0053] The sense strand comprises or is the sequence set forth in SEQ ID NO:55, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:86, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0054] The sense strand comprises or is the sequence set forth in SEQ ID NO:56, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:87, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0055] The sense strand comprises or is the sequence set forth in SEQ ID NO:57, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:88, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0056] The sense strand comprises or is the sequence set forth in SEQ ID NO:58, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:89, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0057] The sense strand comprises or is the sequence set forth in SEQ ID NO:59, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:90, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0058] The sense strand comprises or is the sequence set forth in SEQ ID NO:60, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:91, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0059] The sense strand comprises or is the sequence set forth in SEQ ID NO:61, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:92, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0060] The sense strand comprises or is the sequence set forth in SEQ ID NO:62, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:93, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; or

[0061] The sense strand comprises or is the sequence shown in SEQ ID NO:63, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:94, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom.

[0062] In some embodiments, the RNAi agent comprises duplex 6102, 6103, 6104, 6105, 6106, 6107, 6108, 6109, 6112, 6113, 6115, 6116, 6117, 6119, 6120, 6121, 6122, 6123, 6124, 6125, 6127, 6128, 6129, 6130, 6131, 6132, 6134, 6135, 6137, 6138, or 6140.

[0063] In some embodiments, the sense strand and / or antisense strand of the RNAi agent comprises at least one modified nucleotide independently selected from 2'-deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides (2'-OMe), 2'-fluorine modified nucleotides (2'-F), 2'-deoxy modified nucleotides, locked nucleic acids (LNA), open circle nucleic acids (UNA), bridge nucleic acids (BNA), glycol nucleic acids (GNA), athreose nucleic acids (TNA), conformationally restricted nucleotides, restricted ethyl nucleotides (cEt), 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-O-methoxyethyl modified nucleotides (2'-MOE), abasic nucleotides, inverted nucleotides, nucleotides, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, nucleotides covalently linked to cationic lipids, nucleotides containing 5'-vinylphosphonate (5'-VP) and combinations thereof; preferably selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides containing thiophosphate bonds between nucleotides and combinations thereof; and / or preferably, each nucleotide of the sense chain and / or antisense chain of the RNAi agent is modified.

[0064] In some embodiments, in the RNAi agent, in the 5' to 3' direction, the nucleotides at positions 2, 5, 7 and 14 of the antisense chain are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense chain is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense chain are all 2'-methoxy-modified nucleotides.

[0065] In some embodiments, in the RNAi agent, the antisense strand has at least one phosphorothioate internucleotide linkage; preferably, the phosphorothioate internucleotide linkage is present in one or more of the following: (i) between the first and second nucleotides at the 5' end of the antisense strand; (ii) between the second and third nucleotides at the 5' end of the antisense strand; (iii) between the first and second nucleotides at the 3' end of the antisense strand; and (iv) between the second and third nucleotides at the 3' end of the antisense strand.

[0066] In some embodiments, in the RNAi agent, from the 5' end to the 3' end, the nucleotides at positions 7 and 9 of the sense chain are 2'-fluoro-modified nucleotides, one or two of the nucleotides at positions 5, 8 and 11 of the sense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides.

[0067] In some embodiments, the sense strand has at least one phosphorothioate internucleotide linkage; preferably, the phosphorothioate internucleotide linkage is present at one or more of the following positions: (i) between the first and second nucleotides at the 5' end of the sense strand; (ii) between the second and third nucleotides at the 5' end of the sense strand; (iii) between the first and second nucleotides at the 3' end of the sense strand; and (iv) between the second and third nucleotides at the 3' end of the sense strand.

[0068] In some embodiments, the modification is selected from one of STC, ESC, Advanced ESC, ESC+, AD1-3, AD5, and GalXC.

[0069] In some embodiments, the modification is that the 5'-terminal nucleotide of the antisense strand contains a phosphate or phosphate analog modification, preferably 5'-VP.

[0070] In some embodiments, the RNAi agent further comprises a ligand targeting hepatocytes, preferably, the ligand comprises a galactose moiety, a galactosamine moiety or an N-acetylgalactosamine moiety, further preferably, the ligand is a trivalent or tetravalent N-acetylgalactosamine moiety, and further preferably, the ligand targeting hepatocytes is L96, NAG25 or NAG37; or the RNAi agent further comprises a ligand targeting non-hepatocytes, preferably, the ligand is a lipophilic group or an integrin ligand, a transferrin receptor 1 ligand, and the lipophilic group is preferably selected from: lipids, vitamins, steroids, C5-C 30 Saturated or unsaturated fatty acids, C5-C 30 alkyl, and a polypeptide comprising at least one positively charged amino acid residue; the lipophilic group is more preferably selected from cholesterol, C 16 Saturated or unsaturated fatty acids, C 16 Alkyl, C 22 Saturated or unsaturated fatty acids or C 22 alkyl.

[0071] Another aspect of the present invention provides a pharmaceutical composition comprising any antisense oligonucleotide or any RNAi agent of the present invention and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is formulated as an intravenous or subcutaneous injection.

[0072] Another aspect of the present invention provides the use of any one of the RNAi agents of the present invention in the preparation of the following drugs:

[0073] (i) an agent for reducing the expression level of ACVR2B in cells;

[0074] (ii) a drug for preventing or treating a disease mediated by abnormal expression levels of ACVR2B; or

[0075] (iii) a drug for preventing or treating a disease selected from the group consisting of muscle atrophy, muscular dystrophy, obesity, diabetes, chronic kidney disease, acute kidney injury, postoperative healing of trauma, maxillofacial tumors, eclampsia during pregnancy, liver fibrosis, atherosclerosis, ischemic stroke and Alzheimer's disease.

[0076] Another aspect of the present invention provides the use of an agent that reduces expression of the gene encoding the activin type IIB receptor (ACVR2B) in the preparation of a medicament for treating a disease or condition treatable by ACVR2B knockdown. Preferably, the agent is an antisense oligonucleotide and / or an RNAi agent. Preferably, the disease or condition is selected from the group consisting of muscular dystrophy, muscular dystrophy, obesity, diabetes, chronic kidney disease, acute kidney injury, postoperative traumatic healing, maxillofacial tumors, eclampsia during pregnancy, liver fibrosis, atherosclerosis, ischemic stroke, and Alzheimer's disease.

[0077] Accordingly, the present invention also provides a method for reducing the expression level of ACVR2B in a cell, the method comprising administering to a subject in need thereof a therapeutically effective amount of any antisense oligonucleotide or any RNAi agent of the present invention. The present invention also provides a method for preventing or treating a disease mediated by abnormal expression levels of ACVR2B (e.g., caused by increased levels), the method comprising administering to a subject in need thereof a therapeutically effective amount of any antisense oligonucleotide or any RNAi agent of the present invention. The present invention also provides a method for preventing or treating a disease selected from muscle atrophy, muscular dystrophy, obesity, diabetes, chronic kidney disease, acute kidney injury, postoperative healing of trauma, maxillofacial tumors, eclampsia during pregnancy, liver fibrosis, atherosclerosis, ischemic stroke, and Alzheimer's disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of any antisense oligonucleotide or any RNAi agent of the present invention. The present invention also provides the use of any antisense oligonucleotide or any RNAi agent of the present invention in the preparation of a medicament for preventing or treating the above-mentioned diseases. The present invention also provides any antisense oligonucleotide or any RNAi agent of the present invention for preventing or treating the above-mentioned diseases.

[0078] Other aspects of the present invention will become apparent from the detailed description of the specification which follows. DETAILED DESCRIPTION

[0079] definition

[0080] Activin A Receptor Type 2B (ACVR2B), referred to herein as the activin A receptor type 2B protein or its encoding gene, is also known as HTX4, ACTRIIB, or ActR-IIB, with Gene ID: 93. The NCBI accession number for the human full-length ACVR2B mRNA transcript is NM_001106.4. The NCBI accession number for the cynomolgus monkey ACVR2B mRNA transcript is XM_015445674.2.

[0081] As used herein, "oligonucleotide" refers to a nucleotide sequence that forms a nucleotide chain via internucleotide linkages, wherein each nucleoside and internucleotide linkage may be modified or unmodified. Unless otherwise indicated, an oligonucleotide consists of 12-30 linked nucleotides. As used herein, "antisense oligonucleotide" or "ASO" refers to a single-stranded oligonucleotide that is capable of specifically binding to a target RNA (e.g., mRNA) sequence and modulating protein (e.g., ACVR2B) expression. As described herein, "antisense oligonucleotides" may include non-oligonucleotide portions, such as ligands.

[0082] "Internucleotide linkage" means a covalent linkage between adjacent nucleotides in an oligonucleotide. As used herein, "modified internucleotide linkage" means any internucleotide linkage other than a phosphodiester internucleotide linkage. A "phosphorothioate internucleotide linkage" is a modified internucleotide linkage in which one of the non-bridging oxygen atoms of the phosphodiester internucleotide linkage is replaced by a sulfur atom.

[0083] "Deoxy region" means a region of 5-12 contiguous nucleotides in which at least 70% of the nucleosides are 2'-β-D-deoxynucleosides. In certain embodiments, each nucleoside is selected from 2'-β-D-deoxynucleosides, bicyclic nucleosides, and 2'-substituted nucleosides. In certain embodiments, the deoxy region supports RNase H activity. In certain embodiments, the deoxy region is a gap or internal region of a gapmer.

[0084] "Gap polymer" means an antisense oligonucleotide comprising an internal region, wherein the internal region is located between an external region having one or more nucleosides, has a plurality of nucleosides that support RNase H cleavage, wherein the nucleosides constituting the internal region are chemically different from the one or more nucleosides constituting the external region. The internal region can be referred to as a "gap", and the external region can be referred to as a "wing". The internal region is a deoxy region. The position of the internal region or gap refers to the order of the nucleosides in the internal region and is counted from the 5' end of the internal region. Unless otherwise indicated, "gap polymer" refers to a sugar motif. In certain embodiments, each nucleoside in the gap is a 2'-β-D-deoxynucleoside. In certain embodiments, the gap is included in a 2'-substituted nucleoside at position 1, 2, 3, 4 or 5 of the gap, and the remaining nucleosides in the gap are 2'-β-D-deoxynucleosides. As used herein, the term "MOE gap polymer" indicates a gap polymer having a gap comprising 2'-β-D-deoxynucleosides and a wing comprising 2'-MOE nucleosides. As used herein, the term "mixed-wing gapmer" refers to a gapmer having wings comprising modified nucleosides comprising at least two different sugar modifications. Unless otherwise indicated, a gapmer may comprise one or more modified internucleotide linkages and / or modified nucleobases, and such modifications do not necessarily follow the sugar-modified gapmer pattern.

[0085] As used herein, the term "RNAi agent" refers to an agent comprising an RNA molecule that, when introduced into a cell, is capable of downregulating the expression of a target gene (herein, the ACVR2B gene) through an RNA interference mechanism. The term "RNAi agent of the present invention," "RNAi agent described herein," or similar expressions includes both modified RNAi agents of the present invention, regardless of sequence and target gene, and RNAi agents of the present invention having a specific sequence for interfering with the ACVR2B gene. RNA interference refers to a process in which a nucleic acid molecule induces the cleavage and degradation of a target RNA molecule (such as an mRNA molecule) in a sequence-specific manner, such as through an RNA-induced silencing complex (RISC) pathway. RNAi agents herein include siRNA, shRNA, and DNA / RNA hybrid molecules, sometimes also collectively referred to herein as double-stranded RNA (dsRNA), which comprise two antiparallel continuous nucleotide chains that are sufficiently complementary to each other to hybridize to form a double-stranded region. "Hybridization" refers to the pairing of complementary polynucleotides, typically through hydrogen bonds (e.g., Watson-Crick hydrogen bonds, Wobble hydrogen bonds, Hoogsteen hydrogen bonds, or reversed Hoogsteen hydrogen bonds) between complementary bases in the two polynucleotides. "Double-stranded region" refers to a region in two complementary or substantially complementary polynucleotides that hybridize to form base pairs, thereby forming a double strand between the two polynucleotide chains.

[0086] The term "antisense strand" refers to the strand of a dsRNA that contains a region that is substantially complementary to the target sequence. The term "sense strand" or "sense strand" refers to the strand of a dsRNA that contains a region that is substantially complementary to the antisense strand region as defined herein. The term "substantially complementary region" refers to a region that is fully complementary or incompletely complementary. When the complementary region is not fully complementary to the target sequence, mismatches may be located in the interior or terminal regions of the molecule. Typically, the most tolerable mismatches are located in the terminal regions, for example, 5, 4, 3, or 2 at the 5' and / or 3' ends of the dsRNA.

[0087] "siRNA" refers to a nucleic acid that forms double-stranded RNA that has the ability to reduce or inhibit the expression of a target gene when the siRNA and the target gene are present in the same cell. siRNAs are typically about 15 to about 30 base pairs in length, most typically about 19 to 25 base pairs in length, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs in length.

[0088] shRNA refers to a short hairpin RNA that includes two short inverted repeats and an intermediate stem-loop structure connecting the two. The stem-loop may contain at least one unpaired nucleotide, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. The stem-loop may be 10 or fewer nucleotides. The stem-loop may be 8 or fewer unpaired nucleotides. The stem-loop may be 4 to 10 unpaired nucleotides. The stem-loop may be 4 to 8 nucleotides.

[0089] The two substantially complementary chains of a dsRNA need not but may also be covalently linked. The maximum number of base pairs is the number of nucleotides in the shortest chain of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, the dsRNA may also comprise one or more nucleotide overhangs. Overhanging nucleotides refer to one or more unpaired nucleotides that extend beyond the double-stranded region at the end of the chain. When the 3' end of a chain extends beyond the 5' end of the other chain, or when the 5' end of a line extends beyond the 3' end of the other line, nucleotide overhangs are usually produced. For example, at least one chain comprises a 3' overhang of at least 1 nucleotide, for example, 1 to 4 nucleotides overhang. For another example, at least one chain comprises a 5' overhang of at least 1 nucleotide, for example, 1 to 4 nucleotides overhang. In other embodiments, both the 3' end and the 5' end of a chain of the dsRNA comprise an overhang of at least 1 nucleotide.

[0090] As used herein, the term "blunt end" or "blunt end" with respect to dsRNA refers to the absence of unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., the absence of nucleotide overhangs. One or both ends of a dsRNA may be blunt. If both ends of a dsRNA are blunt, the dsRNA is said to be blunt-ended. It should be noted that a "blunt-ended" dsRNA is a dsRNA with both ends blunt, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such molecules are double-stranded throughout their entire length. As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of a dsRNA. For example, a nucleotide overhang is present when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. Nucleotide overhangs may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhangs may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, overhanging nucleotides may be present at the 5' end, the 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0091] The dsRNA molecule may include chemical modifications to ribonucleotides, including modifications to the ribose, bases, or backbone components of the ribonucleic acid, as described herein or known in the art. Any such modifications, as used in double-stranded ribonucleic acid molecules (e.g., siRNA, shRNA, etc.), are encompassed by the term "dsRNA" for the purposes of this disclosure. "Modified" nucleotides refer to nucleotides that independently have modified sugar moieties, modified internucleotide linkages, and / or modified nucleobases. Thus, the term "modified nucleotides" includes substitutions, additions, or removals of, for example, functional groups or atoms of internucleoside linkages, sugar moieties, or nucleobases.

[0092] The term "ligand" refers to a cell or tissue targeting agent that binds to a specified cell type (e.g., hepatocytes), such as a lectin, glycoprotein, lipid, or protein (e.g., an antibody). Exemplary targeting agents include thyrotropin, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetylgalactosamine (GalNAc), multivalent (e.g., divalent or trivalent) GalNAc, N-acetylglucosamine, multivalent mannose, multivalent trehalose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, cholesterol, steroids, bile acid, folate, vitamin B12, biotin, RGD peptide, and RGD peptide mimetics. In a preferred embodiment, the ligand is a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, or polysaccharide. For example, the ligand can be a derivative comprising GalNAc. In a preferred embodiment, the ligand comprises one or more N-acetylgalactosamine derivatives attached via a bivalent or trivalent branched linker.

[0093] The term "therapeutically effective amount" refers to an amount of a RNAi agent of the invention or composition thereof effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0094] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0095] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or delivering an ASO or RNAi agent from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient.

[0096] The term "treatment" encompasses prevention, therapy, and cure. The patient receiving such treatment is generally any animal in need thereof, including primates (particularly humans) and other mammals such as horses, cattle, pigs, sheep, poultry, and pets.

[0097] Antisense oligonucleotides for inhibiting ACVR2B gene expression

[0098] A first aspect of the invention provides an antisense oligonucleotide comprising an oligonucleotide consisting of 12 to 30 linked nucleotides, wherein the oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 nucleotides that are at least 80%, 85%, 90%, 95% or 100% complementary to an equal length of consecutive nucleotides of a target region of an mRNA encoding an activin type IIB receptor (ACVR2B).

[0099] In some embodiments, the target region is 504-554, 588-617, 648-686, 837-866, 1234-1268, 1360-1403, 1503-1532, or 1545-1574 of the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the target region is 504-533, 510-539, 519-548, 525-554, 588-617, 648-677, 653-682, 657-686, 837-866, 1234-1263, 1239-1268, 1360-1389, 1374-1403, 1503-1532, or 1545-1574 of the nucleotide sequence set forth in SEQ ID NO: 1. In a preferred embodiment, the target region is 509-528, 515-534, 524-543, 530-549, 593-612, 653-672, 658-677, 662-681, 842-861, 1239-1258, 1244-1263, 1365-1384, 1379-1398, 1508-1527 or 1550-1569 of the nucleotide sequence shown in SEQ ID NO: 1.

[0100] Thus, in some embodiments, the present invention provides an antisense oligonucleotide comprising an oligonucleotide consisting of 12 to 30 linked nucleotides, wherein the oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 nucleotides that are at least 80%, 85%, 90%, 95% or 100% complementary to an equal length of consecutive nucleotides in 504-554, 588-617, 648-686, 837-866, 1234-1268, 1360-1403, 1503-1532 or 1545-1574 of the nucleotide sequence shown in SEQ ID NO: 1.

[0101] In some embodiments, the present invention provides an antisense oligonucleotide comprising an oligonucleotide consisting of 12 to 30 linked nucleotides, wherein the oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 sequences corresponding to SEQ ID NO: Nucleotides that are at least 80%, 85%, 90%, 95% or 100% complementary to an equal length of consecutive nucleotides in 504-533, 510-539, 519-548, 525-554, 588-617, 648-677, 653-682, 657-686, 837-866, 1234-1263, 1239-1268, 1360-1389, 1374-1403, 1503-1532 or 1545-1574 of the nucleotide sequence shown in NO:1.

[0102] In some embodiments, the present invention provides an antisense oligonucleotide comprising an oligonucleotide consisting of 12 to 30 linked nucleotides, wherein the oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 sequences corresponding to SEQ ID NO: Nucleotides that are at least 80%, 85%, 90%, 95% or 100% complementary to an equal length of consecutive nucleotides in 509-528, 515-534, 524-543, 530-549, 593-612, 653-672, 658-677, 662-681, 842-861, 1239-1258, 1244-1263, 1365-1384, 1379-1398, 1508-1527 or 1550-1569 of the nucleotide sequence shown in NO:1.

[0103] In some embodiments, the oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleotide sequences of any one of SEQ ID NOs: 3 to 17.

[0104] In some embodiments, the oligonucleotide consists of 12-30, 14-22, 14-20, 14-18, 14-20, 15-17, 15-25, or 16-20 linked nucleotides and has a sequence of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleotides comprising the nucleotide sequence of any one of SEQ ID NOs: 3 to 17, and wherein when measured over the entire nucleotide sequence of the antisense oligonucleotide, the antisense oligonucleotide has a binding affinity to messenger RNA (mRNA) of human and / or cynomolgus monkey activin type IIB receptor (ACVR2B) (NCBI Accession Nos. NM_001106.4, SEQ ID NO. 1 and XM_015445674.2, SEQ ID NO. A nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% (preferably 100%) complementary to the nucleotide sequence of NO. 2).

[0105] In some embodiments, the antisense oligonucleotides provided do not include modified nucleotides, such as modified sugar moieties, modified nucleobase moieties, or modified internucleotide linkages. In such embodiments, for example, the antisense oligonucleotides provided herein can comprise a nucleotide sequence as set forth in any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom.

[0106] In some embodiments, the antisense oligonucleotides provided include modified nucleotides, such as modified sugar moieties and / or modified nucleobase moieties and / or modified internucleotide linkages. In preferred embodiments, the antisense oligonucleotides provided include modified nucleotides, including modified sugar moieties, modified nucleobase moieties, and modified internucleotide linkages.

[0107] In some embodiments, the antisense oligonucleotide comprises an oligonucleotide consisting of 12-30, 14-22, 14-20, 14-18, 14-20, 15-17, 15-25, or 16-20 linked nucleotides, comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleotides of any one of SEQ ID NOs: 3 to 17, wherein the antisense oligonucleotide comprises at least one modification selected from a modified sugar moiety, a modified nucleobase moiety, and a modified internucleotide linkage.

[0108] In some embodiments, the antisense oligonucleotide comprises an oligonucleotide in which each nucleotide is modified, including a modified sugar moiety and a modified internucleotide linkage.

[0109] In some embodiments, the antisense oligonucleotide comprises an oligonucleotide in which each nucleotide is modified, including a modified sugar moiety, a modified nucleobase moiety, and a modified internucleotide linkage.

[0110] In some embodiments, the modified sugar moiety can be at least one bicyclic sugar moiety, at least one non-bicyclic modified sugar moiety, and / or at least one sugar surrogate.

[0111] In some embodiments, the antisense oligonucleotide comprises at least one bicyclic sugar moiety. In some embodiments, two atoms of the substituent bridged furanosyl ring are to form second ring, thereby produce bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety comprises a bridge between 4 ' and 2 ' furanose ring atoms. Examples of such 4' to 2' bridged sugar substituents include, but are not limited to: 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' ("LNA"), 4'-CH2-S-2', 4'-(CH2)2-O-2' ("ENA"), 4'-CH(CH3)-O-2' (referred to as "constrained ethyl" or "cEt"), 4'-CH2-O-CH2-2', 4'-CH2-N(R)-2', 4'-CH(CH2OCH3)-O-2' ("constrained MOE" or "cMOE"), and the like (see, e.g., Seth et al., US Pat. No. 7,399,845; Bhat et al., US Pat. No. 7,569,686; Swayze et al., US Pat. No. 7,741,457; and Swayze et al., US8,022,193), 4'-C(CH3)(CH3)-O-2' and analogs thereof (see, e.g., Seth et al., US8,278,283), 4'-CH2-N(OCH3)-2' and analogs thereof (see, e.g., Prakash et al., US8,278,425), 4'-CH2-ON(CH3)-2' (see, e.g., Allerson et al., US7,696,345 and Allerson et al., US8,124,745), 4'-CH2-C(H)(CH3)-2' (see, e.g., Zhou et al., J. Org. Chem., 2009, 74, 118-134), 4'-CH2-C(=CH2)-2' and analogs thereof (see, e.g., Seth et al., US8,278,426), 4'-C(R a R b )-N(R)-O-2'、4'-C(R a R b )-ON(R)-2', 4'-CH2-ON(R)-2' and 4'-CH2-N(R)-O-2', wherein each R, R a and R b are independently H, a protecting group or a C1-C 12Alkyl (see, for example, Imanishi et al., US7,427,672). In certain embodiments, bicyclic sugar moieties and nucleosides having such bicyclic sugar moieties are further defined by isomeric configurations. For example, LNA nucleosides (described herein) can be in an α-L configuration or in a β-D configuration. In a preferred embodiment, the bicyclic sugar moiety has a 4'-2' bridge, and the 4'-2' bridge is preferably cEt or LNA.

[0112] In some embodiments, the antisense oligonucleotide comprises at least one non-bicyclic modified sugar moiety. Such non-bridging substituents may be located at any position of the furanose moiety, including but not limited to substituents at the 2', 4', and / or 5' positions. In certain embodiments, one or more non-bridging substituents of the non-bicyclic modified sugar moiety are branched. Examples of suitable 2'-substituents for the non-bicyclic modified sugar moiety include, but are not limited to: 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE" or "O-methoxyethyl"). In certain embodiments, the 2'-substituent is selected from: halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O-C1-C 10 Alkoxy, substituted O-C1-C 10 Alkoxy, O-C1-C 10 Alkyl, substituted O-C1-C 10 Alkyl, S-alkyl, N(R m )-alkyl, O-alkenyl, S-alkenyl, N(R m )-alkenyl, O-alkynyl, S-alkynyl, N(R m )-alkynyl, O-alkylene-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(R m )(R n ) or OCH2C(=O)-N(R m )(R n ), where each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10Alkyl, and 2'-substituents described in Cook et al., US Pat. No. 6,531,584; Cook et al., US Pat. No. 5,859,221; and Cook et al., US Pat. No. 6,005,087. Certain embodiments of these 2'-substituents may be further substituted with one or more substituents independently selected from the group consisting of hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl. Examples of suitable 4'-substituents for non-bicyclic modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO 2015 / 106128. Examples of suitable 5'-substituents for non-bicyclic modified sugar moieties include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In certain embodiments, non-bicyclic modified sugar moieties comprise more than one non-bridging sugar substituent, such as 2'-F-5'-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008 / 101157 and Rajeev et al., US 2013 / 0203836.

[0113] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from the group consisting of F, NH2, N3, OCF3, OCH3, O(CH2)3NH2, CH2CH=CH2, OCH2CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(R m )(R n ), O(CH2)2O(CH2)2N(CH3)2 and N-substituted acetamide (OCH2C(=O)-N(R m )(R n )), where each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 alkyl.

[0114] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from the group consisting of F, OCF3, OCH3, OC H2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, and OCH2C(=O)-N(H)CH3 ("NMA"). In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from the group consisting of F (2'-F), OCH3 (2'-OMe), and OCH2CH2OCH3 (2'-MOE).

[0115] In certain embodiments, the modified furanosyl sugar moiety and the nucleoside having such modified furanosyl sugar moiety are further defined by isomeric configurations. For example, the 2'-deoxyfuranosyl sugar moiety can be in seven isomeric configurations except the naturally occurring β-D-deoxyribosyl configuration. Such modified sugar moieties are described in, for example, WO 2019 / 157531, which is incorporated herein by reference. The 2'-modified sugar moiety has another stereocenter at the 2' position relative to the 2'-deoxyfuranosyl sugar moiety; therefore, such sugar moieties have a total of sixteen possible isomeric configurations. Unless otherwise specified, the 2'-modified sugar moieties described herein are in β-D-ribosyl isomeric configurations.

[0116] In some embodiments, the antisense oligonucleotide comprises at least one sugar surrogate. In some such embodiments, the oxygen atom of the sugar moiety is replaced by, for example, a sulfur, carbon, or nitrogen atom. In some such embodiments, such modified sugar moieties also include bridged and / or non-bridged substituents as described herein. For example, some sugar surrogate comprises a 4'-sulfur atom and a substitution at the 2' position (see, for example, Bhat et al., US7,875,733 and Bhat et al., US7,939,677) and / or the 5' position.

[0117] In certain embodiments, the sugar surrogate comprises a ring having other than 5 atoms. For example, in certain embodiments, the sugar surrogate comprises a 6-membered tetrahydropyran ("THP"). Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acids ("HNA"), anitol nucleic acids ("ANA"), mannitol nucleic acids ("MNA") (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), and fluoro-HNA (F-HNA, also referred to as F-THP or 3'-fluorotetrahydropyran).

[0118] In certain embodiments, the sugar surrogate comprises a ring with more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholinyl sugar moieties and their uses in oligonucleotides have been reported. In certain embodiments, the sugar surrogate comprises an acyclic portion. Examples of nucleosides and oligonucleotides comprising such acyclic sugar surrogate include, but are not limited to, peptide nucleic acids ("PNA"), acyclic butyl nucleic acids (see, for example, Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865) and Manoharan et al., nucleosides and oligonucleotides described in WO2011 / 133876. By introducing a modified nucleobase with a cationic lipid or its equivalent covalently attached thereto, PNA can be made highly permeable to mammalian cell membranes (see, for example, WO2019022434 and WO2009113828). In a preferred embodiment, the sugar surrogate is selected from POM, PNA, THP and F-HNA.

[0119] In some embodiments, the antisense oligonucleotide comprises one or more nucleosides comprising unmodified nucleobases. In certain embodiments, the antisense oligonucleotide comprises one or more nucleosides comprising modified nucleobases. In certain embodiments, the antisense oligonucleotide comprises one or more nucleosides that do not comprise nucleobases, referred to as abasic nucleosides.

[0120] In certain embodiments, the modified nucleobase is selected from: a 5-substituted pyrimidine (eg, 5' methylcytosine, m5C), a 6-azapyrimidine, an alkyl or alkynyl substituted pyrimidine, an alkyl substituted purine, and N-2, N-6, and O-6 substituted purines. In certain embodiments, the modified nucleobase is selected from the group consisting of: 2-aminopropyladenine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C≡C-CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, 8-aza and other 8-substituted Purines; 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil and 5-halocytosine; 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 5-methylcytosine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl4-N-benzoylcytosine, 5-methyl4-N-benzoyluracil, universal bases, hydrophobic bases, mixed bases, size-expanded bases and fluorinated bases. Other modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenthiazine-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. In a preferred embodiment, the antisense oligonucleotide comprises one or more 5-methylcytosines (m5C).

[0121] In some embodiments, the modified nucleobase can be a nucleobase covalently attached to a cationic lipid. By introducing a modified nucleobase with a cationic lipid covalently attached thereto or its equivalent, antisense oligonucleotides can be made highly permeable to mammalian cell membranes. Suitable covalent attachment of cationic lipid modifications can be those described in WO2019022434A1, the entire contents of which are incorporated herein by reference.

[0122] In some embodiments, the antisense oligonucleotide comprises at least one modified internucleotide linkage. In preferred embodiments, each internucleotide linkage of the antisense oligonucleotide is a modified internucleotide linkage. In certain embodiments, any internucleotide linkage can be used to link the nucleosides of the antisense oligonucleotide together. Two main categories of internucleoside linking groups are defined based on the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleotide linkages include, but are not limited to, phosphodiester, which contains a phosphodiester bond ("P(O2)=O") (also known as an unmodified linkage or a naturally occurring linkage); phosphotriester; methylphosphonate; phosphoramidate (PN); phosphorothioate ("P(O2)=S", PS) and phosphorodithioate ("HS-P=S"). Phosphoramidates (PN) may have the formula -N(R)P(=X)(OH)O-, -OP(=X)(OH)N(R)-, -OP(NR)(=X)O-, -N(S02R)P(=X)(OH)O-, -OP(=X)(OH)N(S02R)-, or -OP(NS02R)(=X)O- ("MsPA"), wherein X is O or S, R may be an optionally substituted alkyl, aryl, heteroaryl, or heterocycloalkyl group; or NR may be an optionally substituted cycloguanidinyl moiety, an optionally substituted triazolyl, or a Tmg group. More PN structures can be found in WO2023220744, WO 2019 / 032612, and WO2021 / 030778, which are incorporated herein by reference in their entireties.

[0123] Representative non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters, thionocarbamates (-OC(=O)(NH)-S-); siloxanes (-O-SiH2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified internucleotide linkages can be used to alter, and generally increase, the nuclease resistance of oligonucleotides compared to naturally occurring phosphodiester internucleotide linkages. In certain embodiments, internucleotide linkages with chiral atoms can be prepared as racemic mixtures or as separated enantiomers. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleotide linkages are well known to those skilled in the art.

[0124] Representative internucleotide connections with chiral centers include, but are not limited to, alkylphosphonates and phosphorothioates. Antisense oligonucleotides comprising internucleotide connections with chiral centers can be prepared into a population of antisense oligonucleotides comprising stereo-random internucleotide connections, or into a population of antisense oligonucleotides comprising internucleotide connections of phosphorothioates in a specific stereochemical configuration. In certain embodiments, the population of antisense oligonucleotides comprises internucleotide connections of phosphorothioates, wherein all of the internucleotide connections of phosphorothioates are stereo-random. Such antisense oligonucleotides can be generated using a synthetic method that randomly selects the stereochemical configuration of each internucleotide connection of phosphorothioates. Nevertheless, as is well understood by those skilled in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a determined stereo configuration. In certain embodiments, the population of antisense oligonucleotides is enriched with antisense oligonucleotides comprising one or more internucleotide connections of specific phosphorothioates in a specific independently selected stereochemical configuration. In certain embodiments, a specific internucleotide connection of phosphorothioates of a specific configuration is present in at least 65% of the molecules in the population. In certain embodiments, there is a specific phosphorothioate internucleotide connection of a specific configuration in at least 70% of the molecules in the population. In certain embodiments, there is a specific phosphorothioate internucleotide connection of a specific configuration in at least 80% of the molecules in the population. In certain embodiments, there is a specific phosphorothioate internucleotide connection of a specific configuration in at least 90% of the molecules in the population. In certain embodiments, there is a specific phosphorothioate internucleotide connection of a specific configuration in at least 99% of the molecules in the population. Such chiral enriched populations of antisense oligonucleotides can be generated using synthetic methods known in the art, such as the methods described in the following documents: Oka et al., JACS 125, 8307 (2003); Wan et al., Nuc. Acid. Res. 42, 13456 (2014); and WO 2017 / 015555. In certain embodiments, the population of antisense oligonucleotides is enriched with antisense oligonucleotides having at least one indicated phosphorothioate in (Sp) configuration. In certain embodiments, the population of antisense oligonucleotides is enriched for antisense oligonucleotides having at least one phosphorothioate in the (Rp) configuration.

[0125] In some embodiments, the antisense oligonucleotide includes a gap polymer (Gapmer). The gap polymer is defined by two external regions or " wings" and a central or internal region or " gap ". The three regions (5' wing, gap and 3' wing) of the gap polymer motif form a contiguous sequence of nucleosides, wherein at least some sugar moieties of the nucleosides of each wing are different from at least some sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moiety of the nucleosides closest to the gap in each wing (the most 3' end nucleosides of the 5' wing and the most 5' end nucleosides of the 3' wing) is different from the sugar moiety of the adjacent gap nucleosides, thereby defining the boundary between the wing and the gap (that is, the wing / gap boundary). In certain embodiments, the sugar moieties in the gap are identical to each other. In certain embodiments, the gap includes one or more nucleosides, and the sugar moieties possessed by the nucleosides are different from the sugar moieties of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of two wings are identical to each other (symmetrical gap polymers). In certain embodiments, the sugar motifs of 5' wing are different from the sugar motifs of 3' wing (asymmetric gap polymers).

[0126] In some embodiments, the gapmer comprises: (a) a 5' region consisting of 1-6 linked 5' region nucleotides; (b) a central region consisting of 6-10 linked central region nucleotides; and (c) a 3' region consisting of 1-6 linked 3' region nucleotides; wherein each of the nucleotides in the 5' region and the 3' region comprises a sugar moiety modified from 2'-MOE, LNA and cEt, and at least 6 of the nucleotides in the central region comprise a 2'-deoxy sugar moiety.

[0127] In some embodiments, the gapmer has a sugar motif (5' to 3') selected from the following: eekddddddddddkke, ekkddddddddddkke, kkkdyddddddddkkk, kkkddydddddddkkk, kkkdddyddddddkkk, kkkddddddddddddkkk, or eeeeeddddddddddeeeee; wherein e represents a 2'-MOE sugar moiety, k represents a cEt sugar moiety, d represents a 2'-deoxy sugar moiety, and y represents a 2'-OMe sugar moiety.

[0128] In a preferred embodiment, the gapmer has a eeeeeddddddddddeeeee sugar motif (5' to 3'), wherein e represents a 2'-MOE sugar moiety and d represents a 2'-deoxy sugar moiety.

[0129] In a preferred embodiment, the internucleotide linkage of the gapmer is PS. In such an embodiment, the gapmer comprises a nucleotide sequence as shown in any one of SEQ ID NO.18 to 32 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists of any one thereof. In one embodiment, the gapmer comprises a nucleotide sequence as shown in SEQ ID NO.18 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists of it. In one embodiment, the gapmer comprises a nucleotide sequence as shown in SEQ ID NO.19 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists of it. In one embodiment, the gapmer comprises a nucleotide sequence as shown in SEQ ID NO.20 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists of it. In one embodiment, the gapmer comprises a nucleotide sequence as shown in SEQ ID NO.21 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists of it. In one embodiment, the gapmer comprises the nucleotide sequence as shown in SEQ ID NO.22 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists thereof. In one embodiment, the gapmer comprises the nucleotide sequence as shown in SEQ ID NO.23 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists thereof. In one embodiment, the gapmer comprises the nucleotide sequence as shown in SEQ ID NO.24 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists thereof. In one embodiment, the gapmer comprises the nucleotide sequence as shown in SEQ ID NO.25 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists thereof. In one embodiment, the gapmer comprises the nucleotide sequence as shown in SEQ ID NO.26 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists thereof. In one embodiment, the gapmer comprises the nucleotide sequence as shown in SEQ ID NO.27 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists thereof. In one embodiment, the gapmer comprises or consists of the nucleotide sequence shown in SEQ ID NO. 28, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom. In one embodiment, the gapmer comprises or consists of the nucleotide sequence shown in SEQ ID NO. 29, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom. In one embodiment, the gapmer comprises or consists of the nucleotide sequence shown in SEQ ID NO. 30, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom.In one embodiment, the gapmer comprises or consists of the nucleotide sequence shown in SEQ ID NO. 31, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom. In one embodiment, the gapmer comprises or consists of the nucleotide sequence shown in SEQ ID NO. 32, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom.

[0130] RNAi agents for inhibiting ACVR2B gene expression

[0131] One aspect of the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, comprising a sense strand and an antisense strand forming a complementary double-stranded region, wherein the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides that are at least 80%, 85%, 90%, 95%, or 100% complementary to an equal length of a target region of an mRNA encoding ACVR2B, or differs from it by no more than 3 nucleotides.

[0132] In some embodiments, the target region is 508-534, 634-660, 655-695, 733-774, 756-782, 929-955, 1049-1089, 1141-1180, 1189-1215, 1243-1269, 1276-1321, 1377-1403, 1547-1596, 1642-1690, 1744-1773, 1916-1942, or 1949-1975 of the nucleotide sequence shown in SEQ ID NO: 1. 508-534, 634-660, 655-681, 658-684, 669-695, 733-759, 748-774, 756-782, 929-955, 1049-1075, 1052-1078, 1063-1089, 1141-1167, 1154-1180, 1189-1215, 1243-12 69, 1276-1302, 1280-1306, 1283-1309, 1291-1317, 1295-1321, 1377-1403, 1547-1573, 1552-1578, 1570-1596, 1642-1668, 1664-1690, 1744-1770, 1747-1773, 1916-1942 or 1949-1975. In a preferred embodiment, wherein the target region is SEQ ID 511-531, 637-657, 658-678, 661-681, 672-692, 736-756, 751-771, 759-779, 932-952, 1052-1072, 1055-1075, 1066-1086, 1144-1164, 1157-1177, 1192-1212, 1246-12 66, 1279-1299, 1283-1303, 1286-1306, 1294-1314, 1298-1318, 1380-1400, 1550-1570, 1555-1575, 1573-1593, 1645-1665, 1667-1687, 1747-1767, 1750-1770, 1919-1939 or 1952-1972.

[0133] Therefore, in some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, comprising a sense strand and an antisense strand forming a complementary double-stranded region, wherein the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 sequences identical to those of SEQ ID NO: The present invention also comprises a nucleotide sequence of the present invention which is complementary to or differs from the nucleotide sequence of NO: 1 by at least 80%, 85%, 90%, 95% or 100% of an equal length of consecutive nucleotides of 508-534, 634-660, 655-695, 733-774, 756-782, 929-955, 1049-1089, 1141-1180, 1189-1215, 1243-1269, 1276-1321, 1377-1403, 1547-1596, 1642-1690, 1744-1773, 1916-1942 or 1949-1975 of the nucleotide sequence shown in NO: 1, or a nucleotide sequence which differs from the nucleotide sequence by no more than 3 nucleotides.

[0134] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, comprising a sense strand and an antisense strand forming a complementary double-stranded region, wherein the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21 sequences identical to those of SEQ ID NO: 508-534, 634-660, 655-681, 658-684, 669-695, 733-759, 748-774, 756-782, 929-955, 1049-1075, 1052-1078, 1063-1089, 1141-1167, 1154-1180, 1189-1215, 1243-1269, 1276-1302, 1280-1306, 128 1949-1975, or an equal length of consecutive nucleotides that are at least 80%, 85%, 90%, 95% or 100% complementary thereto, or nucleotides that differ by no more than 3 nucleotides.

[0135] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, comprising a sense strand and an antisense strand forming a complementary double-stranded region, wherein the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21 sequences identical to those of SEQ ID NO: 511-531, 637-657, 658-678, 661-681, 672-692, 736-756, 751-771, 759-779, 932-952, 1052-1072, 1055-1075, 1066-1086, 1144-1164, 1157-1177, 1192-1212, 1246-1266, 1279-1299, 1283-1303, 128 an equal length of consecutive nucleotides of 1919-1939, or 1952-1972 that is at least 80%, 85%, 90%, 95% or 100% complementary thereto, or nucleotides that differ by no more than 3 nucleotides.

[0136] In some embodiments, the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 64 to 94, and nucleotide sequences that differ from each of them by 1, 2, or 3 nucleotides.

[0137] In some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, comprising a sense strand and an antisense strand forming complementary double-stranded regions, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides of any one nucleotide sequence selected from SEQ ID NOs: 64 to 94.

[0138] In some embodiments, the present invention provides an RNAi agent for inhibiting ACVR2B gene expression, comprising a sense strand and an antisense strand forming complementary double-stranded regions, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from a nucleotide sequence having 1, 2, or 3 nucleotide differences from any one of SEQ ID NOs: 64 to 94.

[0139] In the present invention, when referring to 1, 2, or 3 nucleotide differences, the 1, 2, or 3 nucleotide differences may be located on the sense strand and / or antisense strand outside the double-stranded region. In some embodiments, the 1, 2, or 3 nucleotide differences are located on the sense strand and / or antisense strand within the double-stranded region. In some embodiments, a portion of the 1, 2, or 3 nucleotide differences are located on the sense strand and / or antisense strand within the double-stranded region, and another portion is located on the sense strand and / or antisense strand outside the double-stranded region. For the antisense strand, in some embodiments, the 1, 2, or 3 nucleotide differences are located at the 3'-most end or the 5'-most end of the antisense strand. In some embodiments, the 1, 2, or 3 nucleotide differences are located between the 3'-most end and the 5'-most end of the antisense strand. In some embodiments, 1 or 2 of the 1, 2, or 3 nucleotide differences are located at the 3'-most end or the 5'-most end of the antisense strand, and the other 1 or 2 are located between the 3'-most end and the 5'-most end of the antisense strand. For the sense strand, in some embodiments, the 1, 2, or 3 nucleotide differences are located at the 3'-most end or the 5'-most end of the sense strand. In some embodiments, the 1, 2, or 3 nucleotide differences are located between the 3'-most end and the 5'-most end of the sense strand. In some embodiments, one or two of the 1, 2, or 3 nucleotide differences are located at the 3'-most end or the 5'-most end of the sense strand, and the other one or two are located between the 3'-most end and the 5'-most end of the sense strand.

[0140] In some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, comprising a sense strand and an antisense strand forming a complementary double-stranded region, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from a nucleotide sequence having one nucleotide difference with any one of SEQ ID NOs: 64 to 94, and the one nucleotide difference is located at the extreme 3' end of the antisense strand. In some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, comprising a sense strand and an antisense strand forming a complementary double-stranded region, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from a nucleotide sequence having two nucleotide differences with any one of SEQ ID NOs: 64 to 94, and the two nucleotide differences are consecutively located at the extreme 3' end of the antisense strand.

[0141] In some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, comprising a sense strand and an antisense strand forming complementary double-stranded regions, wherein the antisense strand is no longer than 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides of any one nucleotide sequence selected from SEQ ID NOs: 64 to 94.

[0142] In some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, comprising a sense strand and an antisense strand forming complementary double-stranded regions, wherein the antisense strand is no longer than 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from a nucleotide sequence having 1, 2, or 3 nucleotide differences from any one of SEQ ID NOs: 64 to 94.

[0143] One aspect of the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, comprising an antisense strand, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20 or 21) consecutive nucleotides selected from SEQ ID NOs: 64 to 94 and nucleotide sequences having 1, 2 or 3 nucleotide differences therefrom.

[0144] In some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, comprising an antisense strand, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides of any one nucleotide sequence selected from SEQ ID NOs: 64 to 94.

[0145] In some embodiments, the present invention provides an RNAi agent for inhibiting ACVR2B gene expression, comprising an antisense strand, wherein the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from a nucleotide sequence having 1, 2, or 3 nucleotide differences from any one of SEQ ID NOs: 64 to 94.

[0146] In some embodiments, the length of the double-stranded region is about 17 to about 23 base pairs. For example, a double-stranded region of suitable length is about 17 to about 22 base pairs, about 17 to about 21 base pairs, about 17 to about 20 base pairs, about 17 to about 19 base pairs, about 17 to about 18 base pairs, about 18 to about 23 base pairs, about 18 to about 22 base pairs, about 18 to about 21 base pairs, about 18 to about 20 base pairs, about 19 to 23 base pairs, about 19 to 22 base pairs, about 19 to 21 base pairs, about 20 to 23 base pairs, about 20 to 22 base pairs, or about 21 to 23 base pairs. In certain embodiments, the length of the double-stranded region is about 18 to about 21 base pairs. In other embodiments, the length of the double-stranded region is about 19 base pairs.

[0147] Therefore, in some embodiments of the present invention, an RNAi agent for inhibiting the expression of the ACVR2B gene is provided, which comprises a sense strand and an antisense strand forming a complementary double-stranded region, the double-stranded region being about 17 to about 23 base pairs in length, and the antisense strand being no longer than 23 nucleotides and comprising at least 15 consecutive nucleotides selected from the group consisting of SEQ ID NOs: 64 to 94 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

[0148] In some embodiments of the present invention, an RNAi agent for inhibiting the expression of the ACVR2B gene is provided, which comprises a sense strand and an antisense strand forming a complementary double-stranded region, the double-stranded region being about 18 to about 21 base pairs in length, and the antisense strand being no longer than 23 nucleotides and comprising at least 15 consecutive nucleotides selected from SEQ ID NOs: 64 to 94 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

[0149] In some embodiments of the present invention, an RNAi agent for inhibiting the expression of the ACVR2B gene is provided, which comprises a sense strand and an antisense strand forming a complementary double-stranded region, wherein the double-stranded region is about 19 base pairs in length, and the antisense strand is no more than 23 nucleotides in length and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 64 to 94 and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom.

[0150] In some embodiments, the sense and antisense strands in the RNAi agents of the invention are each independently about 17 to about 23 nucleotides in length, e.g., about 18 to about 23 nucleotides, about 19 to about 23 nucleotides, about 20 to about 23 nucleotides, about 21 to about 23 nucleotides, about 17 to about 22 nucleotides, about 17 to about 21 nucleotides, about 17 to about 20 nucleotides, about 17 to about 19 nucleotides, about 18 to about 22 nucleotides, about 18 to about 21 nucleotides, about 18 to about 20 nucleotides, about 19 to about 22 nucleotides, about 19 to about 21 nucleotides, or about 20 to about 22 nucleotides. In certain embodiments, the sense and antisense strands are each independently about 17, about 18, about 19, about 20, about 21, about 22, or about 23 nucleotides in length.

[0151] In some embodiments, the sense strand and the antisense strand have the same length but form a double-stranded region that is shorter than the strand, such that the RNAi agent for inhibiting the expression of the ACVR2B gene has a two-nucleotide overhang. For example, in one embodiment, the RNAi agent for inhibiting the expression of the ACVR2B gene comprises (i) a sense strand and an antisense strand that are each 21 nucleotides in length, (ii) a double-stranded region that is 19 base pairs in length, and (iii) a nucleotide overhang of one unpaired nucleotide at the 3' end of the sense strand and the 3' end of the antisense strand. In another embodiment, the RNAi agent for inhibiting the expression of the ACVR2B gene comprises (i) a sense strand and an antisense strand that are each 23 nucleotides in length, (ii) a double-stranded region that is 21 base pairs in length, and (iii) a nucleotide overhang of one unpaired nucleotide at the 3' end of the sense strand and the 3' end of the antisense strand.

[0152] In other embodiments, the sense and antisense strands are of the same length and form a double-stranded region over their entire length, such that no nucleotides protrude from either end of the double-stranded molecule. In one such embodiment, the RNAi agent used to inhibit the expression of the ACVR2B gene is blunt-ended and comprises (i) a sense and antisense strand each of 21 nucleotides in length, and (ii) a double-stranded region of 21 base pairs in length. In another such embodiment, the RNAi agent used to inhibit the expression of the ACVR2B gene is blunt-ended and comprises (i) a sense and antisense strand each of 23 nucleotides in length, and (ii) a double-stranded region of 23 base pairs in length. In another such embodiment, the RNAi agent used to inhibit the expression of the ACVR2B gene is blunt-ended and comprises (i) a sense and antisense strand each of 19 nucleotides in length, and (ii) a double-stranded region of 19 base pairs in length.

[0153] In other embodiments, the sense strand or the antisense strand is longer than the other strand, and the two strands form a double-stranded region equal in length to the length of the shorter strand, such that the RNAi agent for inhibiting expression of the ACVR2B gene includes at least one nucleotide overhang. For example, in some embodiments, the sense strand is 1 to 4 nucleotides longer than the antisense strand, and the double-stranded region formed by the two strands is equal in length to the length of the antisense strand, such that the sense strand forms an overhang with 1 to 4 unpaired nucleotides. In other embodiments, the antisense strand is 1 to 4 nucleotides longer than the sense strand, and the double-stranded region formed by the two strands is equal in length to the length of the sense strand, such that the antisense strand forms an overhang with 1 to 4 unpaired nucleotides. In some embodiments, the nucleotide overhang is 1, 2, 3, or 4 nucleotides in length. In a specific embodiment, the overhang includes 2 nucleotides. In certain embodiments, the overhang includes a single nucleotide.

[0154] The nucleotides that protrude can be ribonucleotides or modified nucleotides as described herein. In some embodiments, the nucleotides that protrude are 2'-modified nucleotides (e.g., 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides) or combinations thereof. For example, in one embodiment, the nucleotides that protrude are deoxyribonucleotides, such as deoxythymidine. In another embodiment, the nucleotides that protrude are 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-methoxyethyl-modified nucleotides, abasic nucleotides, inverted abasic nucleotides, inverted nucleotides or combinations thereof. In other embodiments, the protrusion comprises a 5'-uridine-uridine-3' (5'-UU-3') dinucleotide. In such embodiments, the UU dinucleotide can include a ribonucleotide or modified nucleotide, such as a 2'-modified nucleotide. In other embodiments, the protrusion comprises a 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotide. When there is a nucleotide overhang in the antisense strand, the nucleotides in the overhang may be complementary to the target gene sequence, form a mismatch with the target gene sequence, or contain some other sequence (such as UU, TT, AA, GG, etc.).

[0155] The nucleotide overhang may be at the 5' end or the 3' end of one or both strands. For example, in one embodiment, a RNAi agent for inhibiting the expression of the ACVR2B gene comprises nucleotide overhangs at both the 5' end and the 3' end of the antisense strand. In another embodiment, a RNAi agent for inhibiting the expression of the ACVR2B gene comprises nucleotide overhangs at both the 5' end and the 3' end of the sense strand. In some embodiments, a RNAi agent for inhibiting the expression of the ACVR2B gene includes nucleotide overhangs at both the 5' end of the sense strand and the 5' end of the antisense strand. In other embodiments, a RNAi agent for inhibiting the expression of the ACVR2B gene includes nucleotide overhangs at both the 3' end of the sense strand and the 3' end of the antisense strand. In some embodiments, a RNAi agent for inhibiting the expression of the ACVR2B gene includes only a nucleotide overhang at the 5' end of the sense strand. In some embodiments, a RNAi agent for inhibiting the expression of the ACVR2B gene includes only a nucleotide overhang at the 3' end of the sense strand. In some embodiments, a RNAi agent for inhibiting the expression of the ACVR2B gene includes only a nucleotide overhang at the 3' end of the sense strand. In some embodiments, a RNAi agent for inhibiting the expression of the ACVR2B gene includes only a nucleotide overhang at the 3' end of the antisense strand. In some embodiments, the RNAi agent used to inhibit expression of the ACVR2B gene includes only a nucleotide overhang at the 5' end of the antisense strand. In some embodiments, the RNAi agent used to inhibit expression of the ACVR2B gene includes only a nucleotide overhang at the 5' end of the sense strand.

[0156] The RNAi agent used to inhibit the expression of the ACVR2B gene can include a nucleotide overhang at one end of the double-stranded RNA molecule and a blunt end at the other end. "Blunt end" means that the sense strand and the antisense strand are completely base-paired at the ends of the molecule and that no unpaired nucleotides extend beyond the double-stranded region. In some embodiments, the RNAi agent used to inhibit the expression of the ACVR2B gene includes a nucleotide overhang at the 3' end of the sense strand and a blunt end at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi agent used to inhibit the expression of the ACVR2B gene includes a nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand and the 3' end of the sense strand.

[0157] Specifically, for example, in one embodiment, the RNAi agent for inhibiting the expression of the ACVR2B gene comprises (i) a sense strand of 19 nucleotides in length, (ii) an antisense strand of 21 nucleotides in length, and the two strands form a double-stranded region with a length equal to the length of the sense strand. In another embodiment, the RNAi agent for inhibiting the expression of the ACVR2B gene comprises (i) a sense strand of 21 nucleotides in length, (ii) an antisense strand of 23 nucleotides in length, and the two strands form a double-stranded region with a length equal to the length of the sense strand.

[0158] In some embodiments, an RNAi agent for inhibiting the expression of the ACVR2B gene in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand is no longer than 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 64 to 94, and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom, and the RNAi agent comprises an overhang and a blunt end, and the overhang preferably has 2 unpaired nucleotides.

[0159] In some embodiments, an RNAi agent for inhibiting the expression of the ACVR2B gene in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand being no longer than 23 nucleotides and comprising at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 64 to 94, and nucleotide sequences differing therefrom by 1, 2, or 3 nucleotides, and the RNAi agent comprising an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt end is formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0160] In some embodiments, a RNAi agent for inhibiting the expression of the ACVR2B gene in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is no longer than 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 64 to 94, and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom, and the RNAi agent includes an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt end is formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0161] In some embodiments, a RNAi agent for inhibiting the expression of the ACVR2B gene in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is no longer than 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 64 to 94, and nucleotide sequences differing therefrom by 1, 2, or 3 nucleotides, the sense strand is 19 to 21 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt ends are formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0162] In some embodiments, an RNAi agent for inhibiting the expression of the ACVR2B gene in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is 21 nucleotides in length and is at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 64 to 94 and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom, the sense strand is 19 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt ends are formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0163] In some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense strand is 21 nucleotides in length and is any one nucleotide sequence selected from SEQ ID NOs: 64 to 94, the sense strand is 19 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, the overhang preferably having 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense strand, and the blunt ends are formed at the 3' end of the sense strand and the 5' end of the antisense strand.

[0164] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 64 to 94, and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom, and the sense strand comprising at least 15 (e.g., 15, 16, 17, 18, or 19) consecutive nucleotides selected from any one of SEQ ID NOs: 33 to 63, and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom.

[0165] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand having no more than 23 nucleotides and comprising at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides selected from SEQ ID NOs: 64 to 94, and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom, and the sense strand having no more than 21 nucleotides and comprising at least 15 (e.g., 15, 16, 17, 18, or 19) consecutive nucleotides selected from any one of SEQ ID NOs: 33 to 63, and nucleotide sequences having 1, 2, or 3 nucleotide differences therefrom.

[0166] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand having no more than 23 nucleotides and comprising a nucleotide sequence selected from any one of SEQ ID NOs: 64 to 94, and the sense strand having no more than 21 nucleotides and comprising a nucleotide sequence selected from any one of SEQ ID NOs: 33 to 63.

[0167] In a preferred embodiment, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein:

[0168] The sense strand comprises or is the sequence set forth in SEQ ID NO:33, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:64, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0169] The sense strand comprises or is the sequence set forth in SEQ ID NO:34, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:65, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0170] The sense strand comprises or is the sequence set forth in SEQ ID NO:35, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:66, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0171] The sense strand comprises or is the sequence set forth in SEQ ID NO:36, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:67, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0172] The sense strand comprises or is the sequence set forth in SEQ ID NO:37, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:68, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0173] The sense strand comprises or is the sequence set forth in SEQ ID NO:38, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:69, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0174] The sense strand comprises or is the sequence set forth in SEQ ID NO:39, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:70, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0175] The sense strand comprises or is the sequence set forth in SEQ ID NO:40, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:71, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0176] The sense strand comprises or is the sequence set forth in SEQ ID NO:41, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:72, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0177] The sense strand comprises or is the sequence set forth in SEQ ID NO:42, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:73, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0178] The sense strand comprises or is the sequence set forth in SEQ ID NO:43, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:74, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0179] The sense strand comprises or is the sequence set forth in SEQ ID NO:44, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:75, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0180] The sense strand comprises or is the sequence set forth in SEQ ID NO:45, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:76, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0181] The sense strand comprises or is the sequence set forth in SEQ ID NO:46, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:77, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0182] The sense strand comprises or is the sequence set forth in SEQ ID NO:47, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:78, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0183] The sense strand comprises or is the sequence set forth in SEQ ID NO:48, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:79, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0184] The sense strand comprises or is the sequence set forth in SEQ ID NO:49, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:80, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0185] The sense strand comprises or is the sequence set forth in SEQ ID NO:50, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:81, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0186] The sense strand comprises or is the sequence set forth in SEQ ID NO:51, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:82, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0187] The sense strand comprises or is the sequence set forth in SEQ ID NO:52, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:83, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0188] The sense strand comprises or is the sequence set forth in SEQ ID NO:53, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:84, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0189] The sense strand comprises or is the sequence set forth in SEQ ID NO:54, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:85, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0190] The sense strand comprises or is the sequence set forth in SEQ ID NO:55, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:86, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0191] The sense strand comprises or is the sequence set forth in SEQ ID NO:56, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:87, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0192] The sense strand comprises or is the sequence set forth in SEQ ID NO:57, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:88, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0193] The sense strand comprises or is the sequence set forth in SEQ ID NO:58, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:89, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0194] The sense strand comprises or is the sequence set forth in SEQ ID NO:59, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:90, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0195] The sense strand comprises or is the sequence set forth in SEQ ID NO:60, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:91, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom;

[0196] The sense strand comprises or is the sequence set forth in SEQ ID NO:61, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:92, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0197] The sense strand comprises or is the sequence set forth in SEQ ID NO:62, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence set forth in SEQ ID NO:93, or a nucleotide sequence that has 1, 2, or 3 nucleotide differences therefrom;

[0198] The sense strand comprises or is the sequence shown in SEQ ID NO:63, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:94, or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom.

[0199] In a preferred embodiment, the present invention provides a RNAi agent for inhibiting the expression of the ACVR2B gene, comprising a sense strand and an antisense strand forming complementary double-stranded regions, wherein the sense strand and the antisense strand pair to form any one of duplexes 6102, 6103, 6104, 6105, 6106, 6107, 6108, 6109, 6112, 6113, 6115, 6116, 6117, 6119, 6120, 6121, 6122, 6123, 6124, 6125, 6127, 6128, 6129, 6130, 6131, 6132, 6134, 6135, 6137, 6138, or 6140 as described herein.

[0200] Nucleotide-modified RNAi agents

[0201] For any of the embodiments of the RNAi agent of the present invention described in the section "RNAi agents for inhibiting ACVR2B gene expression", the sense strand and / or antisense strand of the RNAi agent may comprise at least one modified nucleotide.

[0202] In some embodiments, the sense strand and the antisense strand of the RNAi agent for inhibiting ACVR2B gene expression provided by the present invention each contain at least one modified nucleotide.

[0203] In some embodiments, each nucleotide in the sense strand and the antisense strand of the RNAi agent for inhibiting ACVR2B gene expression provided by the present invention is modified.

[0204] In any of the above embodiments, the modified nucleotides are independently selected from 2'-deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides (2'-OMe), 2'-fluorine modified nucleotides (2'-F), 2'-deoxy modified nucleotides, locked nucleic acids (LNA), open ring nucleic acids (UNA), bridge nucleic acids (BNA), glycol nucleic acids (GNA), athreose nucleic acids (TNA), conformationally restricted nucleotides, restricted ethyl nucleotides (cEt), 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-O -methoxyethyl modified nucleotides (2'-MOE), abasic nucleotides, inverted abasic nucleotides, inverted nucleotides, morpholino nucleotides (MOP), phosphoramidates (PN), tetrahydropyran modified nucleotides (THP), 1,5-anhydrohexitol modified (HNA) nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups (PS), nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate, nucleotides containing 5'-phosphate mimetics, nucleotides covalently linked to cationic lipids, nucleotides containing 5'-vinylphosphonate (5'-VP), and combinations thereof.

[0205] In a preferred embodiment, the modified nucleotides are independently selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides comprising thiophosphate internucleotide linkages, and combinations thereof. In a preferred embodiment, each nucleotide of the sense strand and / or antisense strand of the RNAi agent is modified. In a preferred embodiment, each nucleotide of the sense strand and / or antisense strand of the RNAi agent is modified, and the modified nucleotides are independently selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides comprising thiophosphate internucleotide linkages, and combinations thereof.

[0206] In a preferred embodiment, each nucleotide of the sense chain and the antisense chain of the RNAi agent for inhibiting ACVR2B gene expression provided by the present invention is modified, and the modification method is selected from one of STC (Alnylam), ESC (Alnylam), Advanced ESC (Alnylam), ESC+ (Alnylam), AD1-3 (Arrowhead), AD5 (Arrowhead) and GalXC (Dicerna) (see, for example, Hu B, Zhong L, Weng Y, et al. Therapeutic siRNA: state of the art. Signal Transduct Target Ther. 2020; 5(1): 101).

[0207] In a preferred embodiment, the 5'-terminal nucleotide of the antisense strand of the RNAi agent for inhibiting ACVR2B gene expression provided by the present invention contains a phosphate or phosphate analog modification, preferably 5'-VP (see Parmar R, Willoughby JL, Liu J, et al. 5'-(E)-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc Conjugates. Chembiochem. 2016; 17(11): 985-989).

[0208] In some embodiments, each nucleotide of the antisense strand of the RNAi agent for inhibiting ACVR2B gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides.

[0209] In some embodiments, each nucleotide of the antisense strand of the RNAi agent for inhibiting ACVR2B gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7, 12, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides.

[0210] In some embodiments, each nucleotide of the antisense strand of the RNAi agent for inhibiting ACVR2B gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7, 14 and 16 of the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides.

[0211] In some embodiments, each nucleotide of the antisense strand of the RNAi agent for inhibiting ACVR2B gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides, and the antisense strand has at least one phosphorothioate internucleotide linkage. In a preferred embodiment, the phosphorothioate internucleotide linkage exists in one or more of the following: between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand. In a preferred embodiment, the phosphorothioate internucleotide linkage exists between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand.

[0212] In some embodiments, each nucleotide of the sense strand and antisense strand of the RNAi agent for inhibiting ACVR2B gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides; from the 5' end to the 3' end, the nucleotides at positions 7 and 9 of the sense strand are 2'-fluoro-modified nucleotides, one or two of the nucleotides at positions 5, 8, and 11 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides.

[0213] For example, each nucleotide of the sense chain and antisense chain of the RNAi agent for inhibiting the expression of the ACVR2B gene provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7 and 14 of the antisense chain are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense chain is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense chain are all 2'-methoxy-modified nucleotides; from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8 and 9 of the sense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides.

[0214] For example, each nucleotide of the sense chain and antisense chain of the RNAi agent for inhibiting the expression of the ACVR2B gene provided by the present invention is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7 and 14 of the antisense chain are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense chain is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense chain are all 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the nucleotides at positions 7, 8 and 9 of the sense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides.

[0215] For example, each nucleotide of the sense chain and antisense chain of the RNAi agent for inhibiting the expression of the ACVR2B gene provided by the present invention is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7 and 14 of the antisense chain are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense chain is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense chain are all 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the nucleotides at positions 5, 7 and 9 of the sense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides.

[0216] For example, each nucleotide of the sense chain and antisense chain of the RNAi agent for inhibiting the expression of the ACVR2B gene provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7 and 14 of the antisense chain are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense chain is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions of the antisense chain are all 2'-methoxy-modified nucleotides; from the 5' end to the 3' end, the nucleotides at positions 7, 9 and 11 of the sense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides.

[0217] In a preferred embodiment, the sense strand has at least one phosphorothioate internucleotide linkage. In a preferred embodiment, the phosphorothioate internucleotide linkage is present at one or more of the following positions: (i) between the first and second nucleotides at the 5' end of the sense strand; (ii) between the second and third nucleotides at the 5' end of the sense strand; (iii) between the first and second nucleotides at the 3' end of the sense strand; and (iv) between the second and third nucleotides at the 3' end of the sense strand. In a more preferred embodiment, the phosphorothioate internucleotide linkage is present between the first and second nucleotides at the 5' end of the sense strand; and between the second and third nucleotides at the 5' end of the sense strand. In a more preferred embodiment, the phosphorothioate internucleotide linkage exists between the first and second nucleotides at the 5' end of the sense strand; between the second and third nucleotides at the 5' end of the sense strand; between the first and second nucleotides at the 3' end of the sense strand; and between the second and third nucleotides at the 3' end of the sense strand.

[0218] In a preferred embodiment, the antisense strand has at least one phosphorothioate internucleotide linkage. Preferably, the phosphorothioate internucleotide linkage is present in one or more of the following: between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand.

[0219] Therefore, in some embodiments, each nucleotide of the sense strand and the antisense strand of the RNAi agent for inhibiting the expression of the ACVR2B gene provided by the present invention is modified, and in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides, and the antisense strand has at least one phosphorothioate internucleotide linkage, and the phosphorothioate internucleotide linkage is present in one or more of the following: between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; 3 nucleotides; between the 1st nucleotide and the 2nd nucleotide at the 3' end of the antisense chain; and between the 2nd nucleotide and the 3rd nucleotide at the 3' end of the antisense chain; in the 5' to 3' direction, the 7th and 9th nucleotides of the sense chain are 2'-fluoro-modified nucleotides, one or two of the 5th, 8th and 11th nucleotides of the sense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides, and the sense chain has at least one phosphorothioate bond internucleotide connection, and the phosphorothioate bond internucleotide connection exists between the 1st nucleotide and the 2nd nucleotide at the 5' end of the sense chain and / or between the 2nd nucleotide and the 3rd nucleotide at the 5' end of the sense chain.

[0220] In some embodiments, each nucleotide of the sense strand and antisense strand of the RNAi agent for inhibiting ACVR2B gene expression provided by the present invention is modified, and from the 5' end to the 3' end, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro-modified nucleotide, the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy-modified nucleotides, and the antisense strand has at least one phosphorothioate internucleotide linkage, and the phosphorothioate internucleotide linkage exists between: the first nucleotide and the second nucleotide at the 5' end of the antisense strand, and between the second nucleotide and the third nucleotide at the 5' end of the antisense strand. , between the first nucleotide and the second nucleotide at the 3' end of the antisense chain, and between the second nucleotide and the third nucleotide at the 3' end of the antisense chain; in the direction from 5' to 3', the nucleotides at positions 7 and 9 of the sense chain are 2'-fluoro-modified nucleotides, one or two of the nucleotides at positions 5, 8 and 11 of the sense chain are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions of the sense chain are all 2'-methoxy-modified nucleotides, and the sense chain has at least one phosphorothioate bond internucleotide connection, and the phosphorothioate bond internucleotide connection exists between the first nucleotide and the second nucleotide at the 5' end of the sense chain and between the second nucleotide and the third nucleotide at the 5' end of the sense chain.

[0221] Ligand-linked oligonucleotides and RNAi agents

[0222] For any of the embodiments of the antisense oligonucleotide or RNAi agent of the present invention described in the above sections "Antisense oligonucleotides for inhibiting ACVR2B gene expression", "RNAi agents for inhibiting ACVR2B gene expression" and "Nucleotide-modified RNAi agents", the antisense oligonucleotide or RNAi agent may comprise a ligand. As used herein, "ligand" refers to any compound or molecule that is capable of interacting directly or indirectly with another compound or molecule. The interaction of a ligand with another compound or molecule may trigger a biological response (e.g., initiating a signal transduction cascade, inducing receptor-mediated endocytosis), or it may simply be a physical connection. The ligand may alter one or more properties of the attached double-stranded RNA molecule, such as the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and / or clearance of the RNA molecule.

[0223] The ACVR2B gene is expressed in a variety of cells and tissues. Therefore, in certain embodiments, it is desirable to specifically deliver the antisense oligonucleotides or RNAi agents of the present invention to hepatocytes. Therefore, in certain embodiments, ligands are used to specifically target the delivery of antisense oligonucleotides or RNAi agents to hepatocytes using various methods described in more detail below. In certain embodiments, antisense oligonucleotides or RNAi agents are targeted to hepatocytes using ligands that bind to surface-expressed asialoglycoprotein receptors (ASGRs) or components thereof (e.g., ASGR1, ASGR2).

[0224] In some embodiments, antisense oligonucleotides or RNAi agents can be specifically targeted to the liver by using a ligand that binds to or interacts with a protein expressed on the surface of hepatocytes. For example, in certain embodiments, the ligand can include an antigen binding protein (e.g., an antibody or a binding fragment thereof (e.g., Fab, scFv)) that specifically binds to receptors expressed on hepatocytes, such as asialoglycoprotein receptors and LDL receptors. In a specific embodiment, the ligand includes an antibody or a binding fragment thereof that specifically binds to ASGR1 and / or ASGR2. In another embodiment, the ligand includes a Fab fragment of an antibody that specifically binds to ASGR1 and / or ASGR2. In another embodiment, the ligand includes a single-chain variable antibody fragment (scFv fragment) of an antibody that specifically binds to ASGR1 and / or ASGR2. Exemplary antibodies and binding fragments thereof that can be used as ligands for targeting the antisense oligonucleotides or RNAi agents of the present invention to the liver are described in WO 2017 / 058944, which is incorporated herein by reference in its entirety. Other antibodies or binding fragments thereof that specifically bind to ASGR1, the LDL receptor, or other liver surface expressed proteins suitable for use as ligands in the RNAi agents of the present invention are purchased from commercial sources.

[0225] In some embodiments, when used for extrahepatic delivery, including but not limited to, the central nervous system (CNS) (e.g., brain, spine, or eye), muscle, lung, or fat, the antisense oligonucleotide or RNAi agent may comprise one or more lipophilic groups conjugated to one or more positions of the oligonucleotide or one or more positions of the antisense strand and / or sense strand of the RNAi agent. For example, a lipophilic group may be connected to the antisense strand and / or sense strand of the antisense oligonucleotide or RNAi agent via a nucleobase, a sugar moiety, or an internucleoside bond. In some embodiments, a lipophilic group having a phosphoramidite group is coupled to the 3' end or 5' end of the sense strand or antisense strand in the final synthesis cycle. In some embodiments, the octanol-water partition coefficient of the lipophilic group exceeds 0, 1, 1.5, 2, 3, 4, 5, or 10. In some embodiments, the ligand is a lipophilic group, preferably selected from the group consisting of lipids, vitamins, steroids, C5-C 30 Saturated or unsaturated fatty acids, C5-C30 alkyl, and a polypeptide comprising at least one positively charged amino acid residue; the lipophilic group is more preferably selected from cholesterol, C 16 Saturated or unsaturated fatty acids, C 16 Alkyl, C 22 Saturated or unsaturated fatty acids or C 22 Suitable lipophilic groups may be aliphatic, alicyclic, polyalicyclic, steroid, straight chain or branched aliphatic hydrocarbon.

[0226] Exemplary lipophilic groups are, for example, lipophilic groups Y132 to Y135, Y158, Y165 to Y168, L10, L57, L321, L322, Q361 to Q367, Q361s to Q367s, Q370, Q377 to Q379, Q383, etc., described in WO 2021 / 092371. Other ligands that can be linked to the antisense oligonucleotides or RNAi agents of the present invention can be found in WO2017053995, WO2019217459, WO2021092371, WO2017053995, WO2010039548, WO2023064530, WO2022213118, and WO2019079386, the entire contents of each of which are incorporated herein by reference.

[0227] In certain embodiments, part includes carbohydrate." carbohydrate " refers to the compound that is made up of one or more monosaccharide units with at least 6 carbon atoms (can be straight chain, branched or cyclic), and oxygen, nitrogen or sulphur atom is connected on each carbon atom. Carbohydrate includes but is not limited to sugar (for example, monosaccharide, disaccharide, trisaccharide, tetrasaccharide and oligosaccharide containing about 4,5,6,7,8 or 9 monosaccharide units) and polysaccharide (such as starch, glycogen, cellulose and polysaccharide glue). In some embodiments, the carbohydrate that is incorporated into the part is disaccharide and trisaccharide selected from pentose, hexose or heptose and comprises such monosaccharide unit. In other embodiments, the carbohydrate that is incorporated into the part is amino sugar, for example galactosamine, glucosamine, N-acetylgalactosamine and N-acetylglucosamine.

[0228] In some embodiments, the ligand comprises a hexose or a hexosamine. The hexose can be selected from glucose, galactose, mannose, fucose or fructose. The hexosamine can be selected from fructosamine, galactosamine, glucosamine or mannosamine. In certain embodiments, the ligand comprises glucose, galactose, galactosamine or glucosamine. In one embodiment, the ligand comprises glucose, glucosamine or N-acetylglucosamine. In another embodiment, the ligand comprises galactose, galactosamine or N-acetylgalactosamine. In specific embodiments, ligands comprising glucose, galactose and N-acetylgalactosamine (GalNAc) are particularly effective in targeting RNA to hepatocytes because these ligands bind to ASGR expressed on the surface of hepatocytes. Examples of GalNAc- or galactose-containing ligands that can be incorporated into the antisense oligonucleotides or RNAi agents of the invention are described in USP 7,491,805, 8,106,022, and 8,877,917; US Patent Publication No. US20030130186; and WIPO Publication No. WO 2013 / 166155, all of which are incorporated herein by reference in their entirety.

[0229] In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, a "multivalent carbohydrate moiety" refers to a moiety comprising two or more carbohydrate units that are capable of independently binding to or interacting with other molecules. For example, a multivalent carbohydrate moiety comprises two or more binding domains composed of carbohydrates that can bind to two or more different molecules or two or more different sites on the same molecule. The "valency" of a carbohydrate moiety refers to the number of individual binding domains within the carbohydrate moiety. For example, the terms "monovalent," "divalent," "trivalent," and "tetravalent" refer to carbohydrate moieties having one, two, three, and four binding domains, respectively, with respect to a carbohydrate moiety. The multivalent carbohydrate moiety can include a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetylgalactosamine moiety, a multivalent N-acetylglucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety. In some embodiments, the ligand comprises a multivalent galactose moiety. In other embodiments, the ligand comprises a multivalent N-acetyl-galactosamine moiety. In these and other embodiments, the multivalent carbohydrate moiety can be divalent, trivalent, or tetravalent. In such embodiments, the multivalent carbohydrate moiety can be bivalent or trivalent. In a specific embodiment, the multivalent N-acetylgalactosamine moiety is trivalent or tetravalent. In another specific embodiment, the multivalent galactose moiety is trivalent or tetravalent. Exemplary trivalent and tetravalent GalNAc-containing ligands for incorporation into the antisense oligonucleotides or RNAi agents of the present invention are described in detail below.

[0230] The ligand can also include an integrin ligand, such as a ligand that specifically binds to an integrin (including but not limited to αVβ6). Suitable integrin ligands are, for example, those mentioned in WO2019089765A1 or WO2022056286A1, the entire contents of which are incorporated herein by reference.

[0231] The ligand may also include a transferrin receptor 1 (TfR1) ligand, such as an anti-TfR1 antibody or polypeptide. Suitable TfR1 ligands are, for example, the TfR1 antibodies described in WO2022147209A1 or WO2021154476A1, or the bicyclic peptide ligand described in WO2022101633A1. The entire contents of these patent documents are incorporated herein by reference.

[0232] The part can be directly or indirectly connected or conjugated to the RNA molecule of antisense oligonucleotide or RNAi agent.For example, in some embodiments, the part is directly covalently connected to the sense strand or the antisense strand of antisense oligonucleotide or RNAi agent.In other embodiments, the part is covalently connected to the sense strand or the antisense strand of antisense oligonucleotide or RNAi agent by a joint.The part can be connected to the core base, sugar moiety or the internucleotide junction of the sense strand or the antisense strand of antisense oligonucleotide or RNAi agent of the present invention.

[0233] In some embodiments, the ligand can be connected to the 3' or 5' end of the sense strand or antisense strand. In certain embodiments, the ligand is covalently attached to the 5' end of the sense strand. In such embodiments, the ligand is attached to the 5'-terminal nucleotide of the sense strand. In these and other embodiments, the ligand is attached to the 5'-position of the 5'-terminal nucleotide of the sense strand. In other embodiments, the ligand is covalently attached to the 3' end of the sense strand. For example, in some embodiments, the ligand is attached to the 3'-terminal nucleotide of the sense strand. In some such embodiments, the ligand is attached to the 3'-position of the 3'-terminal nucleotide of the sense strand. In alternative embodiments, the ligand is attached near the 3' end of the sense strand, but before one or more terminal nucleotides (i.e., before 1, 2, 3, or 4 terminal nucleotides). In some embodiments, the ligand is attached to the 2'-position of the sugar of the 3'-terminal nucleotide of the sense strand. In other embodiments, the ligand is attached to the 2'-position of the sugar of the 5'-terminal nucleotide of the sense strand.

[0234] In certain embodiments, the part is connected to the sense strand or antisense strand by a joint." joint " refers to the atom or one group of atoms by which the part is covalently connected to the polynucleotide components of antisense oligonucleotides or RNAi agents. The length of the joint can be about 1 to about 30 atoms, about 2 to about 28 atoms, about 3 to about 26 atoms, about 4 to about 24 atoms, about 6 to about 20 atoms, about 7 to about 20 atoms, about 8 to about 20 atoms, about 8 to about 18 atoms, and about 12 to about 18 atoms. In some embodiments, the joint can include a bifunctional linking moiety, which generally includes an alkyl moiety with two functional groups. One of the functional groups is selected to be combined with a compound of interest (such as the sense or antisense strand of an RNAi agent chain), and another functional group is selected to be combined with substantially any selected group, such as a part as described herein. In certain embodiments, the joint includes the oligomer of a chain structure or a repeating unit, such as ethylene glycol or an amino acid unit. Examples of functional groups commonly used in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, the bifunctional linking moiety includes an amino group, a hydroxyl group, a carboxylic acid, a thiol group, an unsaturated bond (e.g., a double bond or a triple bond), and the like.

[0235] Linkers that can be used to attach the ligand to the sense or antisense strand of the antisense oligonucleotide or RNAi agent of the invention include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxooctanoic acid, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid, 6-aminohexanoic acid, substituted C1-C 20 Alkyl, substituted or unsubstituted C2-C 20 Alkenyl or substituted or unsubstituted C2-C 20 Preferred substituents for such linkers include, but are not limited to, hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0236] In certain embodiments, joint is cleavable.Cleaving joint is a kind of enough stable outside cell but is cracked to release two parts that combine together by joint when entering target cell.In some embodiments, cleavable joint is in target cell or under the first reference condition (it can for example be selected as simulation or represent cell condition) than cracking in experimenter's blood fast at least 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least 100 times.

[0237] Cleavable linkers are susceptible to the effects of cleavage agents, such as pH, redox potential, or the presence of degrading molecules. Generally, cleavage agents are more prevalent in cells than in serum or blood, or are found at higher levels or activity in cells. Examples of such cleavage agents include: redox agents selected for specific substrates or having no substrate specificity, including, for example, oxidases or reductases present in cells; esterases; endosomes or reagents that can produce an acidic environment, such as those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linkers as general acids, peptidases (which can be substrate-specific), and phosphatases.

[0238] Cleavable linkers can include pH-sensitive moieties. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, in the range of 5.5 to 6.0, and lysosomes have a more acidic pH (about 5.0). Some linkers will have a cleavable group that is cleaved at a preferred pH, thereby releasing the RNA molecule from the ligand in the cell, or releasing it into the desired organelle of the cell. The linker can include a cleavable group that can be cleaved by a specific enzyme. The type of cleavable group incorporated into the linker can depend on the cell to be targeted. For example, a liver targeting ligand can be connected to the RNA molecule by a linker that includes an ester group. Hepatocytes are rich in esterases, so the linker is more effectively cleaved in hepatocytes than in cell types that are not rich in esterases. Other types of cells rich in esterases include cells of the lung, renal cortex, and testis. When targeting cells rich in peptidases, such as hepatocytes and synovial cells, a linker containing a peptide bond can be used.

[0239] Other types of linkers suitable for attaching ligands to the sense or antisense strands in the antisense oligonucleotides or RNAi agents of the invention are known in the art, such as those described in U.S. Patents 7,723,509, 8,017,762, 8,828,956, 8,877,917, and 9,181,551, all of which are incorporated herein by reference in their entirety.

[0240] In some embodiments, the ligand covalently attached to the sense strand or antisense strand of the antisense oligonucleotide or RNAi agent of the present invention includes a GalNAc moiety, such as a multivalent GalNAc. In some embodiments, the multivalent GalNAc moiety is a trivalent GalNAc and is attached to the 3' end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a trivalent GalNAc and is attached to the 5' end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3' end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5' end of the sense strand.

[0241] In some embodiments, the ligand is L96, NAG25, and NAG37, each having the structural formula shown below, wherein the wavy line represents the position of attachment to the sense or antisense strand of the antisense oligonucleotide or RNAi agent.

[0242] Pharmaceutical composition

[0243] The present invention also includes pharmaceutical compositions and formulations comprising the antisense oligonucleotides or RNAi agents described herein and a pharmaceutically acceptable carrier, excipient, or diluent. Such compositions and formulations can be used to reduce the expression of the ACVR2B gene in patients in need thereof. In the case of clinical applications, pharmaceutical compositions and formulations will be prepared in a form suitable for the intended application. Typically, this will require preparing a composition that is substantially free of pyrogens and other impurities that may be harmful to humans or animals.

[0244] The composition and method for preparing the pharmaceutical composition depend on many standards, including but not limited to route of administration, the type and degree of the disease to be treated or the condition or the dosage to be administered. In some embodiments, the pharmaceutical composition is prepared based on the expected route of delivery. For example, in certain embodiments, the pharmaceutical composition is formulated for parenteral delivery. Parenteral administration forms include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such an embodiment, the pharmaceutical composition may include a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such an embodiment, the pharmaceutical composition may include a targeting ligand (such as a ligand containing GalNAc as described herein or containing an antibody).

[0245] In some embodiments, the pharmaceutical composition comprises an effective amount of an antisense oligonucleotide or RNAi agent as described herein. An "effective amount" refers to an amount sufficient to produce a beneficial or desired clinical outcome. In some embodiments, an effective amount is an amount sufficient to reduce the expression of the ACVR2B gene in a patient's specific tissue or cell type (e.g., liver or hepatocytes). The effective amount of the antisense oligonucleotide or RNAi agent of the present invention can be from about 0.01 mg / kg body weight to about 100 mg / kg body weight and can be administered daily, weekly, monthly, or at longer intervals. Accurately determining a specific effective dosage and dosing frequency may be based on several factors, including the patient's size, age, and general condition, the type of disease to be treated (e.g., myocardial infarction, coronary artery disease, peripheral artery disease, stroke), the specific antisense oligonucleotide or RNAi agent used, and the route of administration.

[0246] The administration of the pharmaceutical composition of the present invention can be carried out by any common route, as long as the target tissue can be obtained by the route. These routes include but are not limited to parenteral (e.g., subcutaneous, intramuscular, intraperitoneal or intravenous), oral, nasal, oral, intradermal, transdermal and sublingual routes, or by direct injection into liver tissue or delivered by the portal vein. In some embodiments, the pharmaceutical composition is parenteral. For example, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously.

[0247] Colloidal dispersion systems can be used as delivery vehicles for antisense oligonucleotides or RNAi agents of the present invention, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Commercially available fat emulsions suitable for transporting nucleic acids of the present invention include Intralipid (Baxter International Inc.), Liposyn (Abbott Pharmaceuticals), Lipsyn II (Hospira), Liposyn III (Hospire), Nutrilipid (B.Braun Medical Inc.), and other similar fat emulsions. A preferred colloidal system for use as an in vivo delivery vehicle is a liposome (i.e., an artificial membrane vesicle). The RNAi agent of the present invention can be encapsulated in a liposome, or a complex can be formed therewith, particularly with a cationic liposome. Alternatively, the RNAi agent of the present invention can be complexed with lipids, particularly with cationic lipids. Suitable cationic lipids are, for example, diol tetramethylaminopropyl (DOTAP) and diol phosphatidylethanolamine (DOTMA).

[0248] Liposomal formulations are particularly well-suited for topical administration, and liposomes offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer antisense oligonucleotides or RNAi agents to the skin. In some embodiments, liposomes are used to deliver antisense oligonucleotides or RNAi agents to epidermal cells and also enhance penetration of the antisense oligonucleotides or RNAi agents into dermal tissue, such as the skin.

[0249] Antisense oligonucleotides of the present invention or RNAi agent can be fully encapsulated in lipid formulations, such as LNP or other nucleic acid-lipid particles. As used herein, term " LNP " refers to stable nucleic acid-lipid particles. LNP generally comprises cationic lipids, non-cationic lipids and the lipid (such as PEG-lipid conjugate) that prevents particle aggregation. LNP is very useful for systemic application because they show prolonged circulation time after intravenous (iv) injection, and accumulate in distal sites (such as the position physically separated from the administration site). LNP includes " pSPLP ", which includes the condensing agent-nucleic acid complex of the encapsulation as described in WO00 / 03683. LNP particles of the present invention generally have an average diameter of about 50nm to about 150nm, more typically about 60nm to about 130nm, more typically about 70nm to about 110nm, most typically about 70nm to about 90nm, and substantially nontoxic. In addition, when nucleic acid is present in nucleic acid-lipid particles of the present invention, it has resistance to the degradation of nuclease in aqueous solution. Nucleic acid-lipid particles and methods for preparing the same are disclosed in, for example, U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication Nos. 2010 / 0324120 and WO 96 / 40964. In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to RNAi agent ratio) will be in the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0250] The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DLenDMA). Dioleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dihydroxypropoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dihydroxypropoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-di Linoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-(N-methylpiperazino)propane (DLin-MPZ) or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenoyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin -K-DMA) or its analogues, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriacontac-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazadiyl)didecadecan-2-ol (Tech G1) or mixtures thereof.The cationic lipid may comprise from about 20 mol% to about 50 mol%, or about 40 mol% of the total lipid present in the particle.

[0251] In some embodiments, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. In some embodiments, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% cholesterol: 10% PEG-C-DOMG (molar percentage), have a particle size of 63.0±20 nm, and a siRNA / lipid ratio of 0.027.

[0252] The ionizable / non-cationic lipids can be anionic lipids or neutral lipids, including but not limited to distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoyl DP-acylglyceroglycerophosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), oleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), oleoylphosphatidylcholine (POPC), palmitoylphosphatidylcholine ...DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC The non-cationic lipid may be present in an amount of about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % (if cholesterol is included) of the total lipids present in the particle.

[0253] In some embodiments, the nucleic acid-lipid particle further comprises cholesterol, for example, 10 mol% to 20 mol% of the total lipid present in the particle, or about 2 mol%. In some embodiments, the nucleic acid-lipid particle further comprises cholesterol, for example, 10 mol% to 60 mol% of the total lipid present in the particle, or about 48 mol%.

[0254] In one embodiment, lipidoid ND98·4HCl (molecular weight 1487) (see U.S. Patent Application No. 12 / 056,230, incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Each stock solution in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-ceramide C16, 100 mg / ml. The stock solutions of ND98, cholesterol, and PEG-ceramide C16 can then be mixed in a molar ratio of, for example, 42:48:10. The combined lipid solution can be mixed with aqueous siRNA (e.g., in sodium acetate at pH 5) to a final ethanol concentration of approximately 35-45% and a final sodium acetate concentration of approximately 100-300 mM. Lipid-siRNA nanoparticles typically form spontaneously upon mixing.

[0255] Other exemplary lipid-siRNA formulations can be found in, for example, WO2009 / 127060 (SNALP), PCT / US2010 / 022614 (XTC), US2010 / 0324120 (MC3), PCT / US09 / 63933 (ALNY-100), and WO2010 / 129709 (C12-200).

[0256] In some embodiments, the antisense oligonucleotides or RNAi agents of the present invention can be linked to a cell penetrating peptide (CPP), which can include at least one positively charged amino acid or ionizable amino acid, such as arginine, to form a peptide-oligonucleotide conjugate. Suitable CPPs for forming peptide-oligonucleotide conjugates can be found, for example, in WO2022213118A1 or WO2019079386A1, the entire contents of which are incorporated herein by reference.

[0257] Pharmaceutical compositions suitable for injection include, for example, sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In general, these preparations are sterile and, to a certain extent, fluid and easy to inject. The preparation should remain stable under production and storage conditions and should be preserved to prevent contamination by microorganisms such as bacteria and fungi. Suitable solvents or dispersion media can include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. For example, suitable fluidity can be maintained by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersion, and by using a surfactant. The effects of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugar or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption in the composition, such as aluminum monostearate and gelatin.

[0258] Sterile injectable solutions can be prepared by adding an appropriate amount of the active compound to a solvent along with any other ingredients (e.g., those listed above) and then sterilizing by filtration. Typically, dispersions are prepared by adding the various sterilized active ingredients to a dispersion medium containing an alkaline dispersion medium and the desired other ingredients, e.g., as described above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods include vacuum drying and freeze drying techniques, which produce a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof.

[0259] The compositions of the present invention can generally be formulated in neutral form or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed by free amino groups) derived from inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (such as sodium, potassium, ammonium, calcium or ferric oxide) or organic bases (such as isopropylamine, trimethylamine, histidine, procaine, etc.). In some embodiments, the antisense oligonucleotides or RNAi agents of the present invention are formulated as sodium salts.

[0260] For example, for parenteral administration in the form of an aqueous solution, the solution is generally appropriately buffered, and the liquid diluent is first made isotonic with, for example, enough saline or glucose. Such an aqueous solution can be used for, for example, intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Preferably, a sterile aqueous medium is used. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion liquid, or injected at the infusion site of the suggestion. For human administration, the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by the local Food and Drug Administration. In certain embodiments, the pharmaceutical composition of the present invention comprises sterile saline solution as described herein and antisense oligonucleotide or RNAi agent or consists of the two. In other embodiments, the pharmaceutical composition of the present invention comprises antisense oligonucleotide or RNAi agent as described herein and sterile water (e.g., water for injection, WFI) or consists of the two. In other embodiments, the pharmaceutical composition of the present invention comprises antisense oligonucleotide or RNAi agent as described herein and phosphate buffered saline (PBS) or consists of it.

[0261] In some embodiments, the pharmaceutical compositions of the present invention are packaged with or stored within a drug delivery device. Devices for injecting formulations include, but are not limited to, injection ports, prefilled syringes, autoinjectors, syringe pumps, intracorporeal syringes, and injection pens. Devices for aerosolizing or powdered formulations include, but are not limited to, inhalers, insufflators, aspirators, and the like. Thus, the present invention includes a drug delivery device containing a pharmaceutical composition of the present invention for use in treating or preventing one or more diseases or conditions described herein.

[0262] Treatment methods and uses

[0263] The present invention provides a method for reducing or inhibiting expression of the ACVR2B gene in a cell by contacting the cell with any of the antisense oligonucleotides or RNAi agents described herein. The cell can be in vitro or in vivo. ACVR2B gene expression can be assessed by measuring the amount or level of ACVR2B mRNA or ACVR2B-C protein. Reduction of ACVR2B expression in cells or animals treated with the antisense oligonucleotides or RNAi agents of the present invention can be determined relative to ACVR2B expression in cells or animals not treated with the RNAi agent or treated with a control RNAi agent. For example, in some embodiments, reduction in ACVR2B expression is assessed by (a) measuring the amount or level of ACVR2B mRNA in cells treated with a RNAi agent of the invention, (b) measuring the amount or level of ACVR2B mRNA in cells treated with a control RNAi agent (e.g., an RNAi agent directed against an RNA molecule not expressed in the cell or an RNAi agent with a nonsense or scrambled sequence) or no RNAi agent, and (c) comparing the ACVR2B mRNA level measured in the treated cells in (a) with the ACVR2B mRNA level in the control cells in (b). Prior to comparison, the ACVR2B mRNA levels in the treated and control cells can be normalized to the RNA level of a control gene (e.g., 18S ribosomal RNA or a housekeeping gene). ACVR2B mRNA levels can be measured by a variety of methods, including Northern blot analysis, nuclease protection assays, fluorescence in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, droplet digital PCR, and the like.

[0264] In some embodiments, the method of assessing ACVR2B expression levels is performed in vitro in cells that naturally express the ACVR2B gene or in cells that have been engineered to express ACVR2B.

[0265] In other embodiments, the method for assessing ACVR2B expression levels is performed in vivo. The antisense oligonucleotide or RNAi agent and any control antisense oligonucleotide or RNAi agent can be administered to an animal (e.g., a transgenic animal or non-human primate expressing the ACVR2B gene), and ACVR2B mRNA or ACVR2B protein levels can be assessed in tissues harvested from the animal after treatment. Alternatively or additionally, biomarkers or functional phenotypes associated with ACVR2B expression can be assessed in the treated animal. For example, ACVR2B protein is an expression product of ACVR2B mRNA in tissue. Therefore, the level of ACVR2B protein can be measured in animals treated with the antisense oligonucleotide or RNAi agent of the invention to assess the functional efficacy of reducing ACVR2B expression.

[0266] In certain embodiments, the expression of ACVR2B in a cell is reduced by at least 40%, at least 45%, or at least 50% by an antisense oligonucleotide or RNAi agent of the invention. In some embodiments, the expression of ACVR2B in a cell is reduced by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% by an antisense oligonucleotide or RNAi agent of the invention. In other embodiments, the expression of ACVR2B in a cell is reduced by about 90% or more, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, by an antisense oligonucleotide or RNAi agent of the invention. The percentage reduction in ACVR2B expression can be measured by any of the methods described herein, as well as other methods known in the art.

[0267] The present invention provides methods for reducing or inhibiting the expression of the ACVR2B gene, thereby reducing or inhibiting the production of ACVR2B protein, in a patient in need thereof, as well as methods for treating or preventing diseases or conditions associated with ACVR2B expression or activity. A "disease or condition mediated by aberrant ACVR2B expression" refers to a disease or condition in which ACVR2B expression levels are altered, or a condition in which elevated ACVR2B expression levels are associated with an increased risk of developing the disease or condition. In certain embodiments, the antisense oligonucleotides or RNAi agents of the present invention are particularly useful for reducing ACVR2B levels.

[0268] Diseases and conditions associated with ACVR2B expression that can be treated or prevented according to the methods of the present invention include, but are not limited to, muscle atrophy, muscular dystrophy, obesity, diabetes, chronic kidney disease, acute kidney injury, post-traumatic healing, maxillofacial tumors, eclampsia during pregnancy, liver fibrosis, atherosclerosis, ischemic stroke, and Alzheimer's disease.

[0269] In certain embodiments, the present invention provides a method for reducing ACVR2B expression in a patient in need thereof, comprising administering to the patient any of the antisense oligonucleotides or RNAi agents described herein. Preferably, after administration of the antisense oligonucleotide or RNAi agent, the expression level of ACVR2B in the patient's cells is reduced compared to the expression level of ACVR2B in the patient who did not receive the antisense oligonucleotide or RNAi agent, or compared to the expression level of ACVR2B in the patient before administration of the antisense oligonucleotide or RNAi agent. In some embodiments, after administration of the antisense oligonucleotide or RNAi agent of the invention, the expression of ACVR2B in the patient is reduced by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The percent reduction in ACVR2B expression can be measured by any of the methods described herein, as well as other methods known in the art. In certain embodiments, the percent reduction in ACVR2B expression is determined by assessing the patient's ACVR2B protein levels according to the methods described herein.

[0270] In certain embodiments, the patient in need of reduced ACVR2B expression is diagnosed with or at risk for a disease mediated by aberrant ACVR2B expression levels. Accordingly, the present invention includes a method for treating or preventing a disease mediated by aberrant ACVR2B expression levels in a patient in need thereof by administering any of the antisense oligonucleotides or RNAi agents of the present invention. In some embodiments, the present invention includes the use of any of the antisense oligonucleotides or RNAi agents described herein in the preparation of a medicament for treating or preventing a disease mediated by aberrant ACVR2B expression levels in a patient in need thereof. In other embodiments, the present invention provides an ACVR2B-targeting antisense oligonucleotide or RNAi agent for use in a method for treating or preventing a disease mediated by aberrant ACVR2B expression levels in a patient in need thereof. As described above, the diseases mediated by aberrant ACVR2B expression levels include, for example, muscle atrophy, muscular dystrophy, obesity, diabetes, chronic kidney disease, acute kidney injury, postoperative traumatic healing, maxillofacial tumors, eclampsia during pregnancy, liver fibrosis, atherosclerosis, ischemic stroke, and Alzheimer's disease. In one embodiment, the disease is muscle atrophy. In one embodiment, the disease is muscular dystrophy. In one embodiment, the disease is obesity. In one embodiment, the disease is diabetes. In one embodiment, the disease is chronic kidney disease. In one embodiment, the disease is acute kidney injury. In one embodiment, the disease is postoperative healing of trauma. In one embodiment, the disease is a maxillofacial tumor. In one embodiment, the disease is gestational eclampsia. In one embodiment, the disease is liver fibrosis. In one embodiment, the disease is atherosclerosis. In one embodiment, the disease is ischemic stroke. In one embodiment, the disease is Alzheimer's disease.

[0271] In certain other embodiments, the patient in need of reduced ACVR2B expression is a patient with elevated ACVR2B levels. Thus, in some embodiments, the present invention provides a method of reducing ACVR2B protein levels in a patient in need thereof by administering to the patient any of the antisense oligonucleotides or RNAi agents described herein. In some embodiments, the present invention includes the use of any of the antisense oligonucleotides or RNAi agents described herein in the preparation of a medicament for reducing ACVR2B protein levels in a patient in need thereof. In other embodiments, the present invention provides an ACVR2B-targeted antisense oligonucleotide or RNAi agent for use in a method of reducing ACVR2B protein levels in a patient in need thereof.

[0272] In some embodiments of the present invention, the present invention provides the use of an agent that reduces expression of the gene encoding the activin type IIB receptor (ACVR2B) in the preparation of a medicament for treating a disease or condition treatable by ACVR2B knockdown. In preferred embodiments, the agent is an antisense oligonucleotide and / or an RNAi agent. In preferred embodiments, the disease or condition is selected from the group consisting of muscular dystrophy, muscular dystrophy, obesity, diabetes, chronic kidney disease, acute kidney injury, postoperative traumatic healing, maxillofacial tumors, eclampsia during pregnancy, liver fibrosis, atherosclerosis, ischemic stroke, and Alzheimer's disease.

[0273] Therefore, in some embodiments, the present invention provides the use of antisense oligonucleotides and / or RNAi agents that reduce the expression of the gene encoding activin type IIB receptor (ACVR2B) in the preparation of a medicament for treating a disease or condition selected from muscle atrophy, muscular dystrophy, obesity, diabetes, chronic kidney disease, acute kidney injury, postoperative healing of trauma, maxillofacial tumors, eclampsia during pregnancy, liver fibrosis, atherosclerosis, ischemic stroke and Alzheimer's disease.

[0274] Sequence Listing

[0275] Human ACVR2B, mRNA (SEQ ID NO. 1, NCBI accession number NM_001106.4)

[0276] Cynomolgus monkey ACVR2B mRNA (SEQ ID NO. 2, NCBI accession number XM_015445674.2) Example

[0277] Experimental methods and materials

[0278] Target sequence screening

[0279] ASOs and siRNAs were designed based on the full-length ACVR2B mRNA sequence (NM_001106.4). All sequences were obtained from the NCBI gene database. Homology to human (Gene ID: 93, NM_001106.4, SEQ ID NO. 1) and cynomolgus macaque (Gene ID: 102118668, XM_015445674.2, SEQ ID NO. 2) sequences was ensured during design.

[0280] Oligonucleotide synthesis

[0281] The oligonucleotide synthesis method is similar to conventional phosphoramidite solid-phase synthesis (all synthesized by Suzhou Beixin Biotechnology Co., Ltd.), consisting of four steps: deprotection, coupling, capping, and oxidation or sulfurization. Starting with a solid-phase support, the nucleoside monomers are sequentially linked from the 3' to the 5' end. Nucleoside phosphoramidite monomer raw materials, such as 2'-OTBDMS, 2'-F RNA, and 2'-OMe RNA, were purchased from Shanghai Zhaowei Technology Development Co., Ltd. After solid-phase synthesis, the solid-phase support was transferred to a centrifuge tube and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours to cleave the oligonucleotide from the solid-phase support into solution. The supernatant was centrifuged and transferred to another centrifuge tube, concentrated and evaporated to dryness, then redissolved in deionized water and purified using a C18 reverse-phase chromatography column with a mobile phase of 0.1 M TEAA and acetonitrile. The target oligonucleotide was collected, lyophilized, identified as the target product by LC-MS, and quantified by UV spectroscopy (260 nm). siRNA is obtained by annealing the sense and antisense strands.

[0282] Dissolution of compounds

[0283] Place the compound powder tube in a centrifuge and centrifuge at 12,000 rpm for 5 min. Add an appropriate amount of RNase-free deionized water and mix well to prepare a 10 μM stock solution. Each compound is dispensed into 3 tubes, 50 μl / tube, and stored in a -20°C refrigerator.

[0284] Reverse transfection

[0285] To dilute the compound, dilute the 10 μM compound stock solution to 12-fold the working concentration in Opti-MEM Reduced Serum Medium and set aside. To dilute the transfection reagent, dilute the transfection reagent in Opti-MEM Reduced Serum Medium at a ratio of 48.5:1.5, mix gently, and let it sit at room temperature for 15 minutes. To prepare the transfection complex, mix equal volumes of the diluted compound and transfection reagent and let it sit at room temperature for 15 minutes. Add 20 μl of the transfection complex to each well of a 96-well cell plate. To seed the cells, select A-673 cells at approximately 80% confluency, trypsinize, resuspend in complete medium without penicillin and streptomycin, and count the cells. Prepare a cell suspension at a concentration of 150,000 cells / ml and add 100 μl of the cell suspension to each well of the cell plate containing the transfection complex. Incubate the cells at 37°C in a 5% CO2 incubator for 24 hours.

[0286] Cell lysis

[0287] Prepare cell lysis buffer according to the following table

[0288] Remove the cell culture plate, discard the culture medium, and wash the cells once with pre-chilled DPBS. Add 50 μl of cell lysis buffer to each well, pipette up and down 5-10 times to mix thoroughly, and let stand at room temperature for 10 minutes. Then, add 5 μl of cell lysis stop buffer to each well, pipette up and down 5-10 times to mix thoroughly, and wait at room temperature for 2 minutes to terminate the reaction. Place the cell plate on ice until ready to use.

[0289] Real-time fluorescence quantitative PCR detection

[0290] Quantitative PCR reaction system

[0291] Prepare a quantitative PCR reaction system according to the table above and add 9 μl to a 384-well plate. Add 1 μl of cell lysate as template. Complete the PCR amplification reaction according to the protocol in the table below.

[0292] Data Analysis

[0293] The relative quantitative PCR method (2 -ΔΔCT ) to analyze the relative expression of genes.

[0294] In order to evaluate the in vitro inhibitory activity of ASO compounds on ACVR2B mRNA in A673 cell lines, the designed ASO compounds were subjected to in vitro functional evaluation. The results are shown in Table 3.

[0295] Table 3. In vitro inhibitory activity of ASO compounds on ACVR2B mRNA in A673 cell line

[0296] In order to evaluate the in vitro inhibitory activity of siRNA compounds on ACVR2B mRNA in A673 cell lines, the designed siRNAs were subjected to in vitro functional evaluation. The results are shown in Table 4.

[0297] Table 4. In vitro inhibitory activity of siRNA compounds against ACVR2B mRNA in A673 cell line

[0298] In order to evaluate the in vitro inhibitory activity of siRNA compounds on ACVR2B mRNA in A673 cell lines, the designed siRNAs were subjected to in vitro functional evaluation. The results are shown in Table 5.

[0299] Table 5 In vitro inhibitory activity of siRNA compounds against ACVR2B mRNA in A673 cell line

Claims

1. An antisense oligonucleotide comprising an oligonucleotide consisting of 12 to 30 linked nucleotides, said oligonucleotide comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides that are at least 80%, 85%, 90%, 95%, or 100% complementary to a continuous nucleotide sequence of equal length to a target region of the mRNA encoding activin type IIB receptor (ACVR2B).

2. The antisense oligonucleotide according to claim 1, wherein the target region is 504 - 554, 588 - 617, 648 - 686, 837 - 866, 1234 - 1268, 1360 - 1403, 1503 - 1532, or 1545 - 1574 of the nucleotide sequence shown in SEQ ID NO:

1.

3. The antisense oligonucleotide according to claim 1 or 2, wherein the oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotide sequences of any one of the nucleotide sequences of SEQ ID NOs: 3 to 17.

4. The antisense oligonucleotide according to any one of claims 1 to 3, wherein when measured over the entire nucleotide sequence of the antisense oligonucleotide, the antisense oligonucleotide has a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the nucleotide sequences shown in SEQ ID NO.1 and / or SEQ ID NO.

2.

5. The antisense oligonucleotide according to any one of claims 1 to 4, wherein the antisense oligonucleotide comprises at least one modification selected from a modified sugar moiety, a modified nucleobase moiety, and a modified internucleotide linkage; preferably, the antisense oligonucleotide comprises one or more of the following modifications: (i) at least one bicyclic sugar moiety, wherein the bicyclic sugar moiety preferably has a 4'-2' bridge, and the 4'-2' bridge is preferably cEt or LNA; (ii) at least one non-bicyclic modified sugar moiety, preferably selected from 2'-deoxy, 2'-F, 2'-MOE, and 2'-OMe sugar moieties; (iii) at least one modified nucleoside, preferably including 5-methylcytosine (m5C); and (iv) at least one sugar substitute, preferably selected from POM, PNA, THP, and F-HNA.

6. The antisense oligonucleotide according to claim 5, wherein the antisense oligonucleotide comprises a gapmer, and preferably, the gapmer comprises: (a) a 5' region consisting of 1 - 6 linked 5' region nucleotides; (b) a central region consisting of 6 - 10 linked central region nucleotides; and (c) a 3' region consisting of 1 - 6 linked 3' region nucleotides; wherein each of the nucleotides in the 5' region and the 3' region comprises a sugar moiety modified with 2'-MOE, LNA, or cEt, and at least 6 of the nucleotides in the central region comprise a 2'-deoxy sugar moiety.

7. The antisense oligonucleotide according to claim 6, wherein the antisense oligonucleotide has a sugar motif (5' to 3') selected from the following: eekddddddddddkke, ekkddddddddddkke, kkkdyddddddddkkk, kkkddydddddddkkk, kkkdddyddddddkkk, kkkddddddddddkkk or eeeeeddddddddddeeeee; wherein e represents a 2'-MOE sugar moiety, k represents a cEt sugar moiety, d represents a 2'-deoxy sugar moiety, and y represents a 2'-OMe sugar moiety.

8. The antisense oligonucleotide according to any one of claims 1 to 7, wherein the antisense oligonucleotide comprises at least one modified internucleotide linkage; preferably, each internucleotide linkage of the antisense oligonucleotide is a modified internucleotide linkage; preferably, the modified internucleotide linkage is preferably selected from phosphorothioate (PS) and phosphoroamidate (PN) (such as methanesulfonylamino phosphoroamidate (MsPA)), more preferably PS.

9. The antisense oligonucleotide according to any one of claims 1 to 8, wherein the antisense oligonucleotide comprises an oligonucleotide consisting of 12 - 30, 14 - 22, 14 - 20, 14 - 18, 14 - 20, 15 - 17, 15 - 25 or 16 - 20 linked nucleotides, preferably, the oligonucleotide consists of 20 linked nucleotides; preferably, the antisense oligonucleotide comprises a nucleotide sequence as shown in any one of SEQ ID NOs. 18 to 32 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or is composed of any one of them.

10. The antisense oligonucleotide according to any one of claims 1 to 9, wherein the antisense oligonucleotide comprises a ligand; wherein the ligand comprises a ligand targeting hepatocytes, preferably, the ligand comprises a galactose moiety, a galactosamine moiety or an N-acetylgalactosamine moiety, more preferably, the ligand is a trivalent or tetravalent N-acetylgalactosamine moiety, still more preferably, the ligand targeting hepatocytes is L96, NAG25 or NAG37; or the ligand comprises a ligand targeting non-hepatocytes, preferably the ligand is a lipophilic group or an integrin ligand, a transferrin receptor 1 ligand, and the lipophilic group is preferably selected from: lipids, vitamins, steroids, C5-C 30 saturated or unsaturated fatty acids, C5-C 30 alkyls, and polypeptides comprising at least one positively charged amino acid residue; the lipophilic group is more preferably selected from cholesterol, C 16 saturated or unsaturated fatty acids, C 16 alkyls, C 22 saturated or unsaturated fatty acids or C 22 alkyls.

11. An RNAi agent for inhibiting the expression of activin type IIB receptor (ACVR2B) gene in cells, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21 consecutive nucleotides of equal length to the target region of the mRNA encoding ACVR2B that are at least 80%, 85%, 90%, 95% or 100% complementary or differ from it by no more than 3 nucleotides.

12. The RNAi agent according to claim 11, wherein the target region is 508 - 534, 634 - 660, 655 - 695, 733 - 774, 756 - 782, 929 - 955, 1049 - 1089, 1141 - 1180, 1189 - 1215, 1243 - 1269, 1276 - 1321, 1377 - 1403, 1547 - 1596, 1642 - 1690, 1744 - 1773, 1916 - 1942 or 1949 - 1975 of the nucleotide sequence shown in SEQ ID NO:

1.

13. The RNAi agent according to claim 11 or 12, wherein the antisense strand comprises at least 15 consecutive nucleotides selected from the nucleotide sequences of SEQ ID NOs: 64 to 94 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

14. The RNAi agent according to any one of claims 11 to 13, wherein the length of the double-stranded region is 17 to 23 base pairs, preferably 18 to 21 base pairs, and more preferably 19 base pairs.

15. The RNAi agent according to any one of claims 11 to 14, wherein the length of each of the sense strand and the antisense strand is 17 to 23 nucleotides, preferably 19 to 21 nucleotides.

16. The RNAi agent according to any one of claims 11 to 15, wherein the RNAi agent comprises one or two blunt ends, preferably one blunt end.

17. The RNAi agent according to any one of claims 11 to 16, wherein the RNAi agent comprises one or two overhangs, preferably one overhang, and each overhang has 1 to 4 unpaired nucleotides, preferably 2 unpaired nucleotides.

18. The RNAi agent according to claim 17, wherein the overhang is located at the 3'-end of the sense strand, the 3'-end of the antisense strand, or both at the 3'-ends of the sense strand and the antisense strand; preferably, the overhang is located at the 3'-end of the antisense strand, and further preferably, the RNAi agent has one blunt end.

19. The RNAi agent according to any one of claims 11 to 18, wherein the sense strand comprises at least 15 consecutive nucleotides selected from any of the nucleotide sequences of SEQ ID NOs: 33 to 63 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

20. The RNAi agent according to any one of claims 11 to 19, wherein the antisense strand has no more than 23 nucleotides and comprises a nucleotide sequence selected from SEQ ID NOs: 64 to 94; the sense strand has no more than 21 nucleotides and comprises a nucleotide sequence of SEQ ID NOs: 33 to 63.

21. The RNAi agent according to claim 20, wherein: the sense strand comprises or is the sequence shown in SEQ ID NO: 33 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 64 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; the sense strand comprises or is the sequence shown in SEQ ID NO: 34 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 65 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; the sense strand comprises or is the sequence shown in SEQ ID NO: 35 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 66 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:36 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:67 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:37 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:68 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:38 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:69 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:39 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:70 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:40 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:71 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:41 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:72 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:42 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:73 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:43 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:74 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:44 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:75 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:45 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:76 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO: 46 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 77 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO: 47 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 78 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO: 48 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 79 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO: 49 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 80 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO: 50 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 81 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO: 51 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 82 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO: 52 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 83 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO: 53 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 84 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO: 54 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 85 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO: 55 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO: 86 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:56 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:87 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:57 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:88 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:58 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:89 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:59 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:90 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:60 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:91 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:61 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:92 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; The sense strand comprises or is the sequence shown in SEQ ID NO:62 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:93 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom; or The sense strand comprises or is the sequence shown in SEQ ID NO:63 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom, and the antisense strand comprises or is the sequence shown in SEQ ID NO:94 or a nucleotide sequence having 1, 2, or 3 nucleotide differences therefrom.

22. The RNAi agent according to claim 21, wherein the RNAi agent comprises duplexes 6102, 6103, 6104, 6105, 6106, 6107, 6108, 6109, 6112, 6113, 6115, 6116, 6117, 6119, 6120, 6121, 6122, 6123, 6124, 6125, 6127, 6128, 6129, 6130, 6131, 6132, 6134, 6135, 6137, 6138 or 6140.

23. The RNAi agent according to any one of claims 11 to 22, wherein the sense strand and / or the antisense strand of the RNAi agent comprises at least one modified nucleotide independently selected from 2'-deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides (2'-OMe), 2'-fluoro modified nucleotides (2'-F), 2'-deoxy modified nucleotides, locked nucleic acids (LNA), unlocked nucleic acids (UNA), bridged nucleic acids (BNA), glycol nucleic acids (GNA), threose nucleic acids (TNA), conformationally restricted nucleotides, constrained ethyl nucleotides (cEt), 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-O-methoxyethyl modified nucleotides (2'-MOE), abasic nucleotides, inverted abasic nucleotides, inverted nucleotides, morpholino nucleotides (MOP), aminophosphates (PN), tetrahydropyran modified nucleotides (THP), 1,5-anhydrohexitol modified (HNA) nucleotides, cyclohexenyl modified nucleotides, nucleotides comprising phosphorothioate linkages (PS), nucleotides comprising methylphosphonate linkages, nucleotides comprising 5'-phosphates, nucleotides comprising 5'-phosphate mimics, nucleotides covalently linked to cationic lipids, nucleotides comprising 5'-vinylphosphonates (5'-VP), and combinations thereof; preferably selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides with phosphorothioate bond internucleotide linkages, and combinations thereof; and / or preferably, each nucleotide of the sense strand and / or the antisense strand of the RNAi agent is modified.

24. The RNAi agent according to claim 23, wherein, in the 5' to 3' direction, the nucleotides at positions 2, 5, 7, and 14 of the antisense strand are 2'-fluoro modified nucleotides, and one of the nucleotides at positions 12 and 16 of the antisense strand is a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions of the antisense strand are all 2'-methoxy modified nucleotides.

25. The RNAi agent according to claim 23 or 24, wherein the antisense strand has at least one phosphorothioate bond internucleotide linkage; preferably, the phosphorothioate bond internucleotide linkage is present in one or more of the following: (i) between the first nucleotide and the second nucleotide at the 5' end of the antisense strand; (ii) between the second nucleotide and the third nucleotide at the 5' end of the antisense strand; (iii) between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and (iv) between the second nucleotide and the third nucleotide at the 3' end of the antisense strand.

26. The RNAi agent according to any one of claims 23 to 25, wherein, in the 5'-to-3' direction, the nucleotides at positions 7 and 9 of the sense strand are 2'-fluoro-modified nucleotides, one or two of the nucleotides at positions 5, 8, and 11 of the sense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides.

27. The RNAi agent according to any one of claims 23 to 26, wherein the sense strand has at least one phosphorothioate internucleotide linkage; preferably, the phosphorothioate internucleotide linkage is present in one or more of the following positions: (i) between the 1st and 2nd nucleotides at the 5'-end of the sense strand; (ii) between the 2nd and 3rd nucleotides at the 5'-end of the sense strand; (iii) between the 1st and 2nd nucleotides at the 3'-end of the sense strand; and (iv) between the 2nd and 3rd nucleotides at the 3'-end of the sense strand.

28. The RNAi agent according to claim 23, wherein the modification is selected from one of STC, ESC, Advanced ESC, ESC+, AD1-3, AD5, and GalXC.

29. The RNAi agent according to any one of claims 23 to 28, wherein the 5'-terminal nucleotide of the antisense strand contains a phosphate or phosphate analogue modification, preferably 5'-VP.

30. The RNAi agent according to any one of claims 23 to 29, wherein the RNAi agent further comprises a ligand targeting hepatocytes. Preferably, the ligand comprises a galactose moiety, a galactosamine moiety or an N-acetylgalactosamine moiety. More preferably, the ligand is a trivalent or tetravalent N-acetylgalactosamine moiety. Even more preferably, the ligand targeting hepatocytes is L96, NAG25 or NAG37; or the RNAi agent further comprises a ligand targeting non-hepatocytes. Preferably, the ligand is a lipophilic group or an integrin ligand, a transferrin receptor 1 ligand. The lipophilic group is preferably selected from: lipids, vitamins, steroids, C5-C 30 saturated or unsaturated fatty acids, C5-C 30 alkyls, and polypeptides comprising at least one positively charged amino acid residue; the lipophilic group is more preferably selected from cholesterol, C 16 saturated or unsaturated fatty acids, C 16 alkyls, C 22 saturated or unsaturated fatty acids or C 22 alkyls.

31. A pharmaceutical composition comprising the antisense oligonucleotide according to any one of claims 1 to 10, or the RNAi agent according to any one of claims 11 to 30; and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is formulated as an intravenous or subcutaneous injection.

32. Use of the antisense oligonucleotide according to any one of claims 1 to 10, the RNAi agent according to any one of claims 11 to 30, or the pharmaceutical composition according to claim 31 in the preparation of the following drugs: (i) A drug for reducing the expression level of ACVR2B in cells; (ii) A drug for preventing or treating a disease mediated by abnormal ACVR2B expression level; or (iii) A drug for preventing or treating a disease selected from muscle atrophy, muscular dystrophy, obesity, diabetes, chronic kidney disease, acute kidney injury, wound healing after trauma, maxillofacial tumor, pregnancy preeclampsia, liver fibrosis, atherosclerosis, ischemic stroke, and Alzheimer's disease.

33. Use of an agent that reduces the expression of the activin receptor type IIB (ACVR2B) gene in the preparation of a medicament for treating a disease or disorder treatable by ACVR2B knockdown; preferably, wherein, The reagent is an antisense oligonucleotide and / or an RNAi agent; preferably, the disease or disorder is selected from muscle atrophy, muscular dystrophy, obesity, diabetes, chronic kidney disease, acute kidney injury, wound healing after trauma, maxillofacial tumor, pregnancy preeclampsia, liver fibrosis, atherosclerosis, ischemic stroke, and Alzheimer's disease.

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