WO2025153061A1 - Nucleic acid for inhibiting the expression of activin receptor type iib gene and use thereof - Google Patents

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 acid for inhibiting activin type IIB receptor gene expression and its use Technical Field

The present invention relates to nucleic acids for inhibiting the expression of activin type IIB receptor (ACVR2b) gene in cells, including antisense oligonucleotides (ASO) and RNAi agents (such as siRNA) and pharmaceutical compositions thereof. The present invention also relates to the medical use of these nucleic acids.

Background Art

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, activin, inhibin (INH), bone morphogenetic protein (BMP), growth differentiation factor (GDP), myostatin (MSTN), follistatin (FST), and the Smad protein family. The mechanism of action of the TGF-β signal transduction pathway is that TGF-β superfamily members bind to ACVR2B on the cell surface, then recruit activin type I receptor (ActRI), and the formed ligand receptor complex activates ActRI kinase (ALK4/5), leading to phosphorylation of the transcription factor Smad protein family (mainly Smad2 and Smad3), which then binds to Smad4 to form a complex and enter the cell nucleus, regulating the target genes of multiple transcription factors, thereby regulating the proliferation, differentiation, migration and apoptosis of multiple cells. The TGF-β signal transduction pathway plays an important regulatory role in embryonic development, bone formation, and muscle growth and development. The study found that the genetic loss of Dystrophin, a key gene in the clinically intractable Duchenne muscular dystrophy (DMD) model mouse, was accompanied by a significant increase in TGF-β expression; similar to this research result, the genetic loss of FBN1, a key gene in the Marfan syndrome (MFS) disease model mouse, was also accompanied by a significant increase in TGF-β expression. The skeletal muscles of both model animals lack the ability to repair and regenerate after injury, but after the TGF-β pathway signal is blocked, the skeletal muscles can restore their repair and regeneration function.

Muscle atrophy is a debilitating and life-threatening disease state 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, muscle-loosening 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 can regulate many muscle-building genes, such as myogenic determinant (MyoD), myogenin and myogenic factor 5 (Myf5), which are involved in the process of cell hypertrophy, proliferation or differentiation. Studies have shown that inhibition of ACVR2B leads to rapid and massive muscle growth, and is therefore often used to treat diseases such as muscle atrophy and muscular dystrophy.

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

Among the many ligands of ACVR2B, activin A can promote the pituitary to synthesize and secrete gonadotropin to regulate reproductive physiological activities; in addition to 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 kidney development and plays an important role in kidney development and repair. It can delay the branching of mouse ureteric buds and reduce the size of mouse metanephros cultured in vitro. It is a negative regulator of kidney development and participates in kidney pathological processes such as renal fibrosis. Elevated levels of activin A in chronic kidney disease can lead to anemia, promote the occurrence of renal fibrosis, and aggravate the progression of the disease. In addition, activin A can also aggravate kidney damage caused by diseases such as systemic lupus erythematosus, vasculitis, and multiple myeloma (MM). Reducing the level of activin A by inhibiting ACVR2A and ACVR2B can prevent acute kidney injury, relieve renal anemia, improve mineral bone abnormalities, and inhibit the occurrence of renal fibrosis.

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

The present invention is committed 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.

One aspect of the invention provides an antisense oligonucleotide comprising an oligonucleotide consisting of 12 to 30 linked nucleotides, the 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 an equal length of consecutive nucleotides of a target region of an mRNA encoding an activin type IIB receptor (ACVR2B).

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.

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.

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 NO:3 to 17.

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.

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) modified nucleosides, preferably including 5-methylcytosine (m5C); and (iv) at least one sugar surrogate, preferably selected from POM, PNA, THP and F-HNA.

In some embodiments, the antisense oligonucleotide comprises a gapmer. In some embodiments, the gapmer comprises: (a) a 5' region consisting of 1-6 connected 5' region nucleotides; (b) a central region consisting of 6-10 connected central region nucleotides; and (c) a 3' region consisting of 1-6 connected 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.

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.

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.

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 of them.

In some embodiments, the antisense oligonucleotide comprises a ligand; preferably, wherein the ligand comprises a ligand targeting hepatocytes, preferably, the ligand comprises a galactose portion, a galactosamine portion or an N-acetylgalactosamine portion, further preferably, the ligand is a trivalent or tetravalent N-acetylgalactosamine portion, and 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 - _C30 saturated or unsaturated fatty acids, _C5 - _C30 alkyls, and polypeptides comprising at least one positively charged amino acid residue; the lipophilic group is more preferably selected from cholesterol, _C16 saturated or unsaturated fatty acids, _C16 alkyls, _C22 saturated or unsaturated fatty acids or _C22 alkyls.

Another aspect of the present invention provides a 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 to a target region of an mRNA encoding ACVR2B that are at least 80%, 85%, 90%, 95% or 100% complementary or differ from it by no more than 3 nucleotides.

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.

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, 1189-1 215, 1243-1269, 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 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, 1192-1 212, 1246-1266, 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.

In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides selected from SEQ ID NO: 64 to 94 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

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.

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.

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

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.

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.

In some embodiments, the positive strand comprises at least 15 consecutive nucleotides selected from any one nucleotide sequence of SEQ ID NO: 33 to 63 and a nucleotide sequence having 1 to 3 nucleotide differences therefrom.

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

In some embodiments, in the RNAi agent:

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 positive strand contains 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 contains or is the sequence shown in SEQ ID NO:94 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom.

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.

In some embodiments, the sense strand and/or antisense strand of the RNAi agent comprises at least one modified nucleotide, the modified nucleotide being 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 The invention relates to a baseless nucleotide, an inverted nucleotide, a morpholino nucleotide (MOP), an aminophosphorate (PN), a tetrahydropyran-modified nucleotide (THP), a 1,5-anhydrohexitol-modified (HNA) nucleotide, a cyclohexenyl-modified nucleotide, a thiophosphate-containing nucleotide (PS), a methylphosphonate-containing nucleotide, a 5'-phosphate-containing nucleotide, a 5'-phosphate-containing nucleotide, a 5'-phosphate mimetic-containing nucleotide, a cationic lipid-covalently linked nucleotide, a 5'-vinylphosphonate-containing nucleotide (5'-VP) and a combination thereof; preferably selected from 2'-O-methyl-modified nucleotides, 2'-fluorine-modified nucleotides, nucleotides containing thiophosphate-bonded nucleotides and a combination thereof; and/or preferably, each nucleotide of the sense strand and/or antisense strand of the RNAi agent is modified.

In some embodiments, in the RNAi agent, in the direction 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.

In some embodiments, in the RNAi agent, the antisense strand has at least one phosphorothioate internucleotide linkage; preferably, the phosphorothioate internucleotide linkage exists 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.

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.

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 nucleotide and the second nucleotide at the 5' end of the sense strand; (ii) between the second nucleotide and the third nucleotide at the 5' end of the sense strand; (iii) between the first nucleotide and the second nucleotide at the 3' end of the sense strand; and (iv) between the second nucleotide and the third nucleotide at the 3' end of the sense strand.

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

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.

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 still 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 - _C30 saturated or unsaturated fatty acids, _C5 - _C30 alkyls, and polypeptides comprising at least one positively charged amino acid residue; the lipophilic group is more preferably selected from cholesterol, _C16 saturated or unsaturated fatty acids, _C16 alkyls, _C22 saturated or unsaturated fatty acids or _C22 alkyls.

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.

Another aspect of the present invention provides the use of any RNAi agent of the present invention 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 expression levels of ACVR2B; or

(iii) a drug for preventing or treating a disease selected from the group consisting of muscular 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.

Another aspect of the present invention provides the use of an agent that reduces the expression of a gene encoding 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 atrophy, muscular dystrophy, obesity, diabetes, chronic kidney disease, acute kidney injury, postoperative healing of trauma, maxillofacial tumors, pregnancy eclampsia, liver fibrosis, atherosclerosis, ischemic stroke and Alzheimer's disease.

Accordingly, the present invention also provides a method for reducing the expression level of ACVR2B in a cell, the method comprising administering a therapeutically effective amount of any antisense oligonucleotide or any RNAi agent of the present invention to a subject in need. 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 a therapeutically effective amount of any antisense oligonucleotide or any RNAi agent of the present invention to a subject in need. The present invention also provides a method for preventing or treating a disease selected from muscular atrophy, muscular dystrophy, obesity, diabetes, chronic kidney disease, acute kidney injury, postoperative healing of trauma, maxillofacial tumors, eclampsia in pregnancy, liver fibrosis, atherosclerosis, ischemic stroke and Alzheimer's disease, the method comprising administering a therapeutically effective amount of any antisense oligonucleotide or any RNAi agent of the present invention to a subject in need. The present invention also provides the use of any antisense oligonucleotide or any RNAi agent of the present invention in the preparation of a drug for preventing or treating the above-mentioned disease. The present invention also provides any antisense oligonucleotide or any RNAi agent of the present invention for preventing or treating the above-mentioned disease.

Other aspects of the present invention will become apparent from the detailed description of the following specification.

DETAILED DESCRIPTION

definition

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

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 (such as mRNA) sequence and modulating protein (such as ACVR2B) expression. As described herein, "antisense oligonucleotide" may include non-oligonucleotide portions, such as ligands.

"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.

"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.

"Gapmer" means an antisense oligonucleotide comprising an internal region, the internal region being located between an external region having one or more nucleosides, having 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 may be referred to as a "gap" and the external region may 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 of the internal region and is counted from the 5' end of the internal region. Unless otherwise indicated, "gapmer" refers to a sugar motif. In certain embodiments, each nucleoside of the gap is a 2'-β-D-deoxynucleoside. In certain embodiments, the gap comprises a 2'-substituted nucleoside at position 1, 2, 3, 4, or 5 of the gap, and the remaining nucleosides of the gap are 2'-β-D-deoxynucleosides. As used herein, the term "MOE gapmer" indicates a gapmer 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 a wing comprising modified nucleosides containing 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.

As used herein, the term "RNAi agent" refers to an agent comprising an RNA molecule that can downregulate the expression of a target gene (ACVR2B gene in this article) through an RNA interference mechanism when introduced into a cell. The term "RNAi agent of the present invention", "RNAi agent described herein" or similar expressions include both modified RNAi agents of the present invention, regardless of sequence and target gene, and RNAi agents with specific sequences for interfering with the ACVR2B gene of the present invention. RNA interference refers to a process in which nucleic acid molecules induce the cutting and degradation of target RNA molecules (such as mRNA molecules) 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 collectively referred to herein as double-stranded RNA (dsRNA), which include two antiparallel continuous nucleotide chains that are fully complementary to each other to hybridize to form a double-stranded region. "Hybridization" refers to the pairing of complementary polynucleotides, usually through hydrogen bonds (e.g., Watson-Crick hydrogen bonds, Wobble hydrogen bonds, Hoogsteen hydrogen bonds, or reverse Hoogsteen hydrogen bonds) between complementary bases in two polynucleotides. The "double-stranded region" refers to a region in two complementary or substantially complementary polynucleotides that form base pairs by hybridization, thereby forming a double strand between the two polynucleotide chains.

The term "antisense strand" refers to a strand in a dsRNA that includes a region that is substantially complementary to a target sequence. The term "sense strand" or "sense strand" refers to a strand in a dsRNA that includes a region that is substantially complementary to an 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, the mismatch may be located in the interior or terminal regions of the molecule. Typically, the most tolerable mismatch is located in the terminal regions, such as 5, 4, 3 or 2 at the 5' and/or 3' ends of the dsRNA.

"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. The siRNA is typically about 15 to about 30 base pairs in length, most typically about 19 to 25 base pairs in length, such as 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs in length.

shRNA refers to short hairpin RNA, which includes two short inverted repeat sequences 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 less nucleotides. The stem-loop may be 8 or less unpaired nucleotides. The stem-loop may be 4 to 10 unpaired nucleotides. The stem-loop may be 4 to 8 nucleotides.

The two substantially complementary chains of dsRNA do not need to be but can 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 can also include one or more nucleotide overhangs. Overhanging nucleotides refer to one or more unpaired nucleotides extending beyond the double-stranded region at the end of the chain. When the 3' end of a chain extends beyond the 5' end of another chain, or when the 5' end of a line extends beyond the 3' end of another line, nucleotide overhangs are usually generated. For example, at least one chain includes a 3' overhang of at least 1 nucleotide, for example, 1 to 4 nucleotides overhang. For another example, at least one chain includes a 5' overhang of at least 1 nucleotide, for example, 1 to 4 nucleotides overhang. In other embodiments, the 3' end and 5' end of a chain of the dsRNA both include an overhang of at least 1 nucleotide.

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

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

The term "ligand" refers to a cell or tissue targeting agent that binds to a specified cell type (such as a hepatocyte), such as a lectin, glycoprotein, lipid or protein (such as 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 (such as 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 mimetic. In a preferred embodiment, the ligand is a carbohydrate, such as a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, a polysaccharide. For example, the ligand can be a derivative comprising GalNAc. In a preferred embodiment, the ligand is comprised of one or more N-acetylgalactosamine derivatives attached via a divalent or trivalent branched linker.

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.

The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms which are within the scope of sound medical judgment and suitable for 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.

The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle 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.

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

Antisense oligonucleotides for inhibiting ACVR2B gene expression

The 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).

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. 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 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.

Therefore, 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 consecutive nucleotides of equal length 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.

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 identical to 504-533, 511-523, 524-536, 537-538 of the nucleotide sequence shown in SEQ ID NO: 1. nucleotides that are at least 80%, 85%, 90%, 95% or 100% complementary to an equal length of consecutive nucleotides in 0-539, 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.

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 identical to 509-528, 510-529 of the nucleotide sequence shown in SEQ ID NO: 1. 5-534, 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 are at least 80%, 85%, 90%, 95% or 100% complementary nucleotides.

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 nucleotide sequence of SEQ ID NO:3 to 17.

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 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 comprising any one of SEQ ID NOs: 3 to 17. column, and wherein the antisense oligonucleotide has 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 the messenger RNA (mRNA) of human and/or cynomolgus monkey activin type IIB receptor (ACVR2B) (NCBI accession numbers NM_001106.4, SEQ ID NO.1 and XM_015445674.2, SEQ ID NO.2, respectively) when measured over the entire nucleotide sequence of the antisense oligonucleotide.

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 may comprise a nucleotide sequence as set forth in any one of SEQ ID NO: 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.

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.

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, 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 20 consecutive nucleotide sequences of any one nucleotide sequence of SEQ ID NO:3 to 17, wherein the antisense oligonucleotide comprises at least one modification selected from a modified sugar portion, a modified nucleobase portion and a modified internucleotide linkage.

In some embodiments, each nucleotide in the oligonucleotide comprised by the antisense oligonucleotide is modified, including a modified sugar moiety and a modified internucleotide linkage.

In some embodiments, each nucleotide in the oligonucleotide comprised by the antisense oligonucleotide is modified, including a modified sugar moiety, a modified nucleobase moiety, and a modified internucleotide linkage.

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.

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 a second ring, thereby producing a 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'("constrainedMOE" or "cMOE") and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 7,399,845; Bhat et al., U.S. Pat. No. 7,569,686; Swayze et al., U.S. Pat. No. 7,741,457; and Swayze et al., U.S. Pat. No. 8,022,193), 4'-C( _CH3 )( _CH3 )-O-2' and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 8,278,283), 4'-CH2-N(OCH3)-2' and analogs thereof (see, e.g., Prakash et al., U.S. Pat. No. 8,278,425), 4'-CH2- _ON ( _CH3 )-2' (see, e.g., Allerson et al., U.S. Pat. No. 7,696,345 and Allerson et al., U.S. Pat. No. 8,124,745), 4'-CH2- _C (H)( _CH3 )-O-2' and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 8,278,283), 4'- _CH2 -N(OCH3)-2' and analogs thereof (see, e.g., Prakash et al., U.S. Pat. No. 8,278,425 ₎ , )-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( _RaRb )-N(R)-O-2', 4'- _C ( _RaRb )-ON(R)-2', 4'- _CH2 -ON(R)-2' and 4'- _CH2 -N(R)-O-2', wherein each R, _Ra and _Rb is independently H, a protecting group or a _C1 - _C12 alkyl group (see, e.g., Imanishi et al., US7,427,672). In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configurations. For example, LNA nucleosides (described herein) may be in the α-L configuration or in the β-D configuration. In a preferred embodiment, the bicyclic sugar moiety has a 4'-2' bridge, which is preferably cEt or LNA.

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 group, including but not limited to substituents located at the 2', 4' and/or 5' positions. In certain embodiments, one or more non-bridging substituents of the non-bicyclic modified sugar moiety have a branched chain. Examples of suitable 2'-substituents for the non-bicyclic modified sugar moiety include but are not limited to: 2'-F, 2'-OCH ₃ ("OMe" or "O-methyl"), and 2'-O(CH ₂ ) ₂ OC H ₃ ("MOE" or "O-methoxyethyl"). In certain embodiments, the 2'-substituent is selected from the group consisting of halo, allyl, amino, azido, SH, CN, OCN, _CF3 , _OCF3 , _OC1 - _C10 alkoxy, substituted _OC1 - _C10 alkoxy, OC1 _{- C10} alkyl, substituted OC1 _{- C10} alkyl, S-alkyl, N( _Rm )-alkyl, O-alkenyl, S-alkenyl, N( _Rm )-alkenyl, O-alkynyl, S-alkynyl, N( _Rm )-alkynyl, O-alkylene-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O( _CH2 ) _2SCH3 , _{O(CH2)2ON ( Rm )} ( _Rn ), or _OCH2C (=O)-N( _Rm )( _Rn ), wherein each _Rm and R _n is independently H, an amino protecting group, or a substituted or unsubstituted C ₁ -C ₁₀ alkyl, and the 2'-substituents described in Cook et al., US6,531,584; Cook et al., US5,859,221; and Cook et al., US6,005,087. 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 (NO ₂ ), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl. Examples of 4'-substituents suitable 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 5'-substituents suitable 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 include more than one non-bridging sugar substituent, such as 2'-F-5'-methyl sugar moieties and modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008/101157 and Rajeev et al., US2013/0203836.

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 ( Rm )} ( _{Rn )} , O( _CH2 ) _2O ( _CH2 ) _2N ( _CH3 ) _{2 , and} N-substituted acetamide ( _OCH2C (=O)-N( _Rm )( _Rn )), wherein each _Rm and _Rn is independently H, an amino protecting group, or a substituted or unsubstituted _C1 - _C10 alkyl.

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 , _OCH2CH2OCH3 , 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).

In certain embodiments, modified furanosyl sugar moieties and nucleosides incorporating such modified furanosyl sugar moieties are further defined by isomeric configurations. For example, a 2'-deoxyfuranosyl sugar moiety can be in seven isomeric configurations in addition to the naturally occurring β-D-deoxyribosyl configuration. Such modified sugar moieties are described, for example, in WO 2019/157531, which is incorporated herein by reference. 2'-modified sugar moieties have an additional 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 the β-D-ribosyl isomeric configuration.

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, 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 2' position (see, for example, Bhat et al., U.S. 7,875,733 and Bhat et al., U.S. 7,939,677) and/or a 5' position substitution.

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), fluoro-HNA (F-HNA, also referred to as F-THP or 3'-fluorotetrahydropyran).

In certain embodiments, sugar substitutes include rings 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, sugar substitutes include acyclic moieties. Examples of nucleosides and oligonucleotides comprising such acyclic sugar substitutes 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, sugar substitutes are selected from POM, PNA, THP and F-HNA.

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.

In certain embodiments, the modified nucleobase is selected from: 5-substituted pyrimidines (e.g., 5'methylcytosine, m5C), 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6, and O-6 substituted purines. In certain embodiments, the modified nucleobase is selected from: 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-CH ₃ )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, especially 5-bromo, 5-trifluoromethyl, 5-halouracil and 5-halocytosine; 7-methylguanine, 7-methyladenine, 2-F-adenine The present invention relates to a nucleobase comprising a purine, a 2-aminoadenine, a 7-deazaguanine, a 7-deazaadenine, a 3-deazaguanine, a 3-deazaadenine, a 6-N-benzoyladenine, a 2-N-isobutyrylguanine, a 5-methylcytosine, a 4-N-benzoylcytosine, a 4-N-benzoyluracil, a 5-methyl4-N-benzoylcytosine, a 5-methyl4-N-benzoyluracil, a universal base, a hydrophobic base, a promiscuous base, a base with an expanded size, and a fluorinated base. Other modified nucleobases include tricyclic pyrimidines such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenathiazin-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazin-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-methylcytosine (m5C).

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 modifications described in WO2019022434A1, the entire contents of which are incorporated herein by reference.

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 major classes of internucleoside linkage 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(O ₂ )=O") (also known as an unmodified linkage or a naturally occurring linkage); phosphotriester; methylphosphonate; phosphoramidate (PN); phosphorothioate ("P(O ₂ )=S", PS) and phosphorodithioate ("HS-P=S"). The phosphoramidate (PN) may have the formula -N(R)P(=X)(OH)O-, -OP(=X)(OH)N(R)-, -OP(NR)(=X)O-, -N(SO2R)P(=X)(OH)O-, -OP(=X)(OH)N(SO ₂ R)-, or -OP(NSO ₂ R)(=X)O- ("MsPA"), wherein X is O or S, R may be an optionally substituted alkyl, aryl, heteroaryl or heterocycloalkyl; or NR may be an optionally substituted cycloguanidine moiety, an optionally substituted triazolyl or a Tmg group. More PN structures may be found in WO2023220744, WO 2019/032612 and WO2021/030778, which are incorporated herein by reference in their entirety.

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, typically increase, the nuclease resistance of an oligonucleotide 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 of skill in the art.

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 prepared 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 phosphorothioate internucleotide connections, wherein all of the phosphorothioate internucleotide connections are stereo-random. Such antisense oligonucleotides can be generated using a synthetic method that randomly selects the stereochemical configuration of each phosphorothioate internucleotide connection. Nevertheless, as fully 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 specific phosphorothioate internucleotide connections in a specific independently selected stereochemical configuration. In certain embodiments, at least 65% of the molecules in the population have specific phosphorothioate internucleotide connections of a specific configuration. In certain embodiments, a specific phosphorothioate internucleotide linkage of a specific configuration is present in at least 70% of the molecules in the population. In certain embodiments, a specific phosphorothioate internucleotide linkage of a specific configuration is present in at least 80% of the molecules in the population. In certain embodiments, a specific phosphorothioate internucleotide linkage of a specific configuration is present in at least 90% of the molecules in the population. In certain embodiments, a specific phosphorothioate internucleotide linkage of a specific configuration is present 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 those 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 a (Sp) configuration. In certain embodiments, the population of antisense oligonucleotides is enriched for antisense oligonucleotides having at least one phosphorothioate in the (Rp) configuration.

In some embodiments, the antisense oligonucleotide includes a gapmer. The gapmer 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 gapmer 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 (i.e., the wing/gap junction). 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 of the nucleosides are different from the sugar moieties of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two wings are identical to each other (symmetrical gapmers). In certain embodiments, the sugar motifs of the 5' wing are different from the sugar motifs of the 3' wing (asymmetric gapmers).

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.

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.

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.

In a preferred embodiment, the internucleotide connection of the gap polymer is PS. In such an embodiment, the gap polymer 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 gap polymer 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 gap polymer 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 gap polymer 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 gap polymer 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 gap polymer comprises a 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 gap polymer comprises a 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 gap polymer comprises a 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 gap polymer comprises a 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 gap polymer comprises a 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 gap polymer comprises a 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 gap polymer comprises a nucleotide sequence as shown in SEQ ID NO.28 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists thereof. In one embodiment, the gap polymer comprises a nucleotide sequence as shown in SEQ ID NO.29 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists thereof. In one embodiment, the gap polymer comprises a nucleotide sequence as shown in SEQ ID NO.30 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists thereof. In one embodiment, the gap polymer comprises a nucleotide sequence as shown in SEQ ID NO.31 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists thereof. In one embodiment, the gap polymer comprises a nucleotide sequence as shown in SEQ ID NO.32 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists thereof.

RNAi agents for inhibiting ACVR2B gene expression

One aspect of the present invention provides a 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 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 ACVR2B or differ from it by no more than 3 nucleotides.

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. 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, 1189-1 215, 1243-1269, 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, the target region is 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, 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.

Therefore, in some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, which comprises a sense strand and an antisense strand that form complementary double-stranded regions, 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 residues that are identical to 508-534, 634-660, 655-695, 733-774, 756-782, An equal length of consecutive nucleotides of 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 is at least 80%, 85%, 90%, 95% or 100% complementary or differs from nucleotides therewith by no more than 3 nucleotides.

In a preferred embodiment, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, which comprises a sense strand and an antisense strand that form complementary double-stranded regions, 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 21 508-534, 634-660, 655-681, 658-684, 669-695, 733-759, 748-774, 756-782, 929-955, 1049-1075, 1052-1078, 1063-1089, 1141-1 167, 1154-1180, 1189-1215, 1243-1269, 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, which are at least 80%, 85%, 90%, 95% or 100% complementary to, or differ from, nucleotides by no more than 3 nucleotides.

In a preferred embodiment, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, which comprises a sense strand and an antisense strand that form complementary double-stranded regions, 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 nucleotides that are identical to 511-531, 637-657, 658-678, 661-681, 672-692, 736-756, 751-771, 759-779, 932-952, 1052-1072, 1055-1075, 1066-1086, 1144-1145 of the nucleotide sequence shown in SEQ ID NO:1. 164, 1157-1177, 1192-1212, 1246-1266, 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 are at least 80%, 85%, 90%, 95% or 100% complementary to, or differ from, nucleotides by no more than 3 nucleotides.

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 NO: 64 to 94 and nucleotide sequences having 1, 2 or 3 nucleotide differences from each of them.

In some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, which comprises a sense chain and an antisense chain forming complementary double-stranded regions, wherein the antisense chain comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20 or 21) consecutive nucleotides of any nucleotide sequence selected from SEQ ID NO: 64 to 94.

In some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, which comprises a sense chain and an antisense chain that form complementary double-stranded regions, wherein the antisense chain 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 with any one of the nucleotide sequences of SEQ ID NO: 64 to 94.

In the present invention, when 1, 2 or 3 nucleotide differences are mentioned, 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' end or the 5' end of the antisense strand. In some embodiments, the 1, 2 or 3 nucleotide differences are located between the 3' end and the 5' end of the antisense strand. In some embodiments, 1 or 2 of the 1, 2 or 3 nucleotide differences are located at the 3' end or the 5' end of the antisense strand, and the other 1 or 2 are located between the 3' end and the 5' 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, 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 sense strand, and the other 1 or 2 are located between the 3'-most end and the 5'-most end of the sense 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 that form 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 1 nucleotide difference with any one of the nucleotide sequences of SEQ ID NO: 64 to 94, and the 1 nucleotide difference is located at the 3'-most 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 that form 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 2 nucleotide differences with any one of the nucleotide sequences of SEQ ID NO: 64 to 94, and the 2 nucleotide differences are continuously located at the 3'-most end of the antisense strand.

In some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, which comprises a sense chain and an antisense chain that form complementary double-stranded regions, wherein the antisense chain does not exceed 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20 or 21) consecutive nucleotides selected from any one nucleotide sequence of SEQ ID NO: 64 to 94.

In some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, which comprises a sense strand and an antisense strand that form complementary double-stranded regions, wherein the antisense strand does not exceed 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 with any one of the nucleotide sequences of SEQ ID NO: 64 to 94.

One aspect of the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, which comprises an antisense chain, wherein the antisense chain comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20 or 21) consecutive nucleotides selected from SEQ ID NO: 64 to 94 and nucleotide sequences having 1, 2 or 3 nucleotide differences therefrom.

In some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, which comprises an antisense chain, wherein the antisense chain comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20 or 21) consecutive nucleotides of any nucleotide sequence selected from SEQ ID NO: 64 to 94.

In some embodiments, the present invention provides an RNAi agent for inhibiting the expression of the ACVR2B gene, which comprises an antisense chain, wherein the antisense chain 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 the nucleotide sequences of SEQ ID NO: 64 to 94.

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.

Therefore, in some embodiments of the present invention, a RNAi agent for inhibiting the expression of the ACVR2B gene is provided, which comprises a sense strand and an antisense strand that form a complementary double-stranded region, the length of the double-stranded region is about 17 to about 23 base pairs, and the length of the antisense strand does not exceed 23 nucleotides and comprises at least 15 consecutive nucleotides selected from SEQ ID NO: 64 to 94 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

In some embodiments of the present invention, a RNAi agent for inhibiting the expression of the ACVR2B gene is provided, which comprises a sense strand and an antisense strand that form a complementary double-stranded region, the length of the double-stranded region is about 18 to about 21 base pairs, and the length of the antisense strand does not exceed 23 nucleotides and comprises at least 15 consecutive nucleotides selected from SEQ ID NO: 64 to 94 and nucleotide sequences having 1 to 3 nucleotide differences therefrom.

In some embodiments of the present invention, a RNAi agent for inhibiting the expression of the ACVR2B gene is provided, which comprises a sense strand and an antisense strand that form a complementary double-stranded region, the length of the double-stranded region is about 19 base pairs, and the length of the antisense strand does not exceed 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20 or 21) consecutive nucleotides selected from SEQ ID NO: 64 to 94 and nucleotide sequences having 1, 2 or 3 nucleotide differences therefrom.

In some embodiments, the length of the sense strand and the antisense strand in the RNAi agent of the invention is each independently about 17 to about 23 nucleotides, such as 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 length of the sense strand and the antisense strand is each independently about 17, about 18, about 19, about 20, about 21, about 22, about 23 nucleotides.

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, so that the RNAi agent for inhibiting the expression of the ACVR2B gene has two nucleotide overhangs. For example, in one embodiment, the RNAi agent for inhibiting the expression of the ACVR2B gene includes (i) a sense strand and an antisense strand each having a length of 21 nucleotides, (ii) a double-stranded region of 19 base pairs in length, and (iii) a nucleotide overhang of 1 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 includes (i) a sense strand and an antisense strand each having a length of 23 nucleotides, (ii) a double-stranded region of 21 base pairs in length, and (iii) a nucleotide overhang of 1 unpaired nucleotide at the 3' end of the sense strand and the 3' end of the antisense strand.

In other embodiments, the sense strand and the antisense strand have the same length and form a double-stranded region over their entire length, so that no nucleotides protrude at 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 includes (i) a sense strand and an antisense strand each having a length of 21 nucleotides, 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 includes (i) a sense strand and an antisense strand each having a length of 23 nucleotides, 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 includes (i) a sense strand and an antisense strand each having a length of 19 nucleotides, and (ii) a double-stranded region of 19 base pairs in length.

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 with a length equal to the length of the short strand, so that the RNAi agent for inhibiting the expression of the ACVR2B gene includes at least one nucleotide protrusion. 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 to the length of the antisense strand, so 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 to the length of the sense strand, so that the antisense strand forms an overhang with 1 to 4 unpaired nucleotides. In some embodiments, the length of the nucleotide protrusion is 1, 2, 3 or 4 nucleotides. In a specific embodiment, the overhang includes 2 nucleotides. In certain embodiments, the overhang includes a single nucleotide.

The protruding nucleotides can be ribonucleotides or modified nucleotides as described herein. In some embodiments, the protruding nucleotides are 2'-modified nucleotides (e.g., 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides) or combinations thereof. For example, in one embodiment, the protruding nucleotides are deoxyribonucleotides, such as deoxythymidine. In another embodiment, the protruding nucleotides 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 protruding comprises 5'-uridine-uridine-3' (5'-UU-3') dinucleotides. In such embodiments, the UU dinucleotides can include ribonucleotides or modified nucleotides, such as 2'-modified nucleotides. In other embodiments, the protruding comprises 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotides. 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.).

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

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 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.

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.

In some embodiments, a RNAi agent for inhibiting the expression of the ACVR2B gene in a cell is provided, comprising a sense chain and an antisense chain forming a double-stranded region, the antisense chain having a length of 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 NO: 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, and the overhang preferably has 2 unpaired nucleotides.

In some embodiments, a RNAi agent for inhibiting the expression of ACVR2B gene in a cell is provided, comprising a sense chain and an antisense chain forming a double-stranded region, the antisense chain having a length of 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 NO: 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 chain, and the blunt end is formed at the 3' end of the sense chain and the 5' end of the antisense chain.

In some embodiments, a RNAi agent for inhibiting the expression of ACVR2B gene in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, the length of the double-stranded region is 19 base pairs, the length of the antisense strand does not exceed 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20 or 21) consecutive nucleotides selected from SEQ ID NO: 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.

In some embodiments, a RNAi agent for inhibiting the expression of ACVR2B gene in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, the length of the double-stranded region is 19 base pairs, the length of the antisense strand does not exceed 23 nucleotides and comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20 or 21) consecutive nucleotides selected from SEQ ID NO: 64 to 94 and nucleotide sequences having 1, 2 or 3 nucleotide differences therefrom, the length of the sense strand is 19 to 21 nucleotides, 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.

In some embodiments, a RNAi agent for inhibiting the expression of ACVR2B gene in a cell is provided, comprising a sense strand and an antisense strand forming a double-stranded region, the double-stranded region having a length of 19 base pairs, the antisense strand having a length of 21 nucleotides and being at least 15 (e.g., 15, 16, 17, 18, 19, 20 or 21) consecutive nucleotides selected from SEQ ID NO: 64 to 94 and nucleotide sequences having 1, 2 or 3 nucleotide differences therefrom, the sense strand having a length of 19 nucleotides, and the RNAi agent including 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.

In some embodiments, the present invention provides an RNAi agent for inhibiting the expression of ACVR2B gene in a cell, comprising a sense chain and an antisense chain forming a double-stranded region, wherein the double-stranded region is 19 base pairs in length, the antisense chain is 21 nucleotides in length and is any one nucleotide sequence selected from SEQ ID NO: 64 to 94, the sense chain is 19 nucleotides in length, and the RNAi agent includes an overhang and a blunt end, wherein the overhang preferably has 2 unpaired nucleotides, wherein the overhang is formed at the 3' end of the antisense chain, and the blunt end is formed at the 3' end of the sense chain and the 5' end of the antisense chain.

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

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

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

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

The positive strand contains 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 contains or is the sequence shown in SEQ ID NO:94 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom.

In a preferred embodiment, the present invention provides a RNAi agent for inhibiting the expression of the ACVR2B gene, which comprises a sense strand and an antisense strand that form complementary double-stranded regions, wherein the sense strand and the antisense strand pair to form a 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,
Any one of 6134, 6135, 6137, 6138 or 6140.

Nucleotide-modified RNAi agents

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

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

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.

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), assueose 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 thiophosphate groups (PS), nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate esters, nucleotides containing 5'-phosphate mimetics, nucleotides covalently linked to cationic lipids, nucleotides containing 5'-vinylphosphonate (5'-VP), and combinations thereof.

In a preferred embodiment, the modified nucleotides are independently selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides comprising thiophosphate bond internucleotide connections, 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 bond internucleotide connections, and combinations thereof.

In a preferred embodiment, each nucleotide of the sense chain and the antisense chain of the RNAi agent for inhibiting the expression of the ACVR2B gene 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).

In a preferred embodiment, the 5'-terminal nucleotide of the antisense strand of the RNAi agent for inhibiting the expression of the ACVR2B gene 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).

In some embodiments, each nucleotide of the antisense chain of the RNAi agent for inhibiting the expression of the ACVR2B gene provided by the present invention is modified, and in the direction 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.

In some embodiments, each nucleotide of the 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, 12 and 14 of the antisense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense chain are all 2'-methoxy-modified nucleotides.

In some embodiments, each nucleotide of the 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, 14 and 16 of the antisense chain are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense chain are all 2'-methoxy-modified nucleotides.

In some embodiments, each nucleotide of 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 direction from the 5' end to the 3' end, the nucleotides at the 2nd, 5th, 7th and 14th positions of the antisense strand are 2'-fluoro-modified nucleotides, and one of the nucleotides at the 12th and 16th positions 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 bond internucleotide connection. In a preferred embodiment, the phosphorothioate bond internucleotide connection exists in one or more of the following: between the first nucleotide and the second nucleotide at the 5' end of the antisense strand; 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 strand; and between the second nucleotide and the third nucleotide 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.

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

For example, each nucleotide of the sense chain and the antisense chain of the RNAi agent for inhibiting the expression of the ACVR2B gene provided by the present invention is modified, and in the direction 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; in the direction 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.

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 direction 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; in the direction from the 5' end to the 3' end, 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.

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 direction 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; in the direction from the 5' end to the 3' end, 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.

For example, each nucleotide of the sense chain and the antisense chain of the RNAi agent for inhibiting the expression of the ACVR2B gene provided by the present invention is modified, and in the direction 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; in the direction 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.

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

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 nucleotide and the second nucleotide at the 5' end of the antisense strand; 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 strand; and between the second nucleotide and the third nucleotide at the 3' end of the antisense strand.

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 direction 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 bond internucleotide connection, and the phosphorothioate bond internucleotide connection exists in one or more of the following: between the first nucleotide and the second nucleotide at the 5' end of the antisense strand; between the second nucleotide and the first nucleotide 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 strand; and between the 2nd nucleotide and the 3rd nucleotide at the 3' end of the antisense strand; in the direction from 5' to 3', the 7th and 9th nucleotides of the sense strand are 2'-fluoro-modified nucleotides, one or two of the 5th, 8th and 11th nucleotides of the sense strand are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions of the sense strand are 2'-methoxy-modified nucleotides, and the sense strand 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 strand and/or between the 2nd nucleotide and the 3rd nucleotide at the 5' end of the sense strand.

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 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 bond internucleotide connection, and the phosphorothioate bond internucleotide connection 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 1st nucleotide and the 2nd nucleotide at the 3' end of the antisense strand, and between the 2nd nucleotide and the 3rd nucleotide at the 3' end of the antisense strand; in the direction from 5' to 3', the 7th and 9th nucleotides of the sense strand are 2'-fluoro-modified nucleotides, one or two of the 5th, 8th and 11th nucleotides of the sense strand are 2'-fluoro-modified nucleotides, the nucleotides at the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides, and the sense strand 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 strand and between the 2nd nucleotide and the 3rd nucleotide at the 5' end of the sense strand.

Ligand-linked oligonucleotides and RNAi agents

For any of the embodiments of the antisense oligonucleotide or RNAi agent of the present invention described in the above sections of "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 contain 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 be just a physical connection. The ligand may change 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.

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, the ligands are targeted to specifically deliver antisense oligonucleotides or RNAi agents to hepatocytes using various methods described in more detail below. In certain embodiments, the antisense oligonucleotides or RNAi agents are targeted to hepatocytes with ligands that bind to surface expressed asialoglycoprotein receptors (ASGR) or components thereof (e.g., ASGR1, ASGR2).

In some embodiments, antisense oligonucleotides or RNAi agents can be specifically targeted to the liver by using ligands that bind to or interact with proteins expressed on the surface of hepatocytes. For example, in certain embodiments, the ligand may 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 particular 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 specifically bind to ASGR1 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, LDL receptor, or other liver surface expressed proteins suitable for use as ligands in the RNAi agents of the invention are purchased from commercial sources.

In some embodiments, when used for extrahepatic delivery, including but not limited to, central nervous system (CNS) (such as brain, spine or eye), muscle, lung or fat, antisense oligonucleotide or RNAi agent may include one or more lipophilic groups, and the lipophilic group is conjugated to one or more positions of oligonucleotide or RNAi agent antisense strand and/or sense strand.For example, lipophilic group can be connected to antisense oligonucleotide or RNAi agent antisense strand and/or sense strand via core base, sugar moiety or internucleoside bond.In certain embodiments, the lipophilic group with phosphoramidite group is coupled with 3' end or 5' end of sense strand or antisense strand in the last synthesis cycle.In certain embodiments, the octanol-water partition coefficient of lipophilic group exceeds 0,1,1.5,2,3,4,5 or 10. In some embodiments, the ligand is a lipophilic group, which is preferably selected from lipids, vitamins, steroids, C ₅ -C ₃₀ saturated or unsaturated fatty acids, C ₅ -C ₃₀ alkyl groups, 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 alkyl groups, C ₂₂ saturated or unsaturated fatty acids or C ₂₂ alkyl groups. Suitable lipophilic groups may be aliphatic, alicyclic, polyalicyclic compounds, steroids, straight-chain or branched aliphatic hydrocarbons.

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.

In certain embodiments, part includes carbohydrate. "Carbohydrate" refers to a compound composed of one or more monosaccharide units with at least 6 carbon atoms (can be straight chain, branched or cyclic), each carbon atom is connected with oxygen, nitrogen or sulfur atoms. 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 selected from pentose, hexose or heptose and includes disaccharides and trisaccharides of such monosaccharide units. In other embodiments, the carbohydrate that is incorporated into the part is amino sugar, for example aminogalactose, glucosamine, N-acetylgalactosamine and N-acetylglucosamine.

In some embodiments, the ligand comprises a hexose or a hexosamine. The hexose may be selected from glucose, galactose, mannose, fucose or fructose. The hexosamine may 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 a specific embodiment, 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; U.S. Patent Publication No. US20030130186; and WIPO Publication No. WO 2013/166155, all of which are incorporated herein by reference in their entirety.

In certain embodiments, the ligand includes a multivalent carbohydrate portion. As used herein, a "multivalent carbohydrate portion" refers to a portion comprising two or more carbohydrate units that can independently bind or interact with other molecules. For example, a multivalent carbohydrate portion includes two or more binding domains consisting of carbohydrates, which can bind to two or more different molecules or two or more different sites on the same molecule. The "valence" of a carbohydrate portion indicates the number of single binding domains within the carbohydrate portion. For example, the terms "monovalent," "divalent," "trivalent," and "tetravalent" refer to carbohydrate portions having one, two, three, and four binding domains, respectively, relative to a carbohydrate portion. The multivalent carbohydrate portion can include a multivalent lactose portion, a multivalent galactose portion, a multivalent glucose portion, a multivalent N-acetylgalactosamine portion, a multivalent N-acetylglucosamine portion, a multivalent mannose portion, or a multivalent fucose portion. In some embodiments, the ligand includes a multivalent galactose portion. In other embodiments, the ligand includes a multivalent N-acetyl-galactosamine portion. In these and other embodiments, the multivalent carbohydrate moiety can be divalent, trivalent or tetravalent. In such embodiments, the multivalent carbohydrate moiety can be bi-branched or tri-branched. 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 antisense oligonucleotides or RNAi agents of the present invention are described in detail below.

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.

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 ligands described in WO2022101633A1. The entire contents of these patent documents are incorporated herein by reference.

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

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.

In certain embodiments, the ligand is connected to a sense strand or an antisense strand by a joint." joint "refers to an atom or a group of atoms by which a ligand is covalently connected to a polynucleotide component of an antisense oligonucleotide or RNAi agent. 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 portion, which generally includes an alkyl portion 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 ligand as described herein. In certain embodiments, the joint includes an 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 amino, hydroxyl, carboxylic acid, thiol, unsaturated bonds (e.g., double bonds or triple bonds), etc.

Linkers that can be used to link 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 C ₁ -C ₂₀ alkyl, substituted or unsubstituted C ₂ -C ₂₀ alkenyl, or substituted or unsubstituted C ₂ -C ₂₀ alkynyl. Preferred substituents for such linkers include, but are not limited to, hydroxyl, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

In certain embodiments, the joint is cleavable. A cleavable joint is one that is sufficiently stable outside the cell but is cleaved to release two parts that are combined together by a joint when entering the target cell. In some embodiments, a cleavable joint is 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 faster than cracking in the blood of the experimenter in the target cell or under the first reference condition (it can, for example, be selected as a simulation or represent intracellular conditions).

Cleavable joints are susceptible to the effects of cleavage agents, such as pH, redox potential, or the presence of degradation molecules. In general, cleavage agents are more prevalent in cells than in serum or blood, or are found at higher levels or activities in cells. Examples of such cleavage agents include: redox agents selected for specific substrates or without 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 joints as general acids, peptidases (which can be substrate-specific), and phosphatases.

Cleaving joints may include pH-sensitive parts. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, and lysosomes have a more acidic pH (about 5.0) in the range of 5.5-6.0. Some joints 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 joint may include a cleavable group that can be cleaved by a specific enzyme. The type of cleavable group incorporated into the joint may depend on the cell to be targeted. For example, a liver targeting ligand may be connected to an RNA molecule by a joint including an ester group. Hepatocytes are rich in esterases, so joints are 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 joint containing a peptide bond may be used.

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. Pat. Nos. 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.

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.

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

Pharmaceutical composition

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

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 delivery route. 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 or an antibody as described herein).

In some embodiments, the pharmaceutical composition comprises an effective amount of antisense oligonucleotides or RNAi agents as described herein. "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 specific tissue or cell type (e.g., liver or hepatocyte) of the patient. The effective amount of the antisense oligonucleotides or RNAi agents 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 body 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 oligonucleotides or RNAi agents used, and the route of administration.

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 by delivery through 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 subcutaneously administered.

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 delivering 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. The preferred colloidal system used 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 can form a complex with it, 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).

Liposomal formulations are particularly suitable for topical administration, and liposomes have several advantages over other formulations. Such 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 the penetration of antisense oligonucleotides or RNAi agents into dermal tissues such as the skin.

Antisense oligonucleotides or RNAi agents of the present invention can be completely encapsulated in lipid formulations, such as LNP or other nucleic acid-lipid particles. As used herein, the term "LNP" refers to stable nucleic acid-lipid particles. LNP generally comprises cationic lipids, non-cationic lipids and lipids (such as PEG-lipid conjugates) that prevent particle aggregation. LNP is very useful for systemic applications because they show extended circulation time after intravenous (i.v.) injection and accumulate in distal sites (such as sites physically separated from the administration site). LNP includes "pSPLP", which includes the encapsulated condensing agent-nucleic acid complex as described in WO00/03683. The 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 are substantially nontoxic. In addition, when nucleic acid is present in nucleic acid-lipid particles of the present invention, it is resistant to the degradation of nucleases in aqueous solution. Nucleic acid-lipid particles and methods for making them 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 ratio of lipid to drug (mass/mass ratio) (e.g., lipid to RNAi agent) 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.

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-dioleylcarbamoyloxy-3-dimethylaminopropane (DLinDMA), 1,2-dioleylcarbamo ...N,N-dimethylaminopropane (DLenDMA), 1,2-dioleylcarbamoyloxy-N,N-trimethylaminopropane (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dioleylcarbamoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dioleylcarbamoyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dioleylcarbamoyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dioleylcar 1,2-dihydroxypropoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dihydroxypropoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleyl-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-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TA P.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-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di ((9Z,12Z)-octadec-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriacontriacontria-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)didecane-2-ol (Tech G1), or a mixture 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.

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), a particle size of 63.0±20nm, and a siRNA/lipid ratio of 0.027.

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-acylglycerol phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), oleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), oleoylphosphatidylcholine (POPC), palmitoylphosphatidylcholine (POPE), oleoylphosphatidylcholine (POPC), palmitoylphosphatidylcholine (POPE), oleoylphosphatidylcholine (POPC), palmitoylphosphatidylcholine (POPE), oleoylphosphatidylcholine (POPC), palmitoylphosphatidylcholine (POPE), oleoylphosphatidylcholine (POPC), palmitoylphosphatidylcholine (DP ... 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 lipid present in the particle.

The coupled lipids that inhibit particle aggregation can be, for example, polyethylene glycol (PEG)-lipids, including but not limited to PEG-diacylglycerols (DAG), PEG-dialkoxypropyls (DAA), PEG-phospholipids, PEG-ceramides (Cer) or mixtures thereof. PEG-DAA conjugates can be, for example, PEG-docosyloxypropyls (C12), PEG-dimyristyloxypropyls (C14), PEG-dipalmityloxypropyls (C14) or PEG-distearyloxypropyls (C18). Prevent particle aggregation and coupled lipids can be 0mol% to about 20mol% of the total lipids present in the particle, or about 2mol%. In some embodiments, nucleic acid-lipid particles further include cholesterol, for example, accounting for about 10mol% to about 60mol% of the total lipids present in the particle, or about 48mol%.

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) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. The lipid-siRNA nanoparticles typically form spontaneously upon mixing.

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).

In some embodiments, the antisense oligonucleotides or RNAi agents of the present invention can be linked to a cell penetrating peptide (CPP), which may 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, which are incorporated herein by reference in their entirety.

Pharmaceutical compositions suitable for injection include, for example, sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injection 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 the contamination of microorganisms such as bacteria and fungi. Suitable solvents or dispersion media may include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), their suitable mixtures and vegetable oils. For example, appropriate 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, isotonic agents are preferably included, such as sugar or sodium chloride. The extended absorption of injectable compositions can be achieved by using agents that delay absorption in the composition, such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by adding an appropriate amount of the active compound to a solvent together with any other ingredients (e.g., those listed above) and then filtering and sterilizing. Typically, dispersions are prepared by adding various sterilized active ingredients to a dispersion medium containing an alkaline dispersion medium and other desired 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.

The compositions of the present invention can be generally prepared 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 iron 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.

For example, for parenteral administration in the form of an aqueous solution, the solution is usually appropriately buffered, and the liquid diluent is first made isotonic with, for example, sufficient 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 standards. In certain embodiments, the pharmaceutical composition of the present invention comprises sterile saline solution and antisense oligonucleotide or RNAi agent as described herein 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.

In some embodiments, the pharmaceutical composition of the present invention is packaged with or stored in a drug delivery device. Devices for injectable formulations include, but are not limited to, injection ports, prefilled syringes, automatic injectors, injection pumps, in vivo injectors, and injection pens. Equipment for atomized or powdered formulations includes, but is not limited to, inhalers, insufflators, aspirators, and the like. Therefore, the present invention includes a drug delivery device containing a pharmaceutical composition of the present invention for treating or preventing one or more diseases or conditions described herein.

Treatment methods and uses

The present invention provides a method for reducing or inhibiting the expression of the ACVR2B gene in a cell by contacting the cell with any one 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. The reduction in ACVR2B expression in cells or animals treated with the antisense oligonucleotides or RNAi agents of the present invention can be determined relative to the expression of ACVR2B in cells and 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 an 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 to 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) to the ACVR2B mRNA level in the control cells in (b). Prior to comparison, the ACVR2B mRNA levels in the treated cells and the 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.

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.

In other embodiments, the method of 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 an ACVR2B gene), and ACVR2B mRNA or ACVR2B protein levels are assessed in tissues harvested from the animal after treatment. Alternatively or additionally, biomarkers or functional phenotypes associated with ACVR2B expression can be assessed in treated animals. For example, ACVR2B protein is the expression product of ACVR2B mRNA in tissues. 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.

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, such as 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 and other methods known in the art.

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 patients in need thereof, as well as methods for treating or preventing diseases or conditions associated with ACVR2B expression or activity. "Diseases or conditions mediated by abnormal expression of ACVR2B" refers to diseases or conditions in which the expression level of ACVR2B is altered, or conditions or conditions in which elevated expression levels of ACVR2B 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 the level of ACVR2B.

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, postoperative healing of trauma, maxillofacial tumors, eclampsia during pregnancy, liver fibrosis, atherosclerosis, ischemic stroke, and Alzheimer's disease.

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 antisense oligonucleotide or RNAi agent 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 present 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.

In certain embodiments, the patient who needs to reduce ACVR2B expression is a patient diagnosed with a disease mediated by abnormal expression levels of ACVR2B or a patient at risk of such a disease. Therefore, the present invention includes a method for treating or preventing a disease mediated by abnormal expression levels of ACVR2B in a patient in need by administering any antisense oligonucleotide or RNAi agent of the present invention. In some embodiments, the present invention includes the use of any antisense oligonucleotide or RNAi agent described herein in the preparation of a medicament for treating or preventing a disease mediated by abnormal expression levels of ACVR2B in a patient in need. In other embodiments, the present invention provides an ACVR2B-targeted antisense oligonucleotide or RNAi agent in a method for treating or preventing a disease mediated by abnormal expression levels of ACVR2B in a patient in need. As described above, the disease mediated by abnormal expression levels of ACVR2B is, for example, muscle atrophy, muscular dystrophy, obesity, diabetes, chronic kidney disease, acute kidney injury, postoperative healing of trauma, maxillofacial tumors, pregnancy eclampsia, 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.

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.

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

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 muscular atrophy, muscular dystrophy, obesity, diabetes, chronic kidney disease, acute kidney injury, postoperative healing of trauma, maxillofacial tumors, eclampsia in pregnancy, liver fibrosis, atherosclerosis, ischemic stroke and Alzheimer's disease.

Sequence Listing

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

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





Example

Experimental methods and materials

Target sequence screening

ASO and siRNA were designed based on the full-length mRNA sequence of ACVR2B (NM_001106.4), and all sequences were derived from the NCBI gene database. All ASO and siRNA were designed to ensure homology with human (Gene ID: 93, NM_001106.4, SEQ ID NO.1) and cynomolgus monkey (Gene ID: 102118668, XM_015445674.2, SEQ ID NO.2) sequences.

Oligonucleotide synthesis

The synthesis method of oligonucleotides is the same as the conventional phosphoramidite solid phase synthesis method (all synthesized by Suzhou Beixin Biotechnology Co., Ltd.), including four steps of deprotection, coupling, capping, oxidation or sulfurization. The solid phase carrier is used as the starting cycle, and the nucleoside monomers are connected one by one from 3'-5'. The raw materials of nucleoside phosphoramidite monomers 2'-OTBDMS, 2'-F RNA, 2'-OMe RNA, etc. were purchased from Shanghai Zhaowei Technology Development Co., Ltd. After the solid phase synthesis is completed, the solid phase carrier is transferred to a centrifuge tube, and a 3:1 28% ammonia and ethanol solution is added to soak the above carrier at 50°C for 16 hours to cleave the oligonucleotide from the solid phase carrier into the solution. The supernatant is transferred to another centrifuge tube by centrifugation, concentrated and evaporated to dryness, and then dissolved in deionized water again, and purified using a C18 reverse chromatographic column with a mobile phase of 0.1M TEAA and acetonitrile. The target oligonucleotide is collected and freeze-dried, and identified as the target product by LC-MS, and then quantified by UV (260nm). siRNA is obtained by annealing the sense strand and the antisense strand.

Dissolution of compounds

Place the compound dry 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 storage solution. Each compound is divided into 3 tubes, 50 μl/tube, and stored in a -20°C refrigerator.

Reverse transfection

Compound dilution: dilute the stock solution of 10μM compound with Opti-MEM reduced serum medium to 12 times the working concentration and let it stand for use. Transfection reagent dilution: dilute the transfection reagent with Opti-MEM reduced serum medium at a ratio of 48.5:1.5, mix gently and let it stand at room temperature for 15 minutes. Transfection complex preparation: mix the diluted compound and transfection reagent in equal volumes and let it stand at room temperature for 15 minutes. Add 20μl of transfection complex to each well in a 96-well cell plate. Inoculate cells: select A-673 cells with a cell confluence of about 80%, trypsinize, resuspend the cells in complete medium without penicillin and streptomycin, and count. Prepare a cell suspension with a concentration of 150,000 cells/ml, and add 100μl of cell suspension to each well in the cell plate containing the transfection complex. The cells were cultured in a 37℃ 5% CO ₂ incubator for 24hrs.

Cell lysis

Prepare cell lysis buffer according to the following table:

Remove the cell culture plate, discard the culture medium, and wash the cells once with pre-cooled DPBS. Add 50μl of cell lysis solution to each well, pipette 5-10 times to mix, and let stand at room temperature for 10 minutes. Then add 5μl of cell lysis termination solution to each well, pipette 5-10 times to mix, and wait for 2 minutes at room temperature to terminate the reaction. Place the cell plate on ice for later use.

Real-time fluorescence quantitative PCR detection

Quantitative PCR reaction system

Prepare quantitative PCR reaction system according to the table above, add 9μl to a 384-well plate. Add 1μl cell lysate as template. Complete PCR amplification reaction according to the procedure in the table below.

Data analysis

The relative expression of genes was analyzed by relative quantitative PCR (2 ^-ΔΔCT ).

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

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

In order to evaluate the in vitro inhibitory activity of siRNA compounds on ACVR2B mRNA in A673 cell line, the designed siRNA was subjected to in vitro functional evaluation. The results are shown in Table 4.

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

In order to evaluate the in vitro inhibitory activity of siRNA compounds on ACVR2B mRNA in A673 cell line, the designed siRNA was subjected to in vitro functional evaluation. The results are shown in Table 5.

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

Claims (33)

An antisense oligonucleotide comprising an oligonucleotide consisting of 12 to 30 linked nucleotides, the 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 an equal length of consecutive nucleotides of a target region of an mRNA encoding an activin type IIB receptor (ACVR2B). 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. 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 nucleotide sequence of SEQ ID NO: 3 to 17. An antisense oligonucleotide according to any one of claims 1 to 3, wherein 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. 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 comprising 5-methylcytosine (m5C); and (iv) at least one sugar substitute, preferably selected from the group consisting of POM, PNA, THP and F-HNA. The antisense-oligonucleotide according to claim 5, wherein the antisense-oligonucleotide comprises a gapmer, 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 from 2'-MOE, LNA and cEt, and at least 6 of the nucleotides in the central region comprise a 2'-deoxy sugar moiety. 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, 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. 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 phosphoramidate (PN) (e.g., methylsulfonyl phosphoramidate (MsPA)), more preferably PS. 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 NO. 18 to 32 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom, or consists of any one thereof. 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, 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 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 - _C30 saturated or unsaturated fatty acids, C5- _{C30 alkyl} groups, and polypeptides comprising at least one positively charged amino acid residue; the lipophilic group is more preferably selected from cholesterol, _C16 saturated or unsaturated fatty acids, _C16 alkyl groups, _C22 saturated or unsaturated fatty acids or _C22 alkyl groups. A RNAi agent for inhibiting the expression of an 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 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 ACVR2B, or differ from it by no more than 3 nucleotides. 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. The RNAi agent according to claim 11 or 12, wherein the antisense chain comprises at least 15 consecutive nucleotides selected from SEQ ID NO: 64 to 94 and nucleotide sequences having 1 to 3 nucleotide differences therefrom. 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. The RNAi agent according to any one of claims 11 to 14, wherein the sense strand and the antisense strand are each 17 to 23 nucleotides in length, preferably 19 to 21 nucleotides in length. 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. The RNAi agent according to any one of claims 11 to 16, wherein the RNAi agent comprises one or two overhangs, preferably one overhang, each overhang having 1 to 4 unpaired nucleotides, preferably 2 unpaired nucleotides. 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 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. The RNAi agent according to any one of claims 11 to 18, wherein the positive chain comprises at least 15 consecutive nucleotides selected from any one nucleotide sequence of SEQ ID NO: 33 to 63 and a nucleotide sequence having 1 to 3 nucleotide differences therefrom. The RNAi agent according to any one of claims 11 to 19, wherein the antisense chain has no more than 23 nucleotides and comprises a nucleotide sequence selected from SEQ ID NO: 64 to 94; the sense chain has no more than 21 nucleotides and comprises a nucleotide sequence selected from SEQ ID NO: 33 to 63. 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 positive strand contains 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 contains or is the sequence shown in SEQ ID NO:94 or a nucleotide sequence having 1, 2 or 3 nucleotide differences therefrom. The RNAi agent of claim 21, wherein 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. The RNAi agent according to any one of claims 11 to 22, wherein the sense strand and/or antisense strand of the RNAi agent comprises at least one modified nucleotide, the modified nucleotide being 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 (MOPs), phosphoramidates (PNs), tetrahydropyran-modified nucleotides (THPs), 1,5-anhydrohexitol-modified (HNA) nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing thiophosphate groups (PS), nucleotides containing methylphosphonate groups, 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 bond internucleotide linkages and combinations thereof; and/or preferably, each nucleotide of the sense strand and/or antisense strand of the RNAi agent is modified. The RNAi agent according to claim 23, wherein in the direction 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. The RNAi agent according to claim 23 or 24, wherein 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 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 from the 3' end of the antisense strand. The RNAi agent according to any one of claims 23 to 25, wherein in the direction 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. 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 first nucleotide and the second nucleotide at the 5' end of the sense strand; (ii) between the second nucleotide and the third nucleotide at the 5' end of the sense strand; (iii) between the first nucleotide and the second nucleotide at the 3' end of the sense strand; and (iv) between the second nucleotide and the third nucleotide at the 3' end of the sense strand. 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. According to any one of claims 23 to 28, the RNAi agent, wherein the 5'-terminal nucleotide of the antisense strand contains a phosphate or phosphate analog modification, preferably 5'-VP. 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, 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 - _C30 saturated or unsaturated fatty acids, _C5 - _C30 alkyls, and polypeptides comprising at least one positively charged amino acid residue; the lipophilic group is more preferably selected from cholesterol, _C16 saturated or unsaturated fatty acids, _C16 alkyls, _C22 saturated or unsaturated fatty acids or _C22 alkyls. 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. 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 expression levels of ACVR2B; or (iii) a drug for preventing or treating a disease selected from the group consisting of muscular 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. Use of an agent that reduces the expression of a gene encoding activin type IIB receptor (ACVR2B) in the preparation of a medicament for treating a disease or condition treatable by ACVR2B knockdown; preferably, wherein the agent is an antisense oligonucleotide and/or an RNAi agent; preferably, the disease or condition is selected from the group consisting of muscular atrophy, muscular dystrophy, obesity, diabetes, chronic kidney disease, acute kidney injury, postoperative healing of trauma, maxillofacial tumors, pregnancy eclampsia, liver fibrosis, atherosclerosis, ischemic stroke and Alzheimer's disease.