ACVR2b-targeted rnai agent and pharmaceutical use thereof

Abstract

Provided are an RNAi agent for ACVR2B, a pharmaceutical composition, and pharmaceutical use thereof. Also provided are a cell or kit comprising the RNAi agent, and a method of the RNAi agent for treating and / or preventing related disorders of a subject.

Description

RNAi agents targeting ACVR2B and medical uses thereof

[0001] The present disclosure claims priority to Chinese patent application No. 202410534446.6, filed on April 30, 2024, which is incorporated herein in its entirety. TECHNICAL FIELD

[0002] The present disclosure belongs to the field of biological medicine, and particularly relates to RNAi agents targeting ACVR2B gene, compositions and medical uses thereof. BACKGROUND

[0003] Activin A receptor type 2B (ACVR2B or ACTRIIB) is a type II activin receptor. Upon binding to ligands, it can form a receptor complex consisting of two type II and two type I transmembrane serine / threonine kinases. Common ligands are activins and growth differentiation factors (GDFs), which, upon binding to ACVR2B, activate the type I receptors ALK4 and ALK5, which subsequently activate downstream molecules SMAD2 / 3. SMADs regulate many myogenic genes associated with cell hypertrophy, proliferation, or differentiation, such as myoD, myogenin, and Myf5. Non-canonical ACVR2B pathways have also been shown to regulate mitogen-activated protein kinases (MAPKs). ACVR2B can also block signaling of myostatin and its homolog GDF11, as well as activin A, activin B, and BMP10. It can thus preserve muscle mass and prolong survival in tumor hosts and increase bone mass in models of osteogenesis imperfecta and muscular dystrophy.

[0004] ACVR2B plays a role in metabolic disorders and cancer. Inhibition of ACVR2B can modulate metabolism, improve conditions such as overweight and obesity, treat muscle disorders, fibrotic diseases, inflammatory diseases, skeletal diseases, ocular diseases, neurological diseases, metabolic diseases, and cancer, etc.

[0005] The present disclosure aims to provide nucleotide drugs that inhibit ACVR2B, which can effectively inhibit the expression of ACVR2B, and provide a new optional way for the treatment of the above diseases. SUMMARY

[0006] The present disclosure provides an RNAi agent targeting ACVR2B.

[0007] In some embodiments, the present disclosure provides an RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprising at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 7; the antisense strand comprising at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide set forth in any one of SEQ ID NO: 83 to SEQ ID NO: 89.

[0008] In some aspects, the "differing by no more than 3 nucleotides" means that there can be 0, 1, 2, 3 nucleotides differing. In some aspects, the "at least 15 contiguous" means that there can be at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides identical. In some aspects, there can be at least 17, 18, 19, 20, or 21 contiguous nucleotides identical.

[0009] In some embodiments, the antisense strand is at least partially complementary to the target sequence to mediate RNA interference. In some embodiments, there are no more than 5, 4, 3, 2, or 1 mismatches between the antisense strand and the target sequence. In some embodiments, the antisense strand is perfectly reverse complementary to the target sequence.

[0010] In some embodiments, the sense strand is at least partially reverse complementary to the antisense strand to form a double-stranded region. In some embodiments, there are no more than 5, 4, 3, 2, or 1 mismatches between the sense strand and the antisense strand. In some embodiments, the sense strand is perfectly reverse complementary to the antisense strand.

[0011] In some embodiments, the RNAi agent of the present disclosure comprises one or two blunt ends.

[0012] In some embodiments, the sense strand and / or the antisense strand of the RNAi agent of the present disclosure each independently comprises 1 or 2 unpaired nucleotides. In some embodiments, the 3' end of the antisense strand comprises an overhang formed by unpaired nucleotides.

[0013] In some embodiments, the sense strand and the antisense strand each independently consists of 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides. In some embodiments, the sense strand and the antisense strand each independently consists of 18, 19, 20, 21, 22, or 23 nucleotides.

[0014] In some embodiments, the sense and antisense strands are the same or different in length, the sense strand is 19-23 nucleotides in length, and the antisense strand is 19-26 nucleotides in length. Thus, the sense and antisense strands of the RNAi agents provided herein can have a length ratio of 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 19, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25, or 23 / 26 in length. In some embodiments, the sense and antisense strands of the RNAi agents have a length ratio of 19 / 19, 19 / 21, 21 / 21, 21 / 23, 23 / 23, or 23 / 25. In some embodiments, the sense and antisense strands have a length ratio of 19 / 21 or 21 / 23.

[0015] In some embodiments, the sense strand comprises at least 15 contiguous nucleotides and differs by no more than 2 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 7. In some embodiments, the sense strand comprises at least 15 contiguous nucleotides and differs by no more than 1 nucleotide from any one of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 7. In some embodiments, the sense strand comprises at least 15 contiguous nucleotides and differs by 1 nucleotide from any one of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 7.

[0016] In some embodiments, the antisense strand comprises at least 15 contiguous nucleotides and differs by no more than 2 nucleotides from any one of the nucleotide sequences of SEQ ID NO: 83 to SEQ ID NO: 89. In some embodiments, the antisense strand comprises at least 15 contiguous nucleotides and differs by no more than 1 nucleotide from any one of the nucleotide sequences of SEQ ID NO: 83 to SEQ ID NO: 89. In some embodiments, the antisense strand comprises at least 15 contiguous nucleotides and differs by 1 nucleotide from any one of the nucleotide sequences of SEQ ID NO: 83 to SEQ ID NO: 89.

[0017] In some embodiments, the sense strand comprises at least 15 contiguous nucleotides of any one of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 7. In some embodiments, the sense strand comprises at least 16 contiguous nucleotides of any one of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 7. In some embodiments, the sense strand comprises at least 17 contiguous nucleotides of any one of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 7. In some embodiments, the sense strand comprises at least 19 contiguous nucleotides of any one of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 7. In some embodiments, the sense strand comprises at least 18 contiguous nucleotides of any one of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 7.

[0018] In some embodiments, the antisense strand comprises at least 15 contiguous nucleotides of any one of the nucleotide sequences of SEQ ID NO: 83 to SEQ ID NO: 89. In some embodiments, the antisense strand comprises at least 16 contiguous nucleotides of any one of the nucleotide sequences of SEQ ID NO: 83 to SEQ ID NO: 89. In some embodiments, the antisense strand comprises at least 17 contiguous nucleotides of any one of the nucleotide sequences of SEQ ID NO: 83 to SEQ ID NO: 89. In some embodiments, the antisense strand comprises at least 18 contiguous nucleotides of any one of the nucleotide sequences of SEQ ID NO: 83 to SEQ ID NO: 89. In some embodiments, the antisense strand comprises at least 19 contiguous nucleotides of any one of the nucleotide sequences of SEQ ID NO: 83 to SEQ ID NO: 89. In some embodiments, the antisense strand comprises at least 20 contiguous nucleotides of any one of the nucleotide sequences of SEQ ID NO: 83 to SEQ ID NO: 89. In some embodiments, at least 21 contiguous nucleotides.

[0019] In some embodiments, the sense strand comprises the nucleotide sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 7; and / or the antisense strand comprises or the nucleotide sequence set forth in any one of SEQ ID NO: 83 to SEQ ID NO: 89.

[0020] In some embodiments, the sense strand is the nucleotide sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 7; and / or the antisense strand is the nucleotide sequence set forth in any one of SEQ ID NO: 83 to SEQ ID NO: 89.

[0021] In some embodiments, the RNAi agent comprises or is selected from any one of the following sets of sense and antisense strands: In some embodiments, the RNAi agent comprises or is selected from any one of the following sets of sense and antisense strands:

[0022] Group 1), the sense strand as shown in SEQ ID NO: 1 and the antisense strand as shown in SEQ ID NO: 83;

[0023] Group 2), the sense strand as shown in SEQ ID NO: 2 and the antisense strand as shown in SEQ ID NO: 84;

[0024] Group 3), the sense strand as shown in SEQ ID NO: 3 and the antisense strand as shown in SEQ ID NO: 85;

[0025] Group 4), the sense strand as shown in SEQ ID NO: 4 and the antisense strand as shown in SEQ ID NO: 86;

[0026] Group 5), the sense strand as shown in SEQ ID NO: 5 and the antisense strand as shown in SEQ ID NO: 87;

[0027] Group 6), the sense strand as shown in SEQ ID NO: 6 and the antisense strand as shown in SEQ ID NO: 88;

[0028] Group 7), the sense strand as shown in SEQ ID NO: 7 and the antisense strand as shown in SEQ ID NO: 89.

[0029] In some embodiments, at least one nucleotide in the sense strand and / or the antisense strand is a modified nucleotide. In some embodiments, all of the nucleotides in the sense strand and / or the antisense strand are modified nucleotides.

[0030] In some embodiments, the sense strand contains three consecutive 2'-fluoro modified nucleotides.

[0031] In some embodiments, the 7th, 8th, and 9th nucleotides from the 5' end to the 3' end of the sense strand are 2'-fluoro modified nucleotides, and the remaining nucleotides are non-2'-fluoro modified nucleotides.

[0032] In some embodiments, the 9th, 10th, and 11th nucleotides from the 5' end to the 3' end of the sense strand are 2'-fluoro modified nucleotides, and the remaining nucleotides are non-2'-fluoro modified nucleotides.

[0033] In some embodiments, the antisense strand contains at least 5 2'-fluoro modified nucleotides.

[0034] In some embodiments, the 2nd, 6th, 12th, 14th, and 16th nucleotides from the 5' end to the 3' end of the antisense strand are each independently 2'-fluoro modified nucleotides.

[0035] In some embodiments, in the 5' end to 3' end direction, wherein the nucleotides in the 2nd, 4th, 6th, 10th, 12th, 14th, 16th, 18th positions of the antisense strand are each independently 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are non-2'-fluoro modified nucleotides.

[0036] In some embodiments, in the 5' end to 3' end direction, wherein the nucleotides in the 2nd, 6th, 12th, 14th, 16th positions of the antisense strand are each independently 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are non-2'-fluoro modified nucleotides.

[0037] In some embodiments, the non-2'-fluoro modified nucleotides are 2'-methoxy modified nucleotides.

[0038] In some embodiments, at least one of the phosphodiester groups in the sense strand and / or the antisense strand is a phosphodiester group with a modification group. The modification group provides increased stability of the RNAi agent in a biological sample or environment. In some embodiments, a plurality of the phosphodiester groups with a modification group are included in the sense strand and / or the antisense strand. In some embodiments, a plurality of the phosphodiester groups with a modification group are included in both the sense strand and the antisense strand. In some embodiments, the phosphodiester group with a modification group is present at one or more of the positions selected from the group consisting of: between any two adjacent nucleotides from the 1st to the 4th nucleotide at the 5' terminal end and / or the 3' terminal end of the sense strand, and / or between any two adjacent nucleotides from the 1st to the 4th nucleotide at the 5' terminal end and / or the 3' terminal end of the antisense strand.

[0039] In some embodiments, the phosphorothioate group is present at at least one of the positions:

[0040] between the 1st and 2nd nucleotides at the 5' terminal end of the sense strand;

[0041] between the 2nd and 3rd nucleotides at the 5' terminal end of the sense strand;

[0042] between the 1st and 2nd nucleotides at the 3' terminal end of the sense strand;

[0043] between the 2nd and 3rd nucleotides at the 3' terminal end of the sense strand;

[0044] between the 1st and 2nd nucleotides at the 5' terminal end of the antisense strand;

[0045] between the 2nd and 3rd nucleotides at the 5' terminal end of the antisense strand;

[0046] between the 1st and 2nd nucleotides at the 5' terminal end of the antisense strand; and

[0047] between the 2nd and 3rd nucleotides at the 5' terminal end of the antisense strand.

[0048] In some embodiments, the sense strand and / or the antisense strand comprises a plurality of phosphorothioate linkages, which are present between:

[0049] between the 1st and 2nd nucleotides at the 5' terminal end of the sense strand; and

[0050] between the 2nd and 3rd nucleotides at the 5' terminal end of the sense strand; and

[0051] between the 1st and 2nd nucleotides at the 3' terminal end of the sense strand; and

[0052] between the 2nd and 3rd nucleotides at the 3' terminal end of the sense strand; and

[0053] between the 1st and 2nd nucleotides at the 5' terminal end of the antisense strand; and

[0054] between the 2nd and 3rd nucleotides at the 5' terminal end of the antisense strand; and

[0055] between the 1st and 2nd nucleotides at the 3' terminal end of the antisense strand; and

[0056] between the 2nd and 3rd nucleotides at the 3' terminal end of the antisense strand.

[0057] In some embodiments, the phosphodiester linkage with a modifying group is a phosphorothioate linkage.

[0058] In some embodiments, the sense strand is selected from or comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 165 to SEQ ID NO: 178.

[0059] In some embodiments, the antisense strand is selected from or comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 253 to SEQ ID NO: 266.

[0060] In some embodiments, the RNAi agent further comprises one or more delivery groups attached to the sense strand and / or the antisense strand.

[0061] In the context of the present disclosure, "attached" includes covalent and non-covalent attachment. The delivery groups are capable of delivering the RNAi agent of the present disclosure to a location where ACVR2B gene expression is present.

[0062] In some embodiments, the delivery group comprises a lipophilic group, the one or more lipophilic groups attached to any one or more nucleotides in the sense strand or the antisense strand of the RNAi agent. In some embodiments, the one or more lipophilic groups are attached to the sense strand of the RNAi agent. In some embodiments, the one or more lipophilic groups are attached to the antisense strand of the RNAi agent. In some embodiments, at least one of the lipophilic groups is attached to the sense strand of the RNAi agent, and at least one of the lipophilic groups is attached to the antisense strand of the RNAi agent.

[0063] In some embodiments, the lipophilic group is attached to the base of a nucleotide. In some embodiments, the lipophilic group is attached to the sugar ring of a nucleotide. In some embodiments, the lipophilic group is attached to the internucleoside linking group between two adjacent nucleotides. In some embodiments, the lipophilic group comprises a saturated or unsaturated C 4-30 hydrocarbon chain, and optionally a functional group selected from the group consisting of halogen, alkoxy, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0064] In some embodiments, the lipophilic group comprises a saturated or unsaturated C 6-22 hydrocarbon chain. In some embodiments, the lipophilic group comprises a saturated or unsaturated C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 hydrocarbon chain. In some embodiments, the lipophilic group comprises a saturated or unsaturated C 16 , C 18 , C 22 hydrocarbon chain.

[0065] In some specific embodiments, the lipophilic group comprises a saturated or unsaturated C 22 hydrocarbon chain.

[0066] In some embodiments, the lipophilic group comprises a saturated or unsaturated C 6-18 hydrocarbon chain. In some embodiments, the lipophilic group comprises a saturated or unsaturated C 16 hydrocarbon chain.

[0067] In some embodiments, the delivery moiety comprises a targeting ligand that targets the liver. In some embodiments, the targeting ligand binds to the asialoglycoprotein receptor (ASGPR). In some embodiments, the targeting ligand comprises a galactose cluster or a cluster of galactose derivatives selected from N-acetyl-galactosamine (GalNAc), N-trifluoroacetyl galactosamine, N-propionyl galactosamine, N-n-butyryl galactosamine, or N-isobutyryl galactosamine.

[0068] In some embodiments, the delivery moiety is attached to the 3' end or the 5' end of the sense strand of the RNAi agent.

[0069] In some embodiments, the delivery moiety is attached to the 3' end or the 5' end of the sense strand of the RNAi agent.

[0070] In some embodiments, the delivery moiety is indirectly attached to the 3' end or the 5' end of the sense strand of the RNAi agent via a phosphodiester group, a phosphorothioate group, or a phosphonate group. In some embodiments, the delivery moiety is indirectly attached to the 3' end or the 5' end of the sense strand of the RNAi agent via a phosphodiester group.

[0071] In some embodiments, the delivery moiety is directly attached to the 3' end or the 5' end of the sense strand of the RNAi agent via a phosphodiester group, a phosphorothioate group, or a phosphonate group. In some embodiments, the delivery moiety is directly attached to the 3' end or the 5' end of the sense strand of the RNAi agent via a phosphodiester group.

[0072] In some embodiments, the delivery moiety is directly attached to the 3' end or the 5' end of the sense strand of the RNAi agent via a phosphodiester group or a phosphorothioate group. In some embodiments, the delivery moiety is directly attached to the 3' end or the 5' end of the sense strand of the RNAi agent via a phosphodiester group.

[0073] In another aspect, the present disclosure provides a pharmaceutical composition comprising an RNAi agent described herein, and one or more pharmaceutically acceptable carriers, such as excipients, vehicles, diluents, and / or delivery systems (e.g., delivery polymers). Various delivery systems are known and can be used to deliver the RNAi agents of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing RNAi agents, receptor-mediated endocytosis, construction of the nucleic acid as part of a retroviral or other vector, and the like.

[0074] In some embodiments, the pharmaceutical composition can further comprise a pharmaceutically acceptable excipient and / or adjuvant, which can be one or more of various agents or compounds conventionally employed in the art. For example, the pharmaceutically acceptable excipient can include at least one of a pH buffering agent, a protective agent, and an osmotic pressure adjusting agent.

[0075] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg to 1000 mg.

[0076] In certain embodiments, the pharmaceutical composition contains 0.01-99.99% of the aforementioned RNAi agent, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned RNAi agent. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned RNAi agent. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned RNAi agent. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned RNAi agent.

[0077] In certain embodiments, the pharmaceutical composition contains 0.01%-99.99% of the aforementioned pharmaceutically acceptable carrier, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1%-99.9% of the aforementioned pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned pharmaceutically acceptable carrier.

[0078] In some embodiments, the RNAi agent or pharmaceutical composition of the present disclosure, when contacted to a cell expressing a target gene, inhibits the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, 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%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, as determined by, for example, psiCHECK activity screening and luciferase reporter assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence assays, for example, Western Blot or flow cytometry.

[0079] In some embodiments, the RNAi agent or pharmaceutical composition of the present disclosure, when contacted to a cell expressing the target gene, causes a percent of remaining expression of the mRNA of the target gene of no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%, as determined by, for example: psiCHECK activity screening and luciferase reporter assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence assays, for example Western Blot, or flow cytometry.

[0080] In some embodiments, the RNAi agent or pharmaceutical composition of the present disclosure, when contacted to a cell expressing the target gene, causes a reduction in off-target activity of at least 20%, at least 25%, 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%, or at least 75%, while maintaining the target activity, as determined by, for example: psiCHECK activity screening and luciferase reporter assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence assays, for example Western Blot, or flow cytometry.

[0081] In some embodiments, the RNAi agent or pharmaceutical composition of the present disclosure, when contacted to a cell expressing the target gene, causes a reduction in off-target activity of at least 20%, at least 25%, 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%, or at least 75%, while reducing the target activity by at most 20%, at most 19%, at most 15%, at most 10%, at most 5%, or more than 1%, as determined by, for example: psiCHECK activity screening and luciferase reporter assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence assays, for example Western Blot, or flow cytometry.

[0082] In some embodiments, the RNAi agent or pharmaceutical composition of the present disclosure, when contacted to a cell expressing a target gene, causes at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, 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%, or at least 80% increase in target activity while causing at least 20%, at least 25%, 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%, or at least 75% decrease in off-target activity, as determined by, for example: psiCHECK activity screening and luciferase reporter assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence assays, for example Western Blot, or flow cytometry.

[0083] In another aspect, the present disclosure provides a method of reducing ACVR2B expression (in vitro, in vivo, or ex vivo), comprising administering to a subject (or target cell) an effective amount or effective dose of the RNAi agent and / or pharmaceutical composition of the present disclosure.

[0084] In another aspect, the present disclosure provides a method of treating and / or preventing a disease associated with ACVR2B gene expression in a subject, comprising administering to the subject an effective amount or effective dose of the RNAi agent and / or pharmaceutical composition of the present disclosure.

[0085] In another aspect, the present disclosure provides the use of the RNAi agent and / or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating and / or preventing a disease associated with ACVR2B gene expression.

[0086] In another aspect, the present disclosure provides the use of the RNAi agent and / or pharmaceutical composition of the present disclosure in the manufacture of a medicament for inhibiting the expression of ACVR2B.

[0087] In some embodiments, the disease is a disease associated with ACVR2B. In some embodiments, the disease is a metabolic disorder disease, including obesity, type 2 diabetes, inflammation, cardiovascular disease, lung disease, liver disease such as fatty liver, nervous system disease, kidney disease, muscle disorder, fibrotic disease, neurological disease, and the like due to metabolic disorder.

[0088] In some embodiments, the disease associated with ACVR2B is cancer.

[0089] In another aspect, the present disclosure provides a method of delivering an RNAi agent that inhibits expression and / or replication of ACVR2B in vivo, the method comprising administering to a subject an RNAi agent and / or a pharmaceutical composition of the present disclosure.

[0090] The RNAi agents or pharmaceutical compositions and methods disclosed herein can reduce the level of a target mRNA in a cell, a population of cells, a tissue, or a subject, comprising: administering to a subject a therapeutically effective amount of an RNAi agent or a pharmaceutical composition of the present disclosure, the RNAi agent linked to the delivery group, thereby inhibiting expression of the target mRNA in the subject.

[0091] In some embodiments, the subject has been identified as having a pathological upregulation of a target gene in a targeted cell or tissue prior to administration of the RNAi agent and / or pharmaceutical composition of the present disclosure.

[0092] A subject described in the present disclosure refers to a subject diagnosed with (or suspected of having, or susceptible to) a disease or disorder that would benefit from a reduction or inhibition of expression of a target mRNA.

[0093] The RNAi agent and / or pharmaceutical composition of the present disclosure can be delivered by local administration (e.g., direct injection, implantation, or topical administration), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration, and the like, by any suitable means of administration commonly used in the art.

[0094] In alternative embodiments, the pharmaceutical composition provided by the present disclosure can be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal injection.

[0095] In another aspect, the present disclosure also provides a pharmaceutical composition comprising an RNAi agent of the present disclosure.

[0096] In some embodiments, the pharmaceutical composition can further comprise a pharmaceutically acceptable excipient and / or adjuvant, which excipient can be one or more of various agents or compounds conventionally employed in the art. For example, the pharmaceutically acceptable excipient can include at least one of a pH buffering agent, a protective agent, and an osmotic pressure adjusting agent.

[0097] In another aspect, the present disclosure provides a cell comprising an RNAi agent of the present disclosure. The cell is not capable of developing into an individual of a plant or animal.

[0098] In another aspect, the present disclosure provides a kit comprising an RNAi agent and / or a pharmaceutical composition of the present disclosure.

[0099] The present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell, the method comprising the step of contacting the cell with an RNAi agent and / or a pharmaceutical composition according to the present disclosure.

[0100] The present disclosure also provides a method for silencing a target gene or mRNA of a target gene in a cell in vivo or in vitro, the method comprising the step of contacting the cell with an RNAi agent and / or a pharmaceutical composition according to the present disclosure.

[0101] The present disclosure also provides a method for inhibiting expression of a target gene or mRNA of a target gene, the method comprising administering to a subject in need thereof an effective amount or effective dose of an RNAi agent and / or a pharmaceutical composition according to the present disclosure.

[0102] In some embodiments, the effective amount or effective dose of the RNAi agent and / or the pharmaceutical composition is about 0.001 mg / kg body weight to about 200 mg / kg body weight, about 0.01 mg / kg body weight to about 100 mg / kg body weight, or about 0.5 mg / kg body weight to about 50 mg / kg body weight.

[0103] In some embodiments, the target gene is an ACVR2B gene and the target mRNA is mRNA of expression of the target gene.

[0104] The present disclosure also provides a method of making an RNAi agent, comprising: synthesizing an RNAi agent described in the present disclosure.

[0105] In another aspect, without wishing to be bound by theory, the compounds of the present disclosure can exist in particular geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)-isomers, as well as racemic mixtures and other mixtures thereof, such as for example, mixtures of enantiomers or diastereomers, all of which are intended to be within the scope of the present disclosure. Additional asymmetric carbon atoms can be present in a substituent group. All such isomers, as well as mixtures thereof, are included within the scope of the present disclosure.

[0106] Additionally, the compounds and intermediates of the present disclosure can exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The term "tautomeric forms" or "tautomers" refers to different energy forms of structural isomers which can interconvert via a low energy barrier.

[0107] The compounds of the present disclosure can be asymmetric, e.g., having one or more stereocenters. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. Compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically active form or as racemic mixtures. Optically active forms can be obtained, for example, by resolution of a racemic mixture or by synthesis from an optically active starting material or reagent.

[0108] Optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomer. Alternatively, when a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group) is present in the molecule, diastereomeric salts are formed with an appropriate optically active acid or base, and the diastereomeric salt is separated by conventional means, and the desired enantiomer is recovered by treating with base or acid, as the case can be. In addition, separation of the enantiomers and diastereomers is often accomplished by chromatography using a chiral stationary phase, optionally in combination with chemical derivatization (e.g., formation of carbamates from amines).

[0109] The present disclosure also includes some isotopically-labeled compounds of the present disclosure which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be present in compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I, and 36 Cl.

[0110] In the chemical structures of the compounds of the present disclosure, where the configuration is not indicated, the bond indicates that the configuration is not specified, i.e., if there is a chiral isomer in the chemical structure, the bond may be or both Two configurations. Although all structural formulas described herein are represented in certain isomer forms for simplicity, this disclosure can include all isomers, such as tautomers, rotatimers, geometric isomers, diastereomers, racemates, and enantiomers. In the chemical structure of the compounds described herein, bonds... No configuration was specified, i.e., key The configuration can be E-type or Z-type, or it can contain both E-type and Z-type configurations.

[0111] This publication incorporates the full text of WO2023274395A.

[0112] Terminology Explanation

[0113] To facilitate understanding of this disclosure, some technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0114] As used herein, “RNAi agent” refers to an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of degrading, inactivating, or inhibiting (e.g., under appropriate conditions, degrading, inactivating, or inhibiting) the translation of a messenger RNA (mRNA) transcript of a target mRNA in a sequence-specific manner. RNAi agents used herein may act via RNA interference mechanisms (i.e., by interacting with RNA interference pathway mechanisms in mammalian cells, such as RNA-induced silencing complexes or RISC) or through any alternative mechanism or pathway. Although the term RNAi agent as used herein is considered to act primarily through RNA interference mechanisms, the disclosed RNAi agents are not bound to or limited to any particular pathway or mechanism of action. The RNAi agents disclosed herein comprise, or consist of, a sense strand and an antisense strand; the RNAi agents disclosed herein are, for example (but not limited to), presented in the form of short (or small) interfering RNA (siRNA). The antisense strand of the RNAi agent described herein is at least partially complementary to the targeted mRNA. RNAi agents may contain one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0115] Unless otherwise specified, in the context of this disclosure, ACVR2B includes, but is not limited to, human ACVR2B, cynomolgus monkey ACVR2B, mouse ACVR2B, and rat ACVR2B, whose amino acid and complete coding sequences and mRNA sequences are readily available from publicly available databases, such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0116] The term "ACVR2B" also refers to naturally occurring DNA sequence variations of the ACVR2B gene, such as single nucleotide polymorphisms (SNPs) in the ACVR2B gene. Exemplary SNPs can be found in the dbSNP database.

[0117] The term "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of ACVR2B, including mRNA that is a processing product of the primary transcript. The portion of the target sequence that is targeted should be long enough to serve as a substrate for iRNA-directed cleavage. In one embodiment, the target sequence is within the protein coding region of ACVR2B. As used herein, in the context of RNA-mediated gene silencing, the sense strand (also referred to as SS, SS strand, or sense strand) refers to the strand comprising a sequence identical or essentially identical to the sequence of the target mRNA; the antisense strand (also referred to as AS or AS strand) refers to the strand having a sequence partially or fully complementary to the sequence of the target mRNA.

[0118] In the context of describing the sense strand of the RNAi agent described herein, the term "at least 15 contiguous nucleotides of a nucleotide sequence differing by no more than 3 nucleotides from the nucleotide sequence set forth in SEQ ID NO: 1" is intended to mean that the sense strand of the RNAi agent described herein comprises at least 15 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, or differs by no more than 3 nucleotides (optionally, no more than 2 nucleotides; optionally, 1 nucleotide) from at least 15 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1. Similar interpretation should be made to other similar descriptions in the context of the disclosure. The disclosure does not include the case where the "difference" contains nucleotides with different modifications, i.e., nucleotides with the same base but different modifications are not considered as "different" in the context of the disclosure. Similar interpretation should be made to other similar descriptions of the sense strand and / or antisense strand in the context of the disclosure.

[0119] In the context of the disclosure, the "5' region" of a sense strand or an antisense strand is used interchangeably with "5' end" or "5' terminal". For example, the 2nd to 8th nucleotide of the 5' region of an antisense strand is used interchangeably with the 2nd to 8th nucleotide of the 5' end of an antisense strand. Similarly, the "3' region", "3' terminal" and "3' end" of a sense strand or an antisense strand are used interchangeably.

[0120] Unless otherwise specified, in the context of this disclosure, "G", "C", "A", "T" and "U" represent nucleotides, which respectively contain the bases of guanine, cytosine, adenine, thymidine, and uracil. It is well known to those skilled in the art that substitution of bases T and U will not significantly affect the properties of the RNAi agent sequence. In the sequences of this disclosure, U can be arbitrarily replaced with T, and the resulting sequences are also within the scope of protection of this disclosure. In the sequences of this disclosure, for the same nucleic acid chain, the direction from the 5' end to the 3' end is considered left-to-right. A lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy modified nucleoside; a lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluoro modified nucleoside; a lowercase letter s indicates that the two nucleosides adjacent to the letter s are linked by a phosphothioester group. Unless otherwise specified, the two nucleosides are linked by a phosphoester group. Unless otherwise specified, the terms "RNAi agent," "nucleotide," "compound," "chemical modification," "oligonucleotide," "double-stranded RNAi inhibitor molecule," "siRNA," "dsRNA," "nucleic acid," and "RNAi" in this disclosure can exist independently as salts, mixed salts, or non-salts (e.g., free acids or free bases). When present as salts or mixed salts, they are pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. When present as salts, some groups may ionize to form anions / cations; for example, phosphodiester groups and thiophosphonate diester groups can exist in anionic form. Unless otherwise specified, the salt forms of the following structures are also within the scope of protection of this disclosure. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each chain is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each chain is a hydroxyl group.

[0121] The above-mentioned modifications and linking groups have the structures shown in the table below, where Base represents the base at the corresponding position:

[0122] Table 1A

[0123] The terms "lipophilic group" or "lipophilic moiety" broadly refer to any compound or chemical part that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is through the octanol-water partition coefficient logK. ow K ow This represents the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the aqueous phase at equilibrium in a two-phase system. In principle, logK... ow When the value exceeds 0, the chemical substance exhibits lipophilicity. Typically, the logK of the lipophilic portion... ow Values ​​exceeding 1, 1.5, 2, 3, 4, 5, or 10, such as the logK of 6-aminohexanol.ow logK of cholesteryl N-(hexan-6-ol)carbamate is about 0.7, and the logK of cholesteryl N-(octan-7-ol)carbamate is about 0.7. ow

[0124] The lipophilicity of a molecule can vary relative to the functional groups it carries. For example, the addition of a hydroxyl or amine group at the end of a lipophilic moiety can increase or decrease the partition coefficient (e.g., logK ow ) value of the lipophilic moiety. For example, the lipophilic moiety can be an aliphatic, cyclic such as alicyclic, or polycyclic such as a polyalicyclic compound, such as a steroid (e.g., a sterol) or a straight chain or branched aliphatic hydrocarbon. The lipophilic moiety can generally comprise a hydrocarbon chain, which can be cyclic or acyclic. The hydrocarbon chain can comprise various substituents and / or one or more heteroatoms, such as oxygen or sulfur atoms. Such lipophilic aliphatic moieties include, but are not limited to, saturated or unsaturated C4-C 30 hydrocarbons (e.g., C 10 -C 30 hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monoalcohol esters of fatty acids and fatty diamides), terpenes (e.g., C 10 terpenes, C 15 sesquiterpenes, C 20 diterpenes, C 30 triterpenes, and C 40 tetra terpenes), and other polyalicyclic hydrocarbons; for example, the lipophilic moiety can be an optionally substituted straight chain alkyl of C 10-30 ; for example, the lipophilic moiety can be an optionally substituted straight chain alkyl of C 14-24 .

[0125] As used herein, the terms "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases on the other strand in a complementary manner. In DNA, the purine base adenine always pairs with the pyrimidine base thymine (or, in RNA, uracil); the purine base guanine always pairs with the pyrimidine base cytosine. Each base pair comprises one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand. Correspondingly, "mismatch" means in the art that the bases at the corresponding positions in a double-stranded nucleic acid are not paired in a complementary manner.

[0126] ​As used herein, the term "inhibit," can be used interchangeably with "reduce," "silence," "down-regulate," "repress," and other similar terms, and includes inhibition of any level or activity. Inhibition can be assessed by a decrease in absolute or relative levels of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-dosing baseline level or a level determined from a subject, cell, or sample that has not been treated or has been treated with a control (e.g., a buffer control or an inert agent control). For example, the degree of inhibition of target gene expression by an RNAi agent can be characterized by the amount of residual mRNA expression, such as no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%. The degree of inhibition of target gene expression can be measured using the Dual-Luciferase Assay System, reading the firefly chemiluminescence value (Fir) and the Renilla chemiluminescence value (Ren), respectively, and calculating the relative value Ratio = Ren / Fir; in the present disclosure, the residual mRNA expression ratio (or the residual activity %) = Ratio (RNAi agent treatment group) / Ratio (no RNAi agent control group), and the inhibition rate (%) = 100% - residual mRNA expression (%). Luciferase Assay System detection, reading the firefly chemiluminescence value (Fir) and the Renilla chemiluminescence value (Ren), respectively, and calculating the relative value Ratio = Ren / Fir; in the present disclosure, the residual mRNA expression ratio (or the residual activity %) = Ratio (RNAi agent treatment group) / Ratio (no RNAi agent control group), and the inhibition rate (%) = 100% - residual mRNA expression (%).

[0127] The term "pharmaceutically acceptable salt" includes a pharmaceutically acceptable acid addition salt and a pharmaceutically acceptable base addition salt.

[0128] A "pharmaceutically acceptable acid addition salt" means a salt that retains the biological effectiveness of the free base and that is not biologically or otherwise undesirable. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, and the like. Organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, mesylate, phenylsulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalene- disulfonate, and the like. These salts can be prepared by methods known in the art.

[0129] "Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and non-toxicity of the free acids and are formed with inorganic or organic bases. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. In some embodiments, the inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion-exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethyl ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. In some embodiments, the organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0130] "Effective amount" or "effective dose" refers to the amount of a RNAi agent, compound, or pharmaceutical composition necessary to effect one or more beneficial or desired therapeutic results. For prophylactic use, beneficial or desired results include eliminating or reducing risk, lessening severity, or delaying onset of a disorder, including biochemical, histological and / or behavioral symptoms of the disorder, its complications, and intermediate pathological phenotypes presenting during development of the disorder. For therapeutic applications, beneficial or desired results include clinical results, such as decreasing the incidence of various disorders associated with the target gene, target mRNA, or target protein of the disclosure, or ameliorating one or more symptoms of the disorder, reducing the dose of other medications required to treat the disorder, enhancing effect of another medication, and / or delaying the progression of the disorder associated with the target gene, target mRNA, or target protein of the disclosure in a patient.

[0131] As used herein, "patient," "subject," or "individual" are used interchangeably and include a human or non-human animal, such as a mammal, for example, a human or a monkey.

[0132] The RNAi agents provided by the present disclosure can be obtained by methods of preparation conventional in the art, such as methods of solid phase synthesis and liquid phase synthesis. Among them, solid phase synthesis has been commercialized as a subscription service. Methods of preparing nucleoside monomers with corresponding modifications and methods of introducing modified nucleotide groups into the RNAi agents described in the present disclosure by using nucleoside monomers with corresponding modifications are also well known to those skilled in the art.

[0133] The term "chemical modification" or "modification" includes all changes to a nucleotide by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0134] The term "base" includes any known DNA and RNA bases, base analogs, such as purines or pyrimidines, which also include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs.

[0135] The terms "blunt end" or "blunt ends" are used interchangeably to mean that there are no unpaired nucleotides or nucleotide analogs at a given end of an RNAi agent, i.e., there is no nucleotide overhang. Most often, an RNAi agent that has both ends blunt ended will be double stranded over its entire length.

[0136] The terms "about," "approximately," mean values within an acceptable error range for the specific value being sought to be measured or obtained, said range being determined based on the limits of detection for the measurement system being employed, i.e., the resolution and / or variability within more or less the limit of detection for the system. For example, "about" can mean within 1 or more than 1 standard deviation, per the measurement system in use / employing. Alternatively, "about" or "approximately" can mean quantities that deviate by as much as 20%, e.g., by as much as 10%, of the stated amount, by as much as 5%, by as much as 1%, by as much as 0.5%, by as much as 0.1% of the stated amount, individual embodiments of the disclosure can include quantities that deviate by as much as 20%, e.g., by as much as 10%, of the stated amount, by as much as 5%, by as much as 1%, by as much as 0.5%, by as much as 0.1% of the stated amount. In the disclosure, each instance of a number or a numerical range, unless otherwise stated, is also intended to include embodiments within about 20%, e.g., within about 10%, of that value, within about 5%, within about 1%, of that value, within about 0.5%, within about 0.1% of that value. Unless otherwise stated, the

[0137] Unless otherwise indicated, "optionally," "optional," "optional" or "may" means that the subsequently described event or circumstance can or can not occur, and the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally, R1and R2are directly connected to form a ring" means that R1and R2may be directly connected to form a ring, but it is not necessary that R1and R2be directly connected to form a ring, and the description includes instances where R1and R2are directly connected to form a ring and instances where R1and R2are not connected to form a ring.

[0138] In the chemical structures of the disclosure, which can be attached to one or more of any of the groups according to the scope of the invention described herein.

[0139] The term "linked," when referring to the association between two molecules, means that the two molecules are connected by a covalent bond or that the two molecules are associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond), including direct linkage, indirect linkage.

[0140] The term "directly linked" means that a first compound or group is connected to a second compound or group without any intervening atoms or atom groups.

[0141] The term "indirectly linked" means that a first compound or group is linked to a second compound or group through an intervening group, compound, or molecule (e.g., a linker group).

[0142] The term "substituted" means that any one or more hydrogen atoms on the designated atom (typically a carbon, oxygen, and nitrogen atom) is replaced with any group as defined herein, provided that the designated atom's normal valence is not exceeded, and the substitution results in a stable compound. Non-limiting examples of substituents include C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, cyano, hydroxyl, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, aryl, ketone, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halogen (e.g., F, Cl, Br, I). When the substituent is a ketone or oxo (i.e., =O), then two (2) hydrogens on the atom are replaced.

[0143] "Substituted with one or more" means that a single or multiple substituents can be present. When multiple substituents are present, they can be multiple identical substituents or a combination of one or multiple different substituents.

[0144] The present disclosure is further described in connection with the following examples, which are not intended to limit the scope of the disclosure. Unless otherwise indicated, the experimental methods used in the examples below generally follow conventional methods, such as those described in Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; or those described in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY. Unless otherwise indicated, the reagents used in the examples below are commercially available in the quality / purity suitable for molecular biology applications. Unless otherwise indicated, the reagents used in the examples below are commercially available in the quality / purity suitable for molecular biology applications. DETAILED DESCRIPTION

[0145] Example 1. Design of ACVR2B RNAi agents

[0146] The sequences of the sense strand, antisense strand of the RNAi agents of the present disclosure are shown in Table 1B below, which are designed according to the general rules of double-stranded oligonucleotides to satisfy the activity, with 19 / 21 nt unmodified sense / antisense strands, and the modified RNAi agents of the present disclosure are shown in Table 2, with the human ACVR2B gene (NM_001106.4) as the target gene.

[0147] Table 1B. Unmodified RNAi agent sequences targeting the ACVR2B gene

[0148] Table 2. Modified RNAi agent sequences targeting the ACVR2B gene

[0149] In Table 1B and Table 2, in the same nucleic acid sequence, G, C, A, U, T represent nucleosides with guanine, cytosine, adenine, uracil, and thymine, respectively, from left to right according to the direction from the 5' end to the 3' end, the sequence from left to right represents the direction from the 5' end to the 3' end, the lower-case letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleoside; the lower-case letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluorinated-modified nucleoside; the lower-case letter s indicates that the phosphodiester bond between the two nucleosides adjacent to the letter s is a phosphorothioate bond, and no special instructions, the two adjacent nucleosides are connected by a phosphodiester bond. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each strand is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each strand is a hydroxyl group.

[0150] Example 2. Synthesis of the RNAi agent of the present disclosure

[0151] The synthesis of the RNAi agent is not different from the general phosphoramidite solid-phase synthesis method. The synthesis process is briefly described as follows: on the Dr. Oligo 48 synthesizer (Biolytic), using a universal CPG carrier as the starting material, according to the synthesis program, connecting the unmodified nucleoside phosphoramidite monomers or the nucleoside phosphoramidite monomers with modifications at the corresponding positions in the sequence one by one. The nucleoside phosphoramidite monomers used in the examples of the present disclosure are purchased from Shanghai Zhiwei Company and Suzhou Jimar Company. 5-ethylthio-1H-tetrazole (ETT) is used as the activator (0.6M acetonitrile solution), 0.22M PADS dissolved in a 1:1 volume ratio of acetonitrile and 3-methylpyridine (Shanghai Lingjiang) solution is used as the sulfuration reagent, and iodopyridine / water solution (Shanghai Lingjiang) is used as the oxidizing agent.

[0152] After the completion of solid-phase synthesis, the oligoribonucleotide is cleaved from the solid support, and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. Then centrifuged, the supernatant is transferred to another centrifuge tube, and after concentrated and evaporated to dryness, purified by C18 reverse phase chromatography with 0.1M TEAA and acetonitrile as the mobile phase. The target oligonucleotide is collected, freeze-dried, and identified as the target product by LC-MS, and then quantified by UV (260nm).

[0153] The obtained single-stranded oligonucleotide is annealed according to the equimolar ratio according to the complementary pairing, and finally the obtained double-stranded RNAi is dissolved in 1xPBS and adjusted to the required concentration for the experiment for standby.

[0154] Example 3. Single point inhibition activity of ACVR2B in human hepatocarcinoma cells (Huh7)

[0155] Huh7 cells were seeded at about 50,000 cells / well in 24-well plates 24h before transfection, each well containing 500mL of culture medium. At the time of transfection, samples were transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150) according to the product manual, with a final concentration of 0.2nM of sample transfection. After 48h of treatment, the mRNA level of human ACVR2B was determined using the RNA extraction kit (RNeasy Mini Kit), followed by the reverse transcription kit (High Capacity cDNA Reverse Transcription Kit) and real-time quantitative PCR (TaqMan Gene Expression Master Mix) detection, and the mRNA level of human ACVR2B was corrected according to the level of the GAPDH internal control gene.

[0156] The instruments involved in this experiment are shown in Table 3:

[0157] Table 3. Experimental instruments

[0158] In the real-time quantitative PCR detection, a probe Q-PCR detection experiment was used, and the primer information is shown in Table 4:

[0159] Table 4. Taqman primer information table

[0160] Result analysis method

[0161] After the Q-PCR detection experiment was completed, the corresponding Ct value was obtained according to the threshold value set by the system automatically, and the expression of a certain gene could be compared by the Ct value: comparing Ct refers to calculating the difference in gene expression by the difference between the Ct values of the internal control gene, also known as 2-△△Ct, △△Ct = [(Ct experimental group target gene-Ct experimental group internal control)-(Ct control group target gene-Ct control group internal control)]. Inhibition rate (%) = (1-remaining amount of target gene expression)*100%.

[0162] The results are expressed as the percentage of the remaining human ACVR2B mRNA expression in cells treated with RNAi agents.

[0163] Table 5 data results show that all RNAi agents of the present disclosure have high levels of on-target inhibition activity against the ACVR2B gene in Huh7 cells.

[0164] Table 5. ACVR2B inhibition activity of the RNAi agents of the present disclosure in Huh7 cells

[0165] Example 4. Inhibition activity of RNAi agents on ACVR2B in Huh7 cells

[0166] Huh7 cells were seeded in 96-well plates at about 10000 cells / well in 90 μL medium per well; cells were transfected with RNAi agents using RNAi MAX transfection reagent (ThermoFisher, 13778150) according to the instructions, 0.3 μL RNAi MAX transfection reagent was used per well, the highest concentration of RNAi agents was 20 nM, 5-fold dilution step by step, a total of 7 concentration points were set. After transfection for 24 h, total RNA extraction, RNA reverse transcription experiment and quantitative real-time PCR detection were performed using high-throughput cell RNA extraction kit, mRNA level of human ACVR2B was detected, and mRNA level of human ACVR2B was corrected according to GAPDH internal reference gene level. Primer information is shown in Table 4.

[0167] After the Q-PCR detection experiment was completed, the corresponding Ct value was obtained according to the threshold value set by the system automatically, the expression of a certain gene could be compared by Ct value, and the expression of a certain gene could be compared by Ct value: comparing Ct refers to calculating the difference of gene expression by the difference between the Ct value of the internal reference gene, also known as 2-△△Ct, △△Ct = [(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group target gene-Ct control group internal reference)]. According to the mRNA residual rate of 7 concentration points, the dose-effect curve was fitted, and the half inhibition concentration (IC 50 ) was calculated. The results are shown in Table 6.

[0168] Table 6. Inhibition activity of RNAi agents on ACVR2B in Huh7 cells

Claims

1. An RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 7; the antisense strand comprising at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide set forth in any one of SEQ ID NO: 83 to SEQ ID NO:

89.

2. The RNAi agent of claim 1, wherein: the sense strand comprises at least 17 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 7; the antisense strand comprises at least 17 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide set forth in any one of SEQ ID NO: 83 to SEQ ID NO: 89; preferably, the sense strand comprises at least 19 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 7; preferably, differing by no more than 1 nucleotide; and / or, the antisense strand comprises at least 21 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NO: 83 to SEQ ID NO: 89; preferably, differing by no more than 1 nucleotide.

3. The RNAi agent of claim 1 or 2, comprising the sense strand and the antisense strand set forth in any one of the following groups: Group 1), the sense strand set forth in SEQ ID NO: 1 and the antisense strand set forth in SEQ ID NO: 83; Group 2), the sense strand set forth in SEQ ID NO: 2 and the antisense strand set forth in SEQ ID NO: 84; Group 3), the sense strand set forth in SEQ ID NO: 3 and the antisense strand set forth in SEQ ID NO: 85; Group 4), the sense strand set forth in SEQ ID NO: 4 and the antisense strand set forth in SEQ ID NO: 86; Group 5), the sense strand set forth in SEQ ID NO: 5 and the antisense strand set forth in SEQ ID NO: 87; Group 6), the sense strand set forth in SEQ ID NO: 6 and the antisense strand set forth in SEQ ID NO: 88; and Group 7), the sense strand set forth in SEQ ID NO: 7 and the antisense strand set forth in SEQ ID NO:

89.

4. The RNAi agent of any one of claims 1 to 3, wherein at least one nucleotide in the sense strand and / or the antisense strand is a modified nucleotide.

5. The RNAi agent of claim 4, wherein: three consecutive nucleotides in the sense strand are 2'-fluoro-modified nucleotides, preferably, the 7th, 8thand 9thnucleotides of the sense strand, in the 5' end to 3' end direction, are 2'-fluoro-modified nucleotides, or, the 9th, 10thand 11thnucleotides of the sense strand, in the 5' end to 3' end direction, are 2'-fluoro-modified nucleotides. and / or, in the 5' end to 3' end direction, the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand are each independently 2'-fluoro-modified nucleotides, or, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, or 18 of the antisense strand are each independently 2'-fluoro-modified nucleotides; the nucleotides at the remaining positions of the sense strand and the antisense strand are 2'-methoxy-modified nucleotides.

6. The RNAi agent of any one of claims 1-5, wherein at least one of the phosphodiester groups of the sense strand and / or the antisense strand is a phosphodiester group with a modifying group, preferably a phosphorothioate group.

7. The RNAi agent of claim 6, wherein the phosphodiester group with a modifying group is present at at least one of the following positions: between the 1st and 2nd nucleotides at the 5' terminal end of the sense strand; between the 2nd and 3rd nucleotides at the 5' terminal end of the sense strand; between the 1st and 2nd nucleotides at the 3' terminal end of the sense strand; between the 2nd and 3rd nucleotides at the 3' terminal end of the sense strand; between the 1st and 2nd nucleotides at the 5' terminal end of the antisense strand; between the 2nd and 3rd nucleotides at the 5' terminal end of the antisense strand; between the 1st and 2nd nucleotides at the 3' terminal end of the antisense strand; between the 2nd and 3rd nucleotides at the 3' terminal end of the antisense strand; Preferably, the sense strand and / or the antisense strand comprises a plurality of phosphorothioate groups, which are present at: between the 1st and 2nd nucleotides at the 5' terminal end of the sense strand; and between the 2nd and 3rd nucleotides at the 5' terminal end of the sense strand; and between the 1st and 2nd nucleotides at the 3' terminal end of the sense strand; and between the 2nd and 3rd nucleotides at the 3' terminal end of the sense strand; and between the 1st and 2nd nucleotides at the 5' terminal end of the antisense strand; and between the 2nd and 3rd nucleotides at the 5' terminal end of the antisense strand; and between the 1st and 2nd nucleotides at the 3' terminal end of the antisense strand; and between the 2nd and 3rd nucleotides at the 3' terminal end of the antisense strand.

8. The RNAi agent of any one of claims 1-7, wherein, the sense strand comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 165 to SEQ ID NO: 178; and / or, the antisense strand comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 253 to SEQ ID NO:

266.

9. The RNAi agent of any one of claims 1-8, further comprising one or more delivery groups attached to the sense strand and / or the antisense strand; Preferably, the delivery group comprises a lipophilic group; More preferably, the delivery group comprises a saturated or unsaturated C 22 hydrocarbon chain.

10. A pharmaceutical composition, comprising: The RNAi agent of any one of claims 1-9, and a pharmaceutically acceptable carrier.

11. A cell comprising the RNAi agent of any one of claims 1-9.

12. A kit comprising the RNAi agent of any one of claims 1-9 and / or the pharmaceutical composition of claim 10.

13. A method of reducing ACVR2B gene expression, comprising administering to a subject an effective amount or dose of the RNAi agent of any one of claims 1-9 and / or the pharmaceutical composition of claim 10.

14. A method of treating and / or preventing a disease in a subject, comprising administering to the subject an effective amount or dose of the RNAi agent of any one of claims 1-9 and / or the pharmaceutical composition of claim 10. Preferably, the disease is a metabolic disorder disease or a cancer. More preferably, the metabolic disorder disease is obesity, type 2 diabetes, inflammation, cardiovascular disease, pulmonary disease, liver disease, fatty liver, nervous system disease, kidney disease, muscle disorder, fibrotic disease, neurological disease.

15. A method of delivering an RNAi agent that inhibits ACVR2B expression and / or replication in vivo, the method comprising administering to a subject the RNAi agent of any one of claims 1-9 and / or the pharmaceutical composition of claim 10.

16. A method of making an RNAi agent comprising: synthesizing the RNAi agent of any one of claims 1-9.

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