ACVR2a-targeting rnai agent and medical use thereof

Abstract

The present invention relates to an ACVR2A-targeting RNAi agent and medical use thereof. Specifically, the present invention relates to an ACVR2A-targeting RNAi agent, a pharmaceutical composition comprising same, and medical use thereof.

Description

RNAi agents targeting ACVR2A and medical uses thereof TECHNICAL FIELD

[0001] The present disclosure belongs to the field of biological medicine, and particularly relates to RNAi agents targeting ACVR2A gene, compositions comprising the same and medical uses thereof. BACKGROUND

[0002] Activin A receptor type 2A (also known as ACVR2A or ACTRIIA) is a type II activin receptor that mediates the function of activin. Its main ligands include activin A, activin B and inhibin A, and myostatin.

[0003] Generally, after the corresponding activin ligand binds to ACVR2A, it further binds to type I receptors to trigger the SMAD pathway for signal transduction. At this time, the ACVR2A receptor is phosphorylated and activates the autophosphorylated type I receptor. ACVR2A can form a complex with different type I receptors to activate different SMAD signaling pathways, such as signaling to Smad2 / 3 (ALK4) or Smad1 / 5 / 8 (ALK2, ALK3, ALK6).

[0004] ACVR2A plays a role in metabolic disorders, muscle diseases and cancer. Inhibition of ACVR2A can regulate metabolism, improve conditions such as overweight and obesity, and treat muscle disorders, fibrotic diseases, inflammatory diseases, skeletal diseases, eye diseases, neurological diseases, metabolic diseases and cancer. This paper aims to provide nucleotide drugs that inhibit ACVR2A, which can effectively inhibit the expression of ACVR2A, and provide a new optional way for the treatment of the above diseases. SUMMARY

[0005] The present disclosure provides an RNAi agent targeting ACVR2A.

[0006] In one aspect, the present disclosure provides an RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises 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: 84; the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide set forth in any one of SEQ ID NO: 85 to SEQ ID NO: 168.

[0007] In some embodiments, the "differ by no more than 3 nucleotides" means that there can be 0, 1, 2, 3 nucleotide differences; in some embodiments, the "at least 15 contiguous" means at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides; in some embodiments, at least 17, 18, 19, 20, or 21 contiguous nucleotides.

[0008] 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.

[0009] 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.

[0010] In some embodiments, the RNAi agent of the disclosure comprises one or two blunt ends.

[0011] In some embodiments, the sense strand and / or the antisense strand of the RNAi agent of the 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.

[0012] 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.

[0013] In some embodiments, the sense strand and the antisense strand 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 ratio of the length of the sense strand to the antisense strand of the RNAi agent provided in the disclosure can be 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 some embodiments, the ratio of the length of the sense strand to the antisense strand of the RNAi agent is 19 / 19, 19 / 21, 21 / 21, 21 / 23, 23 / 23, or 23 / 25. In some embodiments, the ratio of the length of the sense strand to the antisense strand is 19 / 21, 21 / 21, or 21 / 23.

[0014] In some embodiments, the sense strand comprises or consists of the nucleotide sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 84; and / or the antisense strand comprises or consists of the nucleotide sequence set forth in any one of SEQ ID NO: 85 to SEQ ID NO: 168.

[0015] In some embodiments, the sense strand has the nucleotide sequence as set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 84; and / or the antisense strand has the nucleotide sequence as set forth in any one of SEQ ID NO: 85 to SEQ ID NO: 168.

[0016] In some embodiments, the RNAi agent comprises or consists of the sense strand and the antisense strand of any one of the following groups:

[0017] Group 1), the sense strand as set forth in SEQ ID NO: 1 and the antisense strand as set forth in SEQ ID NO: 85; or the sense strand as set forth in SEQ ID NO: 2 and the antisense strand as set forth in SEQ ID NO: 86;

[0018] Group 2), the sense strand as set forth in SEQ ID NO: 3 and the antisense strand as set forth in SEQ ID NO: 87;

[0019] Group 3), the sense strand as shown in SEQ ID NO: 4 and the antisense strand as shown in SEQ ID NO: 88; or the sense strand as shown in SEQ ID NO: 5 and the antisense strand as shown in SEQ ID NO: 89;

[0020] Group 4), the sense strand as shown in SEQ ID NO: 6 and the antisense strand as shown in SEQ ID NO: 90;

[0021] Group 5), the sense strand as shown in SEQ ID NO: 7 and the antisense strand as shown in SEQ ID NO: 91, or the sense strand as shown in SEQ ID NO: 8 and the antisense strand as shown in SEQ ID NO: 92;

[0022] Group 6), the sense strand as shown in SEQ ID NO: 9 and the antisense strand as shown in SEQ ID NO: 93;

[0023] Group 7), the sense strand as shown in SEQ ID NO: 10 and the antisense strand as shown in SEQ ID NO: 94; or the sense strand as shown in SEQ ID NO: 11 and the antisense strand as shown in SEQ ID NO: 95.

[0024] In some embodiments, at least one of the nucleotides 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.

[0025] In some embodiments, the sense strand contains three consecutive 2'-fluoro modified nucleotides. 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-fluoro modified nucleotides. 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-fluoro modified nucleotides. In some embodiments, the non-fluoro modified nucleotides are 2'-methoxy modified nucleotides.

[0026] In some embodiments, the antisense strand contains 5 2’-fluoro-modified nucleotides. In some embodiments, the nucleotides at positions 2, 6, 12, 14, and 16 in the antisense strand, in the direction from the 5’ end to the 3’ end, are each independently 2’-fluoro-modified nucleotides, and the nucleotides at the remaining positions are non-2’-fluoro-modified nucleotides. In some embodiments, the antisense strand contains 8 2’-fluoro-modified nucleotides. In some embodiments, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, or 18 in the antisense strand, in the direction from the 5’ end to the 3’ end, are each independently 2’-fluoro-modified nucleotides, and the nucleotides at the remaining positions are non-2’-fluoro-modified nucleotides. In some embodiments, the non-fluoro-modified nucleotides are 2’-methoxy-modified nucleotides.

[0027] In some embodiments, at least one of the phosphodiester groups in the sense strand and / or the antisense strand is a phosphodiester group having 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 in the sense strand and / or the antisense strand include a modification group. In some embodiments, a plurality of the phosphodiester groups in both the sense strand and the antisense strand include a modification group. In some embodiments, the sense strand contains 4 phosphodiester groups having a modification group, and the antisense strand contains 4 phosphodiester groups having a modification group. In some embodiments, the phosphodiester group having 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’-end and / or 3’-end of the sense strand, and / or between any two adjacent nucleotides from the 1st to the 4th nucleotide at the 5’-end and / or 3’-end of the antisense strand. For example, the phosphodiester group having a modification group can be located between the 1st and 2nd nucleotides at the 5’-end of the sense strand;

[0028] between the 2nd and 3rd nucleotides at the 5’-end of the sense strand;

[0029] between the 1st and 2nd nucleotides at the 3’-end of the sense strand;

[0030] between the 2nd and 3rd nucleotides at the 3’-end of the sense strand;

[0031] between the 1st and 2nd nucleotides at the 5’-end of the antisense strand;

[0032] between the 2nd and 3rd nucleotides at the 5’-end of the antisense strand;

[0033] between the 1st and 2nd nucleotides of the 5' terminus end of the antisense strand; and

[0034] between the 2nd and 3rd nucleotides of the 3' terminus end of the antisense strand.

[0035] In some embodiments, the phosphodiester group with a modifying group is a phosphorothioate group.

[0036] In some embodiments, the sense strand and antisense strand are selected from or comprise a sense strand and an antisense strand as set forth in any one of Table 2.

[0037] In some embodiments, the sense strand is selected from or comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 169 to SEQ ID NO: 285.

[0038] In some embodiments, the antisense strand is selected from or comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 286 to SEQ ID NO: 402. In some embodiments, the RNAi agent further comprises one or more delivery groups attached to the sense strand and / or the antisense strand. 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 ACVR2A gene expression is present.

[0039] In some embodiments, the delivery groups are lipophilic groups, the one or more lipophilic groups are 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.

[0040] In some embodiments, the lipophilic groups are attached to the base of a nucleotide; in some embodiments, the lipophilic groups are attached to the sugar ring of a nucleotide; in some embodiments, the lipophilic groups are attached to the internucleoside linkage between two adjacent nucleotides. In some embodiments, the lipophilic groups comprise 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.

[0041] In some embodiments, the lipophilic groups comprise a saturated or unsaturated C 6-22hydrocarbon chain; in some embodiments, the lipophilic group comprises a saturated or unsaturated C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , or C 22 hydrocarbon chain. In some specific embodiments, the lipophilic group comprises a saturated or unsaturated C 16 , C 18 , or C 22 hydrocarbon chain.

[0042] In some embodiments, the delivery group 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 cluster of galactose or a cluster of a galactose derivative selected from N-acetyl-galactosamine (GalNAc), N-trifluoroacetyl galactosamine, N-propionyl galactosamine, N-n-butyryl galactosamine, or N-isobutyryl galactosamine.

[0043] In some embodiments, the delivery group is linked to the 3’ end of the sense strand of the RNAi agent. In some embodiments, the delivery group is linked to the 5’ end of the sense strand of the RNAi agent.

[0044] In some embodiments, the delivery group is linked to the end of the RNAi agent via a phosphodiester group, a phosphorothioate group, or a phosphonate group; in some embodiments, via a phosphodiester group.

[0045] In some embodiments, the delivery group is indirectly linked to the end of the RNAi agent via a phosphodiester group, a phosphorothioate group, or a phosphonate group; in some embodiments, via a phosphodiester group.

[0046] In some embodiments, the delivery group is directly linked to the end of the RNAi agent via a phosphodiester group, a phosphorothioate group, or a phosphonate group; in some embodiments, via a phosphodiester group.

[0047] In some embodiments, the delivery group is directly linked to the 3' or 5' end of the sense strand of the RNAi agent via a phosphodiester group or a phosphorothioate group; in some embodiments, via a phosphodiester group. In another aspect, the disclosure provides a pharmaceutical composition comprising an RNAi agent described herein, and one or more pharmaceutically acceptable excipients, such as vehicles, carriers, diluents, and / or delivery polymers. Various drug delivery systems are known and can be used with the RNAi agents of the 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.

[0048] In some embodiments, the pharmaceutical composition can further comprise a pharmaceutically acceptable excipient and / or adjuvant, which can be one or more of various formulations 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. In the context of the present disclosure, the term "pharmaceutical composition" means a mixture of one or more of the compounds described herein or physiologically acceptable salts or prodrugs thereof with other chemical components, such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of an active ingredient to an organism and to facilitate absorption of the active ingredient into the organism in order to exhibit the biological activity.

[0049] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg to 1000 mg.

[0050] In certain embodiments, the pharmaceutical composition contains 0.01 to 99.99% of the aforementioned RNAi agent or a pharmaceutically acceptable salt thereof or an isotopically substituted version thereof, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1 to 99.9% of the aforementioned RNAi agent or a pharmaceutically acceptable salt thereof or an isotopically substituted version thereof. In certain embodiments, the pharmaceutical composition contains 0.5 to 99.5% of the aforementioned RNAi agent or a pharmaceutically acceptable salt thereof or an isotopically substituted version thereof. In certain embodiments, the pharmaceutical composition contains 1 to 99% of the aforementioned RNAi agent or a pharmaceutically acceptable salt thereof or an isotopically substituted version thereof. In certain embodiments, the pharmaceutical composition contains 2 to 98% of the aforementioned RNAi agent or a pharmaceutically acceptable salt thereof or an isotopically substituted version thereof.

[0051] In certain embodiments, the pharmaceutical composition contains 0.01% to 99.99% of a pharmaceutically acceptable excipient, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1% to 99.9% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 0.5% to 99.5% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 1% to 99% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 2% to 98% of a pharmaceutically acceptable excipient.

[0052] 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.

[0053] In some embodiments, the RNAi agent or pharmaceutical composition of the present disclosure, when contacted to a cell expressing a target gene, causes the remaining percentage of expression of the mRNA of the target gene to be 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.

[0054] In some embodiments, the RNAi agent or pharmaceutical composition of the present disclosure, when contacted to a cell expressing the target gene, reduces the off-target activity by 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%, as determined by, for example: psiCHECK activity screening and luciferase reporter assay, other such as PCR or branched DNA (bDNA)-based methods, or protein-based methods, such as immunofluorescence assays, for example Western Blot, or flow cytometry, while maintaining the target activity.

[0055] In some embodiments, the RNAi agent or pharmaceutical composition of the present disclosure, when contacted to a cell expressing the target gene, reduces the off-target activity by 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%, as determined by, for example: psiCHECK activity screening and luciferase reporter assay, other such as PCR or branched DNA (bDNA)-based methods, or protein-based methods, such as immunofluorescence assays, for example Western Blot, or flow cytometry, 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%.

[0056] In some embodiments, the RNAi agent or pharmaceutical composition of the present disclosure, when contacted to a cell expressing the target gene, reduces the off-target activity by 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%, as determined by, for example: psiCHECK activity screening and luciferase reporter assay, other such as PCR or branched DNA (bDNA)-based methods, or protein-based methods, such as immunofluorescence assays, for example Western Blot, or flow cytometry, while increasing the target activity by 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%.

[0057] In another aspect, the present disclosure provides a method of reducing ACVR2A expression, comprising administering to a subject the RNAi agent and / or pharmaceutical composition of the present disclosure.

[0058] In another aspect, the present disclosure provides a method of treating and / or preventing a disease associated with ACVR2A gene expression in a subject, comprising administering to the subject an RNAi agent and / or a pharmaceutical composition of the present disclosure.

[0059] In another aspect, the present disclosure provides use of an RNAi agent and / or a pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating and / or preventing a disease associated with ACVR2A gene expression.

[0060] In another aspect, the present disclosure provides use of an RNAi agent and / or a pharmaceutical composition of the present disclosure in the manufacture of a medicament for inhibiting expression of ACVR2A.

[0061] In some embodiments, the disease is a disease associated with ACVR2A. In some embodiments, the disease is a metabolic disorder disease, including type 2 diabetes, inflammation, cardiovascular disease, lung disease, liver disease such as fatty liver, nervous system disease, kidney disease, muscle disorder, fibrosis disease, neurological disease, etc. caused by metabolic disorder.

[0062] In another aspect, the present disclosure provides a method of delivering an RNAi agent that inhibits expression and / or replication of ACVR2A in vivo, the method comprising administering to a subject an RNAi agent and / or a pharmaceutical composition of the present disclosure.

[0063] 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 the subject a therapeutically effective amount of an RNAi agent or a pharmaceutical composition of the present disclosure, the RNAi agent linked to the delivery moiety, thereby inhibiting expression of the target mRNA in the subject.

[0064] In some embodiments, the subject has been identified as having a pathological upregulation of the target gene in the targeted cell or tissue prior to administration of the RNAi agent and / or pharmaceutical composition of the present disclosure.

[0065] 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.

[0066] 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 known in the art.

[0067] In an alternative embodiment, the pharmaceutical composition provided by the present disclosure can be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal injection.

[0068] In an alternative embodiment, when the targeting ligand is linked to the RNAi agent to form a conjugate, the conjugate can be packaged in a kit.

[0069] In another aspect, the present disclosure also provides a pharmaceutical composition comprising the RNAi agent of the present disclosure.

[0070] 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 preparations or compounds conventionally employed in the art. For example, the pharmaceutically acceptable excipient can include at least one of a pH buffer, a protective agent, and an osmotic pressure adjusting agent.

[0071] In another aspect, the present disclosure provides a cell comprising the RNAi agent of the present disclosure.

[0072] In another aspect, the present disclosure provides a kit comprising the RNAi agent and / or the pharmaceutical composition of the present disclosure. In an alternative embodiment, when the delivery group is linked to the RNAi agent to form a conjugate, the conjugate can be packaged in a kit.

[0073] The present disclosure also provides a method for silencing mRNA of a target gene in a cell, the method comprising the step of introducing the RNAi agent and / or the pharmaceutical composition of the present disclosure into the cell.

[0074] 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 introducing the RNAi agent and / or the pharmaceutical composition according to the present disclosure into the cell.

[0075] 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 the RNAi agent and / or the pharmaceutical composition according to the present disclosure.

[0076] 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.

[0077] In some embodiments, the target gene is an ACVR2A gene, and the target mRNA is mRNA expressed by the target gene.

[0078] The present disclosure also provides a method of preparing an RNAi agent, comprising: synthesizing an RNAi agent described in the present disclosure.

[0079] In the context of the present disclosure, the "compounds" include the RNAi agents of the present disclosure, and pharmaceutically acceptable salts of the compounds are selected from inorganic or organic salts, and the compounds of the present disclosure can be reacted with acidic or basic substances to form the corresponding salts.

[0080] On the other hand, without specifying the configuration, the compounds of the present disclosure include the RNAi agents of the present disclosure can exist in specific 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 the racemic mixtures and other mixtures thereof, such as those that are enantiomeric or diastereomeric pure, all falling within the scope of the present disclosure. Additional asymmetric carbon atoms can be present in a substituent group such as an alkyl group. All such isomers, as well as mixtures thereof, are included within the scope of the present disclosure.

[0081] In addition, without specifying the configuration, the compounds of the present disclosure include the RNAi agents and intermediates of the present disclosure can also exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The term "tautomeric" or "tautomeric forms" refers to different energy structures that are interconvertible via a low energy barrier.

[0082] The compounds of the present disclosure include the RNAi agents of the present disclosure can be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers, both optical and stereoisomeric, are included. Compounds of the present disclosure having an asymmetric carbon atom 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.

[0083] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound disclosed herein, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the desired enantiomer in pure form. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., from amines to carbamates).

[0084] This disclosure also includes compounds of this disclosure that are identical to those described herein, but in which one or more atoms are replaced by isotopes with atomic weights or mass numbers different from those commonly found in nature, including the RNAi agents of this disclosure. Examples of isotopes that can bind to the compounds of this disclosure, including the RNAi agents of this disclosure, include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, 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, etc.

[0085] Without specifying the configuration, in the chemical structure of this disclosure, the bonds are... This indicates that the configuration is not specified; that is, if chiral isomers exist in the chemical structure, the bond... It can be or Or simultaneously include and 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... The configuration is not specified, i.e., the configuration of the bond The configuration can be E or Z, or both E and Z.

[0086] Terminology

[0087] For the purposes of the present disclosure, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in the present disclosure have the meanings that are commonly understood by one of ordinary skill in the art in the field of the present disclosure.

[0088] As used herein, "RNAi agent" (also referred to as "RNAi trigger") means an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that contains a sequence that is capable of degrading or inhibiting (e.g., under appropriate conditions) the translation of a messenger RNA (mRNA) transcript in a sequence-specific manner. As used herein, an RNAi agent can act through an RNA interference mechanism (i.e., by interacting with the machinery of the RNA interference pathway of a mammalian cell (the RNA-induced silencing complex or RISC) to induce RNA interference) or through any alternative mechanism or pathway. While the term RNAi agent as used herein is believed to act primarily through an RNA interference mechanism, the disclosed RNAi agents are not bound or limited to any particular mechanism or pathway of action. The RNAi agents disclosed herein consist of a sense strand and an antisense strand, and include, but are not limited to, short (or small) interfering RNA (siRNA). The antisense strand of the RNAi agent described herein is at least partially complementary to the targeted mRNA. The RNAi agent can comprise one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0089] Unless otherwise specified, in the context of the present disclosure, ACVR2A includes, but is not limited to, human ACVR2A, cynomolgus monkey ACVR2A, mouse ACVR2A, rat ACVR2A, the amino acid and complete coding sequences, mRNA sequences of which are readily available using publicly available databases, e.g., GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0090] The term "ACVR2A" also refers to naturally occurring DNA sequence variations of the ACVR2A gene, such as single nucleotide polymorphisms (SNPs) in the ACVR2A gene. Exemplary SNPs can be found in the dbSNP database.

[0091] The term "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of ACVR2A, including mRNA that is a product of RNA processing 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 ACVR2A. 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 complementary to the sequence of the target mRNA.

[0092] In the context of describing the sense strand of the RNAi agents described herein, the term "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: 11" 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 to SEQ ID NO: 11 as 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 to SEQ ID NO: 11 as set forth in SEQ ID NO: 1. Similar interpretation should be made for other similar descriptions in the context of the disclosure. The disclosure does not include the case where the "differing" nucleotides comprise different modifications, i.e., nucleotides having the same base but different modifications are not considered as "differing" nucleotides in the context of the disclosure. Similar interpretation should be made for other similar descriptions of the sense strand and / or antisense strand in the context of the disclosure.

[0093] In the context of the disclosure, the "5' region" of the sense strand or antisense strand, i.e., "5' end", "5' terminal", can be used interchangeably. For example, the nucleotides at positions 2 to 8 of the 5' region of the antisense strand can be used interchangeably with the nucleotides at positions 2 to 8 of the 5' end of the antisense strand. Similarly, the "3' region", "3' terminal", and "3' end" of the sense strand or antisense strand can be used interchangeably.

[0094] As used herein, "G," "C," "A," "T," and "U" represent nucleotides comprising the bases guanine, cytosine, adenine, thymine, and uracil, respectively, unless otherwise specified. As is well known in the art, the substitution of base T for U does not significantly affect the properties of an RNAi agent sequence, and sequences of the disclosure can have U replaced by T at any position, and the resulting sequence is within the scope of the disclosure. In the sequences of the disclosure, for a given nucleic acid strand, the direction from left to right is from the 5' end to the 3' end, and, unless otherwise specified, the lower case letter m indicates that the nucleotide immediately to the left of the letter m is a 2'-methoxy modified nucleotide; the lower case letter f indicates that the nucleotide immediately to the left of the letter f is a 2'-fluoro modified nucleotide; and the lower case letter s indicates that the two nucleotides immediately flanking the letter s are linked by a phosphorothioate linkage, and, unless otherwise specified, the two nucleotides are linked by a phosphodiester linkage. As used herein, "RNAi agent," "nucleotide," "compound," "chemical modification," "oligonucleotide," "double-stranded RNAi inhibitor molecule," "siRNA," "dsRNA," "nucleic acid," and "RNAi" can each be present independently as a salt, a mixed salt, or a non-salt (e.g., free acid or free base), unless otherwise specified. When present as a salt or a mixed salt, it can be a pharmaceutically acceptable salt. As used herein, "compound" includes an RNAi agent of the disclosure. The term "pharmaceutically acceptable salt" includes both pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. When present as a salt, some groups can ionize to form an anion / cation, e.g., a phosphodiester group can be present as an anion, and, unless otherwise specified, the following structures also represent the salt forms of the structures. As used herein, unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each strand is a hydroxyl group; and the 5' position of the first nucleotide at the 5' end of each strand is a hydroxyl group.

[0095] The above modifications and linkers have the following structures, respectively, as shown in Table 1, wherein Base represents the base at the corresponding position, and, unless otherwise specified, the following structures also represent the salt forms of the structures.

[0096] Table 1.

[0097] The term "lipophilic group" or "lipophilic moiety" broadly refers to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient, logK ow where K ow is the ratio of the concentration of a chemical in the octanol phase to its concentration in the water phase of a two-phase system at equilibrium. In principle, logK owA chemical substance is lipophilic when the logK ow exceeds 1, exceeds 1.5, exceeds 2, exceeds 3, exceeds 4, exceeds 5, or exceeds 10, such as the logK ow of cholesteryl N-(hexan-6-ol)carbamate is about 0.7. ow

[0098] The lipophilicity of a molecule can vary with respect 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 .

[0099] 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 the adenine on one strand always pairs with the thymine (or uracil) on the other strand, and the guanine always pairs with the 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.

[0100] ​As used herein, the term "inhibit," can be used interchangeably with "reduce," "silence," "down-regulate," "repress," and other similar terms, and includes inhibition at any level. 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 rate of inhibition of target gene expression can be characterized by the fold decrease in mRNA expression, such as a decrease of no more than 2-fold, no more than 3-fold, no more than 4-fold, no more than 5-fold, no more than 6-fold, no more than 7-fold, no more than 8-fold, no more than 9-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 35-fold, no more than 40-fold, no more than 45-fold, no more than 50-fold, no more than 55-fold, no more than 60-fold, no more than 65-fold, no more than 70-fold, no more than 75-fold, no more than 80-fold, no more than 85-fold, no more than 90-fold, no more than 95-fold, or no more than 100-fold. The rate of inhibition of target gene expression can also be characterized by the fold increase in the level of a reporter gene, such as a luciferase gene, in the presence of an RNAi agent, such as no more than 2-fold, no more than 3-fold, no more than 4-fold, no more than 5-fold, no more than 6-fold, no more than 7-fold, no more than 8-fold, no more than 9-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 35-fold, no more than 40-fold, no more than 45-fold, no more than 50-fold, no more than 55-fold, no more than 60-fold, no more than 65-fold, no more than 70-fold, no more than 75-fold, no more than 80-fold, no more than 85-fold, no more than 90-fold, no more than 95-fold, or no more than 100-fold. 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 (%).

[0101] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0102] 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-2-disulfonate, and the like. These salts can be prepared by methods known in the art.

[0103] "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.

[0104] "Effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition, which is necessary to achieve a desired therapeutic result. For prophylactic use, a desired result includes eliminating or reducing a risk, lessening the severity, or delaying the onset of a disorder, including biochemical, histological, and / or behavioral symptoms of a disorder, its complications, and intermediate pathological phenotypes presenting during the course of a disorder. For therapeutic applications, a desired result includes a clinical result, such as reducing the incidence of, or ameliorating one or more symptoms of, a disorder associated with a target gene, target mRNA, or target protein of the present disclosure, reducing the dosage of other medications required to treat a disorder, enhancing the effect of another agent, and / or delaying the progression of a disorder associated with a target gene, target mRNA, or target protein of the present disclosure in a patient.

[0105] As used herein, "patient," "subject," or "individual" are used interchangeably and include a human or non-human animal, such as a mammal, e.g., a human or a monkey.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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 of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the measurement system in use / For example, "about" can mean within 20%, e.g., within 10%, e.g., within 1%, e.g., within 0.5%, e.g., within 0.1%, of the stated value. In the disclosure, each instance in which a numerical value or range of values is preceded by the term "about" also includes embodiments in which the exact numerical value is used. Unless otherwise indicated, the use of "about" or "approximately" in connection with a recited parameter means that the parameter can vary from the recited value by up to an acceptable limit for the parameter at hand, as would be understood by one of ordinary skill in the art.

[0111] 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 does not necessarily exist, 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.

[0112] In the chemical structures of the disclosure, Or Or which can be attached to one or more of any of the groups according to the scope of the invention described herein.

[0113] 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.

[0114] The term "directly connected" means that a first compound or group is connected to a second compound or group without any intervening atoms or atom groups.

[0115] The term "indirectly connected" means that a first compound or group is connected to a second compound or group through an intervening group, compound or molecule (e.g., a linker group).

[0116] 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 that 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.

[0117] "Substituted by one or more" means that a single or multiple substituents can be substituted. When substituted by multiple substituents, it can be a plurality of the same substituents or a combination of one or more different substituents. DETAILED DESCRIPTION

[0118] 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 noted, experimental methods in the Examples of the present disclosure were carried out according to conventional conditions, such as Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; or Molecular Cloning: A Laboratory Manual. Reagents for which specific sources are not identified were obtained from any of a variety of commercial suppliers of molecular biology grade reagents. Unless otherwise noted, reagents used in the following examples were commercially available.

[0119] Example 1. Design of ACVR2A RNAi agents

[0120] The sequences of the sense strand, antisense strand in the RNAi agents of the present disclosure are shown in Table 2 below, which are designed to be 19 / 21 nt of the sense strand / antisense strand according to the general rules of double-stranded oligonucleotides satisfying the activity, with the human ACVR2A gene (NM_001278579.2) as the target gene.

[0121] Table 2. Sequences of modified RNAi agents targeting the ACVR2A gene

[0122] In Table 2, in the same nucleic acid sequence, the direction from 5' end to 3' end is from left to right, G, C, A, U respectively represent nucleosides with guanine, cytosine, adenine, uracil, the sequence from left to right represents the direction from 5' end to 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 two nucleosides adjacent to the letter s are connected by phosphorothioate linkage, and no special instructions, the two adjacent nucleosides are connected by phosphodiester linkage. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each strand is hydroxyl; the 5' position of the first nucleotide at the 5' end of each strand is hydroxyl.

[0123] The 2'-methoxy modified nucleoside, 2'-fluorinated modified nucleoside, phosphorothioate, and phosphodiester structure are shown in the following table, and when the RNAi agent of the present disclosure exists in the form of a salt, for example, in the form of a sodium salt, the structure of the salt form corresponding to the structure in Table 3 is also within the protection scope of the present disclosure:

[0124] Table 3

[0125] Example 2. Synthesis of the RNAi agent of the present disclosure

[0126] The synthesis of the RNAi agent of the present disclosure is not different from the general phosphoramidite solid-phase synthesis method. The synthesis process is briefly described as follows: on a Dr. Oligo 48 synthesizer (Biolytic), using a universal CPG carrier as the starting material, according to the synthesis procedure, the unmodified nucleoside phosphoramidite monomers or the nucleoside phosphoramidite monomers with modifications at the corresponding positions in the sequence are connected 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.

[0127] After the solid phase synthesis is completed, the oligoribonucleotide is cleaved from the solid support by soaking in 28% ammonia in ethanol solution at a ratio of 3:1 at 50°C for 16 hours. Then centrifugation is performed, the supernatant is transferred to another centrifuge tube, and after concentrated evaporation, C18 reverse phase chromatography is used for purification 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 (260 nm).

[0128] The obtained single-stranded oligonucleotide is annealed according to the equimolar ratio of complementary pairing, and finally the obtained double-stranded RNAi agent is dissolved in 1xPBS and adjusted to the required concentration for the experiment.

[0129] Example 3. Double-concentration point inhibition activity of ACVR2A in human liver cancer cells (Huh7)

[0130] 24 hours before transfection, Huh7 cells were seeded in a 24-well plate at about 50000 cells / well with 500 μL of culture medium per well. At the time of transfection, Lipofectamine RNAi MAX (ThermoFisher, 13778150) was used to transfect the sample according to the product manual, and the final concentration of sample transfection was 2nM and 0.2nM. After 48 hours of treatment, the mRNA level of human ACVR2A was detected using the RNA extraction kit (RNeasy Mini Kit), and then using 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 ACVR2A was corrected according to the level of GAPDH internal control gene.

[0131] The instruments involved in this experiment are shown in Table 4:

[0132] Table 4. Experimental instruments

[0133] Among them, when real-time quantitative PCR detection is used, probe Q-PCR detection experiment is used, and the primer information is shown in Table 5:

[0134] Table 5. Taqman primer information table

[0135] Result analysis method

[0136] After the Q-PCR detection experiment is completed, the corresponding Ct value is obtained according to the threshold value set by the system, and the expression of a certain gene can be relatively quantified by comparing the Ct value: comparing Ct refers to calculating the gene expression difference by the difference between the Ct values of the reference genes, also known as 2-△△Ct, △△Ct = [(Ct experimental group target gene-Ct experimental group reference)-(Ct control group target gene-Ct control group reference)]. Inhibition rate (%) = (1-remaining amount of target gene expression) * 100%.

[0137] The results are expressed as the percentage of the remaining human ACVR2A mRNA expression relative to the cells treated with the RNAi agent.

[0138] The data results in Table 6 show that all the RNAi agents of the present disclosure have high levels of on-target inhibition activity against the ACVR2A gene in Huh7 cells.

[0139] Table 6. Percentage of ACVR2A mRNA expression remaining in Huh7 cells

[0140] Example 4. Single concentration point inhibition activity of ACVR2A in A673 cells

[0141] A673 cells were seeded in a 96-well plate at about 15000 cells / well in 90 μL of culture medium per well; the cells were transfected with the RNAi agent using the RNAi MAX transfection reagent (ThermoFisher, 13778150) according to the instructions, 0.3 μL of RNAi MAX transfection reagent was used per well, and the RNAi agent was set at a concentration of 10 nM; 24 h after transfection, total cell RNA extraction, RNA reverse transcription experiment (Takara, RR037B) and quantitative real-time PCR detection (ThermoFisher, 4444557) were performed using the high-throughput cell RNA extraction kit (ThermoFisher, A27828) to determine the mRNA level of human ACVR2A, which was corrected according to the level of the GAPDH reference gene. The primers are shown in Table 7.

[0142] Table 7. Taqman primer information table

[0143] After the Q-PCR detection experiment is completed, the corresponding Ct value is obtained according to the threshold value set by the system, and the expression of a certain gene can be relatively quantified by comparing the Ct value: comparing Ct refers to calculating the gene expression difference by the difference between the Ct values of the reference genes, 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)]. Inhibition rate (%) = (1 - residual amount of target gene expression) * 100%. The results are expressed as the percentage of the residual amount of human ACVR2A mRNA expression relative to the cells treated with dsRNA. As shown in Table 8, the results of Table 8 show that the RNAi agents of the present disclosure all have excellent inhibitory activity on human ACVR2A mRNA.

[0144] Table 8. Percentage of residual ACVR2A mRNA expression in A673 cells

[0145] Example 5. Endogenous inhibitory activity of RNAi agents in A673 cells

[0146] A673 cells were seeded in a 96-well plate at about 15000 cells / well in 90 μL of culture medium per well; the cells were transfected with RNAi agents using RNAi MAX transfection reagent (ThermoFisher, 13778150) according to the instructions, 0.3 μL of RNAi MAX transfection reagent was used per well, the highest concentration of RNAi agent 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 a high-throughput cell RNA extraction kit, the mRNA level of human ACVR2A was determined, and the mRNA level of human ACVR2A was corrected according to the level of GAPDH internal reference gene. The primer information is shown in Table 6.

[0147] 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 can be compared by the Ct value: comparing Ct refers to calculating the difference in 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 residual rate of mRNA at 7 concentration points, the dose-effect curve was fitted, and the half inhibitory concentration (IC 50 ) was calculated. The results are shown in Table 9. The results of Table 9 show that the RNAi agents of the present disclosure have good inhibitory activity on ACVR2A mRNA.

[0148] Table 9. IC of RNAi agents of the present disclosure in A673 cells 50

[0149] Example 6. Endogenous inhibitory activity of RNAi agents in A673 cells

[0150] The endogenous inhibition activity of the RNAi agents of the present disclosure in A673 cells was detected in the same manner as in Example 5, and the results are shown in Table 10, which indicates that the RNAi agents of the present disclosure have good inhibition activity.

[0151] Table 10. IC50 of the RNAi agents of the present disclosure in A673 cells 50

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: 11; 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: 85 to SEQ ID NO:

95.

2. The RNAi agent of claim 1, the sense strand comprising at least 17 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: 11; the antisense strand comprising at least 17 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide set forth in any one of SEQ ID NO: 85 to SEQ ID NO: 95; preferably, the sense strand comprising at least 19 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: 11; preferably, differing by no more than 1 nucleotide; and / or, the antisense strand comprising at least 21 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence set forth in any one of SEQ ID NO: 85 to SEQ ID NO: 95; preferably, differing by no more than 1 nucleotide.

3. The RNAi agent of claim 1 or 2, comprising or selected from the sense strand and the antisense strand of 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: 85; or the sense strand set forth in SEQ ID NO: 2 and the antisense strand set forth in SEQ ID NO: 86; Group 2), the sense strand set forth in SEQ ID NO: 3 and the antisense strand set forth in SEQ ID NO: 87; Group 3), the sense strand set forth in SEQ ID NO: 4 and the antisense strand set forth in SEQ ID NO: 88; or the sense strand set forth in SEQ ID NO: 5 and the antisense strand set forth in SEQ ID NO: 89; Group 4), the sense strand set forth in SEQ ID NO: 6 and the antisense strand set forth in SEQ ID NO: 90; Group 5), the sense strand set forth in SEQ ID NO: 7 and the antisense strand set forth in SEQ ID NO: 91, or the sense strand set forth in SEQ ID NO: 8 and the antisense strand set forth in SEQ ID NO: 92; Group 6), the sense strand set forth in SEQ ID NO: 9 and the antisense strand set forth in SEQ ID NO: 93; and Group 7), the sense strand set forth in SEQ ID NO: 10 and the antisense strand set forth in SEQ ID NO: 94; or the sense strand set forth in SEQ ID NO: 11 and the antisense strand set forth in SEQ ID NO:

95.

4. The RNAi agent of any one of claims 1-3, 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, 8th, and 9th nucleotides of the sense strand, in the 5' end to 3' end direction, are each independently a 2'-fluoro modified nucleotide; or, the 9th, 10th, and 11th nucleotides of the sense strand, in the 5' end to 3' end direction, are each independently a 2'-fluoro modified nucleotide.

6. The RNAi agent of claim 4 or 5, wherein the antisense strand contains 5 2'-fluoro modified nucleotides, preferably, the 2nd, 6th, 12th, 14th, and 16th nucleotides of the antisense strand, in the 5' end to 3' end direction, are each independently a 2'-fluoro modified nucleotide; or, the antisense strand contains 8 2'-fluoro modified nucleotides, preferably, the 2nd, 4th, 6th, 10th, 12th, 14th, 16th, or 18th nucleotides of the antisense strand, in the 5' end to 3' end direction, are each independently a 2'-fluoro modified nucleotide.

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

8. The RNAi agent of claim 7, wherein the phosphodiester group with a modification group is present at least at one of: any two adjacent nucleotides between the 1st and 4th nucleotides of the 5' end terminal end and / or between the 1st and 4th nucleotides of the 3' end terminal end of the sense strand, and / or any two adjacent nucleotides between the 1st and 4th nucleotides of the 5' end terminal end and / or between the 1st and 4th nucleotides of the 3' end terminal end of the antisense strand; preferably, the phosphodiester group with a modification group is present at: the 1st and 2nd nucleotides and the 2nd and 3rd nucleotides of the 5' end terminal end of the sense strand; the 1st and 2nd nucleotides and the 2nd and 3rd nucleotides of the 3' end terminal end of the sense strand; the 1st and 2nd nucleotides and the 2nd and 3rd nucleotides of the 5' end terminal end of the antisense strand; and the 1st and 2nd nucleotides and the 2nd and 3rd nucleotides of the 3' end terminal end of the antisense strand; more preferably, the phosphodiester group with a modification group is a phosphorothioate group.

9. The RNAi agent of any of claims 1-8, wherein, the sense strand comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 169-192; and / or the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 286-309; Preferably, the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 169, and / or the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 286; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 170, and / or the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 287; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 171, and / or the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 288; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 172, and / or the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 289; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 173, and / or the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 290; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 174, and / or the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 291; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 175, and / or the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 292; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 176, and / or the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 293; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 177, and / or the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 294; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 178, and / or the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 295; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 179, and / or the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 296; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 180, and / or the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 297; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 181, and / or the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 298; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO: 182, and / or the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO: 299; the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 300; the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 301 ; the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 302; the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 303; the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 304; the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 305; the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 306; the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 307; the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 308; or the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO:

309.

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

11. A pharmaceutical composition comprising the RNAi agent of any one of claims 1-10, and a pharmaceutically acceptable carrier thereof.

12. A cell comprising the RNAi agent of any one of claims 1-10.

13. A kit comprising the RNAi agent of any one of claims 1-10 and / or the pharmaceutical composition of claim 11.

14. A method of reducing expression of an ACVR2A gene, comprising administering to a subject the RNAi agent of any one of claims 1-10 and / or the pharmaceutical composition of claim 11.

15. A method of treating and / or preventing a disease in a subject, comprising administering to the subject an RNAi agent of any one of claims 1-10 and / or a pharmaceutical composition of claim 11; preferably, the disease is a metabolic disorder disease; Preferably, the metabolic disorder disease is selected from the group consisting of type 2 diabetes, inflammation, cardiovascular disease, pulmonary disease, liver disease, nervous system disease, kidney disease, muscle disorder, fibrotic disease, neurological disease caused by metabolic disorder.

16. A method of making an RNAi agent comprising: synthesizing an RNAi agent of any one of claims 1-10 or a pharmaceutical composition of claim 11.

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