SiRNA for inhibiting acvr2a and use thereof

Abstract

The present application relates to siRNA for inhibiting ACVR2A and application thereof, and belongs to the technical field of biotechnology.The present application provides siRNA for inhibiting ACVR2A gene expression, research shows that the siRNA all has high inhibitory activity to the expression of ACVR2A gene and protein.The present application also provides modified siRNA for inhibiting ACVR2A gene expression, the modification includes 2'-methoxy modification, 2'-fluorine modification, phosphorothioate group connection, reverse baseless nucleotide modification, deoxyribonucleotide substitution and / or vinyl phosphate modification, research shows that the modified siRNA all has good ACVR2A inhibitory effect at the concentration of 1nM-10nM.

Description

Cross-references to related applications

[0001] This application claims priority to Chinese Patent Application No. 202510233594.9, filed on February 28, 2025, entitled "siRNA for inhibiting ACVR2A and its modifications and applications," the entire contents of which are incorporated herein by reference. This application also claims priority to Chinese Patent Application No. 202511378959.3, filed on September 25, 2025, entitled "siRNA for inhibiting ACVR2A and its modifications and applications," the entire contents of which are incorporated herein by reference. Finally, this application claims priority to Chinese Patent Application No. 202511378418.0, filed on September 25, 2025, entitled "siRNA for inhibiting ACVR2A and its modifications and applications," the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to siRNA for inhibiting ACVR2A and its applications, belonging to the field of biotechnology. Background Technology

[0003] Activins are dimeric growth and differentiation factors belonging to the transforming growth factor-β (TGF-β) superfamily of structure-associated signaling proteins. Activins transmit signals via heteromeric complexes of receptor serine kinases, comprising at least two type I (I and IB) receptors and two type II (II and IIB) receptors. These receptors are transmembrane proteins, consisting of an extracellular domain for ligand binding to a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain predicting serine / threonine specificity. Type I receptors are essential for signal transduction, while type II receptors are essential for ligand binding and type I receptor expression. Upon ligand binding, type I and type II receptors form a stable complex, leading to phosphorylation of type I receptors by type II receptors.

[0004] Activin receptor type 2A (ACVR2A) is a key type II receptor mediating activin function and various other biological activities. Together with another subtype, ACVR2B (ActRIIB), it forms the core type II receptor of the activin signaling pathway. ACVR2A is expressed in different tissues and cell types, and its dysfunction is associated with various diseases such as cancer, osteoporosis, and neurodegenerative diseases. Therefore, ACVR2A and its related signaling pathways are important targets for the development of drugs to treat these diseases.

[0005] Small interfering RNA (siRNA) is an emerging and highly promising candidate for research. Based on RNA interference mechanisms, it can inhibit or block the expression of target genes of interest in a sequence-specific manner, thereby achieving the goal of treating diseases. Specifically, siRNA is loaded into the RNA-induced silencing complex (RISC). Its guide strand, also known as the antisense strand, pairs complementaryly with the target nucleic acid in the mRNA of the target gene, causing the mRNA of the target gene to degrade, thereby inhibiting or blocking the expression of the target gene. Since siRNA recognizes the target RNA sequence-specifically through base pairing, thereby achieving the cleavage of the target RNA and downregulating the level of the target RNA and inhibiting the expression of the target gene, developing siRNA that can effectively inhibit the expression of the ACVR2A gene would be a more effective and targeted treatment for pathological conditions or diseases caused by the expression of the ACVR2A gene. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides an siRNA for inhibiting ACVR2A gene expression, wherein the siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region; the sense strand of the siRNA comprises at least 15 consecutive nucleotides, with any one of the nucleic acid sequences shown in any of the nucleic acid molecules represented by SEQ ID NO.1~SEQ ID NO.147 or SEQ ID NO.295~SEQ ID NO.391 differing by no more than 3 nucleotides; the antisense strand of the siRNA comprises at least 15 consecutive nucleotides, with any one of the nucleic acid sequences shown in any of the nucleic acid molecules represented by SEQ ID NO.148~SEQ ID NO.294 or SEQ ID NO.392~SEQ ID NO.488 differing by no more than 3 nucleotides.

[0007] In one embodiment of the present invention, the nucleotide sequence of the sense strand of the siRNA is shown in any one of SEQ ID NO.1~SEQ ID NO.147 or SEQ ID NO.295~SEQ ID NO.391; and the nucleotide sequence of the antisense strand of the siRNA is shown in any one of SEQ ID NO.148~SEQ ID NO.294 or SEQ ID NO.392~SEQ ID NO.488.

[0008] In one embodiment of the present invention, at least one nucleotide in the sense strand and / or antisense strand of the siRNA is a modified nucleotide.

[0009] In one embodiment of the present invention, all nucleotides in the sense strand and / or antisense strand of the siRNA are modified nucleotides, and these modifications on the nucleotide groups do not cause a significant weakening or loss of the function of the siRNA disclosed herein in inhibiting ACVR2A gene expression.

[0010] In one embodiment of the present invention, the modified nucleotide includes compounds formed by modifying the ribose of nucleotides, compounds formed by modifying the backbone between nucleotides, compounds formed by modifying the bases on nucleotides, and / or compounds formed by modifying the ends of siRNA.

[0011] In one embodiment of the present invention, the modified nucleotide includes nucleotides modified with 2'-fluorination, nucleotides modified with 2'-methoxy groups, nucleotides linked with thiophosphate groups, nucleotides modified with reverse-nucleotide abase-free nucleotides, nucleotides substituted with deoxyribonucleotides, nucleotides modified with vinyl phosphate groups, 2'-deoxynucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, inosine nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, abase-free nucleotides, and nucleotides modified with reverse-nucleotides. Nucleotides modified with 2'-amino, 2'-O-allyl, 2'-C-alkyl, 2'-hydroxy, 2'-O-methoxyethyl, 2'-O-alkyl, morpholinonucleotide, aminophosphate, nucleotides containing non-natural bases, tetrahydropyran, 1,5-anhydrohexyl, cyclohexenyl, glycolic acid (GNA), uncyclic nucleotides (UNA), methylphosphate, 5'-phosphate, 5'-phosphate analogs, terminal nucleotides linked to cholesterol derivatives, and / or terminal nucleotides linked to dodecanoic acid bis(decyl) groups.

[0012] In one embodiment of the present invention, the 2'-fluorinated nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with fluorine, having the following structure (where Base represents a base): .

[0013] In one embodiment of the present invention, the 2'-methoxy modified nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with a methoxy group, which has the following structure (where Base represents a base): .

[0014] In one embodiment of the present invention, the nucleotides linked by thiophosphate groups refer to two adjacent nucleotides being linked by thiophosphate groups. The thiophosphate group has the following structure: ; The nucleotide linked by a thiophosphate group has the following structure (where Base represents a base): .

[0015] In one embodiment of the present invention, the nucleotide modified with a reverse abase nucleotide refers to a nucleotide coupled with a reverse abase nucleotide (invAb), wherein the reverse abase nucleotide has the following structure: .

[0016] In one embodiment of the present invention, the deoxyribonucleotide-substituted nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a hydrogen atom, which has the following structure (where Base represents a base): .

[0017] In one embodiment of the present invention, the 5' position of the sugar ring of the vinyl phosphate-modified nucleotide is modified with vinyl phosphate; the vinyl phosphate-modified nucleotide includes VPAms, VPUms, VPGms and / or VPCms (VP ​​represents (E)-vinyl phosphate). The VPAms have the following structure: ; The VPUms have the following structure: ; The VPGms has the following structure: ; The VPCms has the following structure: .

[0018] In one embodiment of the present invention, the glycol nucleic acid (GNA) modified nucleotide is a polymer similar to DNA or RNA, but its "backbone" is composed of repeating glycerol units linked by phosphodiester bonds; the glycol nucleic acid (GNA) modified nucleotide includes A (GNA), G (GNA), C (GNA), U (GNA) and / or T (GNA); The A(GNA) has the following structure: ; The G(GNA) has the following structure: ; The C(GNA) has the following structure: ; The U(GNA) has the following structure: ; The T(GNA) has the following structure: .

[0019] In one embodiment of the present invention, the 2'-fluorinated nucleotide is located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 10, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The 2'-fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The 2'-fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The 2'-fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 5th, 7th, 8th, and 9th nucleotides of the sense strand are fluorinated nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides.

[0020] In one embodiment of the present invention, the 2'-fluorinated nucleotide is located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 6, 10, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The 2'-fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The 2'-fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The 2'-fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides.

[0021] In one embodiment of the present invention, the nucleotides modified with 2'-methoxy are located in the antisense and sense strands of the nucleotide sequence, and in the sense strand, the nucleotides not modified with 2'-fluoride and the nucleotides not substituted with deoxyribonucleotides are all nucleotides modified with 2'-methoxy, and in the antisense strand, the nucleotides not modified with 2'-fluoride and the nucleotides not substituted with deoxyribonucleotides are all nucleotides modified with 2'-methoxy.

[0022] In one embodiment of the invention, the nucleotide modified with an inverse abase-free nucleotide is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 3' end of the first nucleotide in the positive strand is attached to an inverse abase-free nucleotide (invAb group); and / or, The inverse abase-free nucleotide is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of at least the penultimate nucleotide of the positive strand is attached to an inverse abase-free nucleotide (invAb group).

[0023] In one embodiment of the invention, the nucleotide modified with inverse abase nucleotide is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 3' end of the first nucleotide of the positive strand is attached to an inverse abase nucleotide (invAb group), and the 5' end of at least the penultimate nucleotide of the positive strand is attached to an inverse abase nucleotide (invAb group).

[0024] In one embodiment of the invention, the deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotide at the 12th position of the antisense strand is a deoxyribonucleotide-substituted nucleotide.

[0025] In one embodiment of the invention, the vinyl phosphate-modified nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of at least the first nucleotide of the antisense strand is attached to a vinyl phosphate group (VP group).

[0026] In one embodiment of the invention, the vinyl phosphate-modified nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of the nucleotide at position 1 of the antisense strand is attached to a vinyl phosphate group (VP group).

[0027] In one embodiment of the invention, the nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotide at position 1 of the sense strand is linked to the inverted non-base nucleotide by a thiophosphate group, and the nucleotide at position 1-1-3 is linked to the inverted non-base nucleotide by a thiophosphate group; at least the nucleotides at positions 1-2, 2-3, 1-1-2, and 2-1-3 of the antisense strand are linked by thiophosphate groups; or, Nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the first and second, and the penultimate and penultimate nucleotides of the sense strand are linked by thiophosphate groups, and at least the first and second, second and third, penultimate and penultimate, and penultimate and penultimate nucleotides of the antisense strand are linked by thiophosphate groups; or, The nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by thiophosphate groups, and at least the nucleotides at positions 1 and 2, positions 2 and 3, positions 1 to 1 and 2 to 1 ...

[0028] In one embodiment of the invention, the nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotide at position 1 of the sense strand is linked to the inverted non-base nucleotide by a thiophosphate group, and the nucleotide at position 1 to 1 is linked to the inverted non-base nucleotide by a thiophosphate group; the nucleotides at positions 1 to 2, 2 to 3, 1 to 1 and 2 to 1, and 2 to 1 to 1 in the antisense strand are linked by thiophosphate groups; or, Nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and from the 1st to the 2nd, and from the 1st to the 2nd, and from the 1st to the 2nd, and from the 1st to the 2nd, and from the 1st to the 2nd, and from the 1st to the 2nd, and from the 1st to the 2nd, and from the 2nd to the 3rd, nucleotides in the antisense strand are linked by thiophosphate groups; or, Nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence. Furthermore, in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by thiophosphate groups, and the nucleotides at positions 1 and 2, positions 2 and 3, positions 1 to 1 and 2 to 1 ...

[0029] In one embodiment of the present invention, the siRNA is truncated; the truncation includes removing 1 to 3 nucleotides starting from the 5' end of the siRNA's positive strand.

[0030] In one embodiment of the present invention, the siRNA is truncated; the truncation includes removing two nucleotides starting from the 5' end of the siRNA's positive strand.

[0031] In one embodiment of the present invention, the siRNA is coupled with a functional molecule; the functional molecule includes a lipophilic molecule, a polypeptide, a small molecule drug, an antibody, a ligand formed from a targeting molecule or a derivative of a targeting molecule; the targeting molecule includes galactose and / or N-acetylgalactosamine (GalNAc).

[0032] In one embodiment of the present invention, the conjugating group is N-glucose amino acid (N-Acetylgalactosamine, GalNAc).

[0033] In one embodiment of the present invention, the conjugation site of the siRNA and the conjugation group is located at the 3' end of the siRNA sense strand, the 5' end of the siRNA sense strand, the internal sequence of the siRNA sense strand, the 5' end of the siRNA antisense strand, or the internal sequence of the siRNA antisense strand.

[0034] In one embodiment of the present invention, the conjugation site of the siRNA and the conjugating group is located at the 3' end of the positive strand of the siRNA.

[0035] In one embodiment of the present invention, the conjugation group is conjugated to the 3' end of the siRNA positive strand via a phosphodiester bond.

[0036] In one embodiment of the present invention, the siRNA conjugate has the following structure: .

[0037] The present invention also provides a recombinant plasmid expressing the above-mentioned siRNA.

[0038] In one embodiment of the present invention, the vector of the recombinant plasmid includes at least one of a viral vector or a non-viral vector; the viral vector includes at least one of a flavivirus vector, a retrovirus vector, a bacteriophage vector, adenovirus vector, adeno-associated virus vector, vaccinia virus vector, hybrid virus vector, baculovirus vector, herpes simplex virus vector, or lentivirus vector; the non-viral vector includes a plasmid vector.

[0039] In one embodiment of the present invention, the recombinant plasmid is prepared by: designing shRNA based on siRNA; and ligating the shRNA with a linearized vector to obtain the recombinant plasmid.

[0040] The present invention also provides a host cell whose genome is integrated with the above-mentioned siRNA; or, the host cell carries the above-mentioned recombinant plasmid.

[0041] In one embodiment of the present invention, the host cell includes fungi, bacteria, plant cells and / or animal cells.

[0042] The present invention also provides the use of the above-mentioned siRNA, the above-mentioned recombinant plasmid or the above-mentioned host cell in the preparation of medicaments for the prevention and / or treatment of pathological conditions or diseases caused by ACVR2A gene expression.

[0043] In one embodiment of the present invention, the pathological conditions or diseases caused by ACVR2A gene expression include cancer, osteoporosis and / or neurodegenerative diseases.

[0044] The present invention also provides a medicament for preventing and / or treating pathological conditions or diseases caused by ACVR2A gene expression, wherein the medicament comprises the above-mentioned siRNA, the above-mentioned recombinant plasmid and / or the above-mentioned host cell.

[0045] In one embodiment of the present invention, the pathological conditions or diseases caused by ACVR2A gene expression include cancer, osteoporosis and / or neurodegenerative diseases.

[0046] In one embodiment of the present invention, the drug further comprises pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include drug carriers, pH buffers, lyophilization protectants, osmotic pressure regulators, excipients, stabilizers and / or preservatives.

[0047] In one embodiment of the present invention, the drug carrier includes magnetic nanoparticles (such as Fe3O4 or Fe2O3-based nanoparticles), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), and poly(2-aminoethyl ethylene phosphate). One or more of phosphate), PPEEA and poly(2-dimethylaminoethylmethacrylate) (PDMAEMA) and its derivatives.

[0048] In one embodiment of the present invention, the pH buffer includes a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.5 to 8.5 and / or a phosphate buffer with a pH of 5.5 to 8.5.

[0049] In one embodiment of the present invention, the protective agent includes one or more of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose.

[0050] In one embodiment of the present invention, the osmotic pressure regulator includes sodium chloride and / or potassium chloride.

[0051] The technical solution of this invention has the following advantages: This invention provides an siRNA for inhibiting ACVR2A gene expression, wherein the siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region; the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO.1~SEQ ID NO.147 or SEQ ID NO.295~SEQ ID NO.391; the antisense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO.148~SEQ ID NO.294 or SEQ ID NO.392~SEQ ID NO.488. Studies have shown that the siRNA exhibits high inhibitory activity against the expression of the ACVR2A gene and protein. Therefore, the siRNA shows great promise in the preparation of drugs for the prevention and / or treatment of pathological conditions or diseases (e.g., cancer, osteoporosis, and / or neurodegenerative diseases) caused by ACVR2A gene expression.

[0052] Furthermore, at least one nucleotide in the sense and / or antisense strands of the siRNA is a modified nucleotide; the modification includes 2'-methoxy modification, 2'-fluoro modification, thiophosphate linkage, reverse abasic nucleotide modification, deoxyribonucleotide substitution, and / or vinyl phosphate modification. Studies have shown that the modified siRNA exhibits good ACVR2A inhibitory effects at concentrations of 1 nM to 10 nM. Therefore, the modified siRNA shows great promise in the preparation of drugs for the prevention and / or treatment of pathological conditions or diseases (e.g., cancer, osteoporosis, and / or neurodegenerative diseases) caused by ACVR2A gene expression. Detailed Implementation

[0053] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0054] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0055] In the following embodiments, ACVR2A mRNA refers to mRNA having the sequences shown in the Genbank accessions for human NM_001616.5, human NM_001278580.2, human NM_001278579.2, cynomolgus monkey XM_005573111.3, cynomolgus monkey XM_015432187.2, cynomolgus monkey XM_045368236.1, mouse NM_007396.4, and rat NM_031571.3. Further, unless otherwise specified, the term "target gene" as used in this disclosure refers to the gene that transcribes the aforementioned ACVR2A mRNA, and the term "target mRNA" refers to the aforementioned ACVR2A mRNA.

[0056] The nucleic acid electrophoresis, real-time PCR and other operations used in the following examples were performed in accordance with the methods described in Molecular Biology (4th Edition) (Alexander McLennan et al., 2019).

[0057] The siRNAs involved in the following examples are siRNA sequences synthesized via phosphoramide solid-phase synthesis.

[0058] In the examples described below, when transfecting cells with ACVR2A-targeting siRNA, modified siRNA, or siRNA used as a negative control, Lipo3000 or RNAiMAX (purchased from Invitrogen) were used as transfection reagents, and the specific procedures were performed according to the manufacturer's instructions. For qPCR detection, HiScript III RT SuperMix for qPCR (purchased from Vazyme) was used as the reverse transcription reagent, and the specific procedures were performed according to the manufacturer's instructions.

[0059] Example 1: A siRNA for inhibiting ACVR2A gene expression This embodiment provides an siRNA for inhibiting ACVR2A gene expression. The nucleotide sequence of the siRNA is designed based on the target mRNA, as shown in Tables 1 and 2.

[0060] Table 1. siRNAs and their sequences that inhibit ACVR2A gene expression

[0061] Table 2. siRNAs and their sequences that inhibit ACVR2A gene expression

[0062] Example 2: A modified siRNA for inhibiting ACVR2A gene expression This embodiment provides a modified siRNA for inhibiting ACVR2A gene expression. The modified siRNA is based on the siRNA of Example 1, with the 7th, 9th, 10th, and 11th nucleotides of the positive strand having a 2' fluorination, and the nucleotides at other positions having a 2' methoxylation. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the 1st nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The 1st nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group. The penultimate nucleotide is linked to the inverse abase nucleotide via a phosphate thioester group. The nucleotides at positions 2, 6, 10, 14, and 16 of the antisense strand are fluorinated at the 2' position. The nucleotide at position 12 is substituted with a deoxyribonucleotide (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide replaces uracil ribonucleotide, and so on). The nucleotides at other positions are methoxylated at the 2' position. Phosphothioester bonds connect the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate. The modified siRNA is based on the siRNA of Example 1, with the 7th, 9th, 10th, and 11th nucleotides of the sense strand having a 2' fluorination, and the other nucleotides having a 2' methoxylation. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the 1st nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The 1st nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group, and the penultimate nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group. The nucleotides at positions 2, 6, 10, 14, and 16 of the chain are fluorinated at the 2' position; the nucleotide at position 12 is substituted with a deoxyribonucleotide (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide is used instead of uracil ribonucleotide, and so on); the nucleotides at other positions are methoxylated at the 2' position; a trans-vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1; and the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by thiophosphate bonds; or, The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 9, 10, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. The nucleotides at positions 1 and 2, and the penultimate and penultimate positions are linked by thiophosphate groups. The nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated at the 2' position, and the nucleotides at positions 1 and 2, 2 and 3, the penultimate and penultimate positions, and the penultimate and penultimate positions are linked by thiophosphate bonds; or, The modified siRNA is based on the siRNA of Example 1, with the 7th, 9th, 10th, and 11th nucleotides of the sense strand having a 2' fluorination, and the other nucleotides having a 2' methoxylation. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the 1st nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The 1st nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group, and the penultimate nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group. The 2nd, 6th, 12th, 14th, and 16th nucleotides of the antisense strand have a 2' fluorination, and the other nucleotides have a 2' methoxylation. Thiophosphate bonds connect the 1st and 2nd, 2nd and 3rd, penultimate and penultimate, and penultimate and penultimate nucleotides; or... The modified siRNA is based on the siRNA of Example 1, with the 7th, 9th, 10th, and 11th nucleotides of the sense strand having a 2' fluorination, and the other nucleotides having a 2' methoxylation. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the 1st nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The 1st nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group, and the penultimate nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group. The 2nd, 6th, 12th, 14th, and 16th nucleotides of the antisense strand have a 2' fluorination, and the other nucleotides have a 2' methoxylation. A trans-vinyl phosphate group (VP group) is attached to the 5' end of the 1st nucleotide. Thiophosphate bonds connect the 1st and 2nd, 2nd and 3rd, penultimate and penultimate, and penultimate and penultimate nucleotides; or... The modified siRNA is based on the siRNA of Example 1, wherein the first and second nucleotides starting from the 5' end of the sense strand are deleted, the 5th, 7th, 8th, and 9th nucleotides of the sense strand are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. The 1st and 2nd nucleotides, and the penultimate and penultimate nucleotides are linked by a thiophosphate group. The 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A trans-vinyl phosphate group (VP group) is attached to the 5' end of the 1st nucleotide. The 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the penultimate and penultimate nucleotides, and the penultimate and penultimate nucleotides are linked by a thiophosphate bond.

[0063] The modified siRNAs are shown in Tables 3-6.

[0064] Table 3. Modified siRNAs and their sequences that inhibit ACVR2A gene expression

[0065] Table 4. Modified siRNAs and their sequences that inhibit ACVR2A gene expression

[0066] Table 5. Modified siRNAs and their sequences that inhibit ACVR2A gene expression

[0067] Table 6. Modified siRNAs and their sequences that inhibit ACVR2A gene expression

[0068] In Tables 3-6, uppercase letters C, G, U, and A represent ribonucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated nucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by a thiophosphate group; string (d) indicates that the nucleotide adjacent to the left of the string is replaced by a deoxyribonucleotide; string (invAb) indicates that the nucleotide adjacent to the left or right of the string is linked to an inverse abase nucleotide (invAb group); string VP indicates that the nucleotide adjacent to the right of the string is a vinyl phosphate-modified nucleotide.

[0069] Experimental Example 1: Validation of the activity of siRNA used to inhibit ACVR2A gene expression in 293T cells This experimental example provides an experiment to verify the activity of siRNA used to inhibit ACVR2A gene expression in 293T cells. The experimental procedure is as follows: 293T cells (human embryonic kidney cells, purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into MEM medium (purchased from Gibco, catalog number 11095-080) containing 10% (v / v) fetal bovine serum (FBS, purchased from Hyclone) and 1% (v / v) penicillin-streptomycin mixture (Penicillin-Streptomycin, purchased from Gibco, catalog number 15140122) and cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 48 h. After culture, the 293T cells were digested with trypsin (purchased from GIBCO, catalog number 25200-072). After digestion, the cells were rinsed with PBS buffer and then resuspended in MEM medium to obtain a cell concentration of 3 × 10⁻⁶ cells / year. 5 Cell suspension of cells / mL; Different siRNAs were diluted separately using opti-MEM (Gibco, catalog number 31985-070) to obtain siRNA dilutions containing different siRNAs. 25 μL of opti-MEM was mixed with 0.25 μL of Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, catalog number 11668-019) to obtain transfection reagent dilutions. 25 μL of each siRNA dilution was then mixed with the transfection reagent dilutions and incubated at room temperature (25℃) for 15 min to obtain transfection solutions containing different siRNAs. After seeding the cell suspension into 96-well plates at a seeding rate of 50 μL / well, the 96-well plates were divided into NC group (the siRNA used in the NC group was a negative control siRNA that was not related to ACVR2A) and siRNA experimental group (the siRNA used in the siRNA experimental group was a different siRNA used to inhibit ACVR2A gene expression), with 3 replicates in each group. After setup, 50 μL of transfection buffer containing different siRNAs was added to the wells of the siRNA experimental group (the hACVR2A_1022AM2 experimental group was given transfection buffer containing ACVR2A_1022AM2, the ACVR2A_1023AM2 experimental group was given transfection buffer containing ACVR2A_1023AM2, and so on. The final concentration of siRNA in the wells was 1 nM, 5 nM, or 10 nM, and the final concentration of siRNA was adjusted by using Opti-MEM dilution). The NC experimental group was given transfection buffer containing NC. The cells were cultured in a 5% (v / v) CO2, 37℃ cell culture incubator for 48 h for transfection. After transfection, the liquid in the wells was discarded, the cells were collected, and total RNA was extracted from the cells in each well using a magnetic bead method total RNA extraction kit (purchased from Suzhou Genegene Co., Ltd., catalog number E31008) according to the method described in the kit instructions. The experimental procedure was performed using HiScript III RT SuperMix for qPCR (purchased from Novizan, catalog number R323-01) according to the product instructions. A 20 μL reverse transcription reaction system was prepared according to the reverse transcription procedure in the kit instructions to reverse transcribe total RNA from cells. The reverse transcription conditions were as follows: the reverse transcription reaction system was incubated at 37°C for 15 min, then at 85°C for 5 s. 80 μL of DEPC water was added to each reverse transcription reaction system to obtain a solution containing cDNA. For each reverse transcription reaction system, 4 μL of the above-mentioned cDNA-containing solution was used as a template. Using the reagents provided in the AceQ Universal SYBR qPCR Master Mix kit (purchased from Vazyme, catalog number Q511-02), 20 μL of qPCR reaction system was prepared on an ice box according to Table 7. Primer 1 and Primer 2 are the PCR primer sequences for amplifying the target gene ACVR2A and the internal reference gene GAPDH, respectively (as shown in Table 8). Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument, and amplification was performed using a three-step method. The amplification program was: 95℃ pre-denaturation for 10 min, then 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. This denaturation, annealing, and extension process was repeated 40 times to obtain product W containing amplified target gene ACVR2A and internal reference gene GAPDH. Product W was then sequentially subjected to 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 1 min. After 15 seconds of incubation, the melting curves of the target gene ACVR2A and the internal reference gene GAPDH in product W were collected by real-time quantitative PCR instrument to obtain the Ct values ​​of the target gene ACVR2A and the internal reference gene GAPDH. The relative quantification of the target gene ACVR2A in each test group was performed using the Ct(ΔΔCt) method, as follows: ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group); ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group); ΔCt(test group) = ΔCt(test group) - ΔCt(control group average); ΔCt(control group) = ΔCt(control group) - ΔCt(control group average); Wherein, ΔCt (control group mean) is the arithmetic mean of ΔCt (control group) for each sample in the control group; thus, each sample in the test group and the control group corresponds to a ΔCt value; Using the control group as a baseline, the expression level of ACVR2A mRNA in the test group was normalized, and the ACVR2A mRNA expression level in the control group was defined as 100%. The relative expression level of ACVR2A mRNA in the test group was 2. -ΔΔCt(测试组) ×100%; ACVR2A mRNA inhibition rate in the test group = 1 - relative expression level of ACVR2A mRNA in the test group; The ACVR2A mRNA level was compared with the internal reference gene GAPDH, and the value was normalized to the mean of the saline control group. The data are expressed as a percentage relative to the saline control group and presented as the mean plus the standard deviation. The experimental results are shown in Table 9.

[0070] As shown in Table 9, the modified siRNAs in Example 2 all exhibited good ACVR2A inhibition effects.

[0071] Table 7. DNA Amplification Reaction System

[0072] Table 8. Primer Information

[0073] Table 9. Inhibitory levels of different modified siRNAs (5 nM) on ACVR2A mRNA in 293T cells

[0074] Experimental Example 2: Validation of the activity of siRNA used to inhibit ACVR2A gene expression in Huh-7 cells This experimental example provides an experiment to verify the activity of siRNA used to inhibit ACVR2A gene expression in Huh-7 cells. The experimental procedure is as follows: Huh-7 cells (human hepatocellular carcinoma cells, purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into DMEM medium (purchased from Gibco, catalog number 11966025) containing 10% (v / v) fetal bovine serum (FBS, purchased from Hyclone), 1% (v / v) sodium pyruvate (purchased from Gibco, catalog number 11360070), and 1% (v / v) penicillin-streptomycin mixture (Penicillin-Streptomycin, purchased from Gibco, catalog number 15140122). The cells were cultured for 48 h in a 5% (v / v) CO2 incubator at 37°C. After culture, the Huh-7 cells were digested with trypsin (purchased from GIBCO, catalog number 25200-072). After digestion, the cells were rinsed with PBS buffer and then resuspended in DMEM medium to obtain a cell concentration of 3 × 10⁶ cells / year. 5 Cell suspension of cells / mL; Different siRNAs were diluted separately using opti-MEM (Gibco, catalog number 31985-070) to obtain siRNA dilutions containing different siRNAs. 25 μL of opti-MEM was mixed with 0.25 μL of Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, catalog number 11668-019) to obtain transfection reagent dilutions. 25 μL of each siRNA dilution was then mixed with the transfection reagent dilutions and incubated at room temperature (25℃) for 15 min to obtain transfection solutions containing different siRNAs. After seeding the cell suspension into 96-well plates at a seeding rate of 50 μL / well, the 96-well plates were divided into NC group (NC group used siRNA which was a negative control siRNA unrelated to ACVR2A), BLANK group (BLANK group was blank control group), MOCK group (MOCK group was transfection reagent control group) and siRNA experimental group (siRNA experimental group used different siRNAs to inhibit ACVR2A gene expression), with 3 replicates in each group. After setup, 50 μL of transfection buffer containing different siRNAs was added to the wells of the siRNA experimental groups (the hACVR2A_317AM7 experimental group was given transfection buffer containing ACVR2A_317AM7, the ACVR2A_2167AM7 experimental group was given transfection buffer containing ACVR2A_2167AM7, and so on, with the final concentration of siRNA in the wells being 1 nM, 5 nM, or 10 nM, respectively, and the final concentration of siRNA was adjusted by using opti-MEM dilution). The NC experimental group was given transfection buffer containing NC, and 50 μL of transfection buffer without any siRNA was added to the wells of the transfection reagent control group (MOCK). The cells were cultured in a 5% (v / v) CO2, 37℃ cell culture incubator for transfection. After 6 h of transfection, the transfection complex was aspirated (i.e., the liquid in the wells was discarded), 100 μL of DMEM medium was added to each well, and the cells were placed in a 5% (v / v) CO2, 37℃ cell culture incubator for further transfection. After transfection for 24 or 48 hours, the cell supernatant was discarded, and cells were washed in wells with an equal volume of 1×PBS buffer pre-cooled to 4°C. After washing, the 1×PBS buffer was removed, and total RNA was extracted from the cells in each well using lysis working solution. The lysis working solution was prepared according to the required volume of the plate (see Table 9 for the lysis working solution formula), and the proportions of each component were: FlysisAmp Cells Lysis Buffer (purchased from Vazyme, catalog number CL101-01): DNase I (purchased from Vazyme, catalog number GMP4104PC): Enhancer Solution = 23:1:1; After preparation, invert and mix 10-15 times, avoiding vigorous vortexing; Add 50 μL of the prepared lysis buffer to each well of the cell culture plate, gently pipette 8-10 times to mix thoroughly, and let stand at room temperature for 5 min to lyse the cells; After lysis, add 5 μL of stop solution to each well of the cell culture plate, gently pipette 8-10 times to mix thoroughly, and let stand at room temperature for 2 min to stop the reaction, obtaining the cell lysis buffer; For each reverse transcription reaction system, 50 μL of the cell lysis buffer was diluted with 150 μL of DEPC H2O to prepare RNA template. Using the reagents provided by the AceQ Universal SYBR qPCR Master Mix kit (purchased from Vazyme, catalog number Q511-02), 13 μL of qPCR reaction system was prepared on an ice box according to Table 10. The qPCR primer and probe sequences are shown in Table 11. 9.75 μL of each prepared qPCR reaction system was dispensed into each well of a 384-well plate. 3.25 μL of RNA template was added to each well, followed by 3 μL of paraffin oil. After sealing with a sealing film, the plate was centrifuged at 3000 rpm for 1 min. After centrifugation, a real-time quantitative PCR reaction was performed on an LC480. The PCR reaction program is shown in Table 12. During the PCR reaction, the melting curves of the target gene ACVR2A and the internal reference gene GAPDH in product W were collected using a real-time quantitative PCR instrument to obtain the Ct values ​​of the target gene ACVR2A and the internal reference gene GAPDH. The relative quantification of the target gene ACVR2A in each test group was performed using the Ct(ΔΔCt) method, as follows: ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group); ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group); ΔCt(test group) = ΔCt(test group) - ΔCt(control group average); ΔCt(control group) = ΔCt(control group) - ΔCt(control group average); Wherein, ΔCt (control group mean) is the arithmetic mean of ΔCt (control group) for each sample in the control group; thus, each sample in the test group and the control group corresponds to a ΔCt value; Using the control group as a baseline, the expression level of ACVR2A mRNA in the test group was normalized, and the ACVR2A mRNA expression level in the control group was defined as 100%. The relative expression level of ACVR2A mRNA in the test group was 2. -ΔΔCt(测试组) ×100%; ACVR2A mRNA inhibition rate in the test group = 1 - relative expression level of ACVR2A mRNA in the test group; The ACVR2A mRNA level was compared with the internal reference gene GAPDH, and the value was normalized to the mean of the saline control group. The data are expressed as a percentage relative to the saline control group and presented as the mean plus the standard deviation. The experimental results are shown in Tables 13-16.

[0075] As shown in Tables 13-16, the siRNA in Example 1 and the modified siRNA in Example 2 both exhibited good ACVR2A inhibition effects.

[0076] Table 9. Cell lysis reaction system

[0077] Table 10. Reaction system for one-step RT-qPCR probe method

[0078] Table 11. Primer and probe information

[0079] Table 12. RT-qPCR reaction procedure

[0080] Table 13. Inhibitory levels of different modified siRNAs (1 nM) on ACVR2A mRNA in Huh-7 cells

[0081] Table 14. Inhibitory levels of different modified siRNAs (10 nM) on ACVR2A mRNA in Huh-7 cells

[0082] Table 15. Inhibitory levels of different modified siRNAs (1 nM) on ACVR2A mRNA in Huh-7 cells

[0083] Table 16. Inhibition levels of different siRNAs and modified siRNAs on ACVR2A mRNA in Huh-7 cells

[0084] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A siRNA for inhibiting ACVR2A gene expression, characterized in that, The siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region; the sense strand of the siRNA contains at least 15 consecutive nucleotides of any one of the nucleic acid sequences shown in any one of SEQ ID NO.1~SEQ ID NO.147 or SEQ ID NO.295~SEQ ID NO.391, differing by no more than 3 nucleotides; the antisense strand of the siRNA contains at least 15 consecutive nucleotides of any one of the nucleic acid sequences shown in any one of SEQ ID NO.148~SEQ ID NO.294 or SEQ ID NO.392~SEQ ID NO.488, differing by no more than 3 nucleotides.

2. The siRNA as described in claim 1, characterized in that, At least one nucleotide in the sense strand and / or antisense strand of the siRNA is a modified nucleotide.

3. The siRNA as described in claim 2, characterized in that, The modified nucleotides include compounds formed by modifying the ribose of nucleotides, compounds formed by modifying the backbone between nucleotides, compounds formed by modifying the bases on nucleotides, and / or compounds formed by modifying the ends of siRNA.

4. The siRNA according to any one of claims 2 to 3, characterized in that, The modified nucleotides include nucleotides modified with 2'-fluorination, nucleotides modified with 2'-methoxy, nucleotides linked by a thiophosphate group, nucleotides modified with a reverse-free nucleotide, nucleotides substituted with deoxyribonucleotides, nucleotides modified with vinyl phosphate, 2'-deoxy-nucleotides, 3'-terminal deoxy-thymidine nucleotides, inosine nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, baseless nucleotides, nucleotides modified with a reverse nucleotide, nucleotides modified with 2'-amino-, nucleotides modified with 2'-O-allyl-, nucleotides modified with 2'-C-alkyl-, and nucleotides modified with 2'- Hydroxyl-modified nucleotides, nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2'-O-alkyl, nucleotides modified with morpholinonucleotide, nucleotides modified with aminophosphate, nucleotides containing non-natural bases, nucleotides modified with tetrahydropyran, nucleotides modified with 1,5-dehydrohexyl alcohol, nucleotides modified with cyclohexenyl, nucleotides modified with ethylene glycol nucleic acid, nucleotides modified with open-ring nucleotides, nucleotides containing methyl phosphate groups, nucleotides containing 5'-phosphate, nucleotides containing 5'-phosphate mimics, terminal nucleotides linked to cholesterol derivatives, and / or terminal nucleotides linked to dodecanoic acid bisdecamide groups.

5. The siRNA as described in claim 4, characterized in that, The 2'-fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 10, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The 2'-fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The 2'-fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The 2'-fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 5th, 7th, 8th, and 9th nucleotides of the sense strand are fluorinated nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides.

6. The siRNA as described in claim 4 or 5, characterized in that, The nucleotides modified with 2'-methoxy are located in the antisense and sense strands of the nucleotide sequence, and in the sense strand, the nucleotides not modified with 2'-fluoride and the nucleotides not substituted with deoxyribonucleotides are all nucleotides modified with 2'-methoxy, and in the antisense strand, the nucleotides not modified with 2'-fluoride and the nucleotides not substituted with deoxyribonucleotides are all nucleotides modified with 2'-methoxy.

7. The siRNA according to any one of claims 4 to 6, characterized in that, The nucleotide modified with a reverse abase-free nucleotide is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 3' end of the first nucleotide of the positive strand is attached to a reverse abase-free nucleotide; and / or, The reverse abase-free nucleotide is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of at least the penultimate nucleotide of the positive strand is attached to a reverse abase-free nucleotide.

8. The siRNA according to any one of claims 4 to 7, characterized in that, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotide at position 12 of the antisense strand is a deoxyribonucleotide-substituted nucleotide.

9. The siRNA according to any one of claims 4 to 8, characterized in that, The vinyl phosphate-modified nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of at least the first nucleotide of the antisense strand is attached to a vinyl phosphate group.

10. The siRNA according to any one of claims 4 to 9, characterized in that, Nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotide at position 1 of the sense strand is linked to the inverse abase nucleotide by a thiophosphate group, and the nucleotide at position 1-1 is linked to the inverse abase nucleotide by a thiophosphate group; at least the nucleotides at positions 1-2, 2-3, 1-1-2, and 2-1-3 of the antisense strand are linked by thiophosphate groups; or, Nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the first and second, and the penultimate and penultimate nucleotides of the sense strand are linked by thiophosphate groups, and at least the first and second, second and third, penultimate and penultimate, and penultimate and penultimate nucleotides of the antisense strand are linked by thiophosphate groups; or, The nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by thiophosphate groups, and at least the nucleotides at positions 1 and 2, positions 2 and 3, positions 1 to 1 and 2 to 1 ...

11. The siRNA according to any one of claims 1 to 10, characterized in that, The siRNA is conjugated to a functional molecule; the functional molecule includes lipophilic molecules, peptides, small molecule drugs, antibodies, ligands formed from targeting molecules or derivatives of targeting molecules; the targeting molecule includes galactose and / or N-glucose amino acids.

12. A recombinant plasmid, characterized in that, The recombinant plasmid expresses the siRNA according to any one of claims 1 to 11.

13. A host cell, characterized in that, The host cell genome is integrated with the siRNA according to any one of claims 1 to 11; or, the host cell carries the recombinant plasmid according to claim 12.

14. The use of the siRNA according to any one of claims 1 to 11, the recombinant plasmid according to claim 12, or the host cell according to claim 13 in the preparation of a medicament for the prevention and / or treatment of pathological conditions or diseases caused by ACVR2A gene expression.

15. A medicament for preventing and / or treating pathological conditions or diseases caused by ACVR2A gene expression, characterized in that, The drug comprises the siRNA as described in any one of claims 1 to 11, the recombinant plasmid as described in claim 12, and / or the host cell as described in claim 13.

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