SiRNA for inhibiting acvr1c and its modifications and applications

Abstract

The present application relates to a kind of siRNA and its modifier for inhibiting ACVR1C and application, belong to biotechnology field.The present application provides the siRNA for inhibiting ACVR1C, the siRNA all has high inhibitory activity to ACVR1C.The present application also provides the modified siRNA for inhibiting ACVR1C, the modified siRNA all has good inhibitory efficiency to ACVR1C.

Description

[0001] This application claims priority to Chinese Patent Application No. 202510374298.0, filed on March 27, 2025, entitled "siRNA for inhibiting ACVR1C 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 ACVR1C, its modifications, and applications, and belongs to the field of biotechnology. Background Technology

[0003] Activin receptor type-1C (ACVR1C), also known as activin receptor-like kinase 7 (ALK7), is a single-transmembrane type I protein. Its extracellular region functions as a receptor, while its intracellular region functions as a kinase. Upon ligand binding, it forms a receptor complex, which consists of two type II and two type I transmembrane serine / threonine kinases. Type II receptor phosphorylation activates type I receptor, which then autophosphorylates and binds to and activates the SMAD transcriptional regulators SMAD2 and SMAD3.

[0004] Small interfering RNA (siRNA) is typically a double-stranded RNA of 20 to 25 nucleotides in length. It primarily uses RNA interference (RNAi) to specifically regulate gene expression, thereby achieving the goal of treating diseases. Summary of the Invention

[0005] To address the aforementioned problems, the present invention provides an siRNA for inhibiting ACVR1C, wherein the siRNA contains a sense strand and an antisense strand, the sense strand and the antisense strand being at least partially anticomplementary to form a double-stranded region; the sense strand of the siRNA comprises at least 17 consecutive nucleotides whose nucleotide sequences differ by no more than 3 nucleotides from any one of the nucleic acid sequences shown in any one of SEQ ID NO. 1 to 159; the antisense strand of the siRNA comprises at least 17 consecutive nucleotides whose nucleotide sequences differ by no more than 3 nucleotides from any one of the nucleic acid sequences shown in any one of SEQ ID NO. 160 to 318.

[0006] Furthermore, at least one nucleotide in the sense or antisense strand of the siRNA is a modified nucleotide.

[0007] Furthermore, the modified nucleotide is a compound formed by modifying the ribose of the nucleotide, or a compound formed by modifying the backbone between nucleotides, or a compound formed by modifying the bases on the nucleotide, or a compound formed by modifying the ends of the siRNA.

[0008] Further, the modified nucleotide is selected from at least one of the following: 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, phosphate thioester modified nucleotides, deoxy-nucleotides, 2'-deoxy-nucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, baseless nucleotides, reverse nucleotides, reverse baseless nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinonucleotides, aminophosphates, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, glycol nucleic acids (GNA), open-ring nucleotides (UNA), nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimics, or terminal nucleotides linked to cholesterol derivatives or terminal nucleotides linked to dodecanoic acid bisdecamide groups.

[0009] Furthermore, the 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, 8, and 9, or positions 5, 7, 8, and 9, or positions 9, 10, and 11, or positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, while the nucleotide groups at the remaining positions in the sense strand are each non-fluorinated nucleotide groups; the nucleotides at positions 2, 6, 12, 14, and 16, or positions 2, 8, 12, 14, and 16, or positions 2, 9, 12, 14, and 16, or positions 2, 10, and 12 of the antisense strand are fluorinated nucleotides. The nucleotides at positions 14 and 16, or positions 2, 6, 10, 12, and 14, or positions 2, 8, 10, 14, and 16, or positions 2, 6, 10, 14, and 16, or positions 2, 4, 12, 14, and 16, or positions 2, 6, 14, and 16, or positions 2, 10, 12, 14, 16, 18, and 20, or positions 2, 6, 8, 14, and 16, or positions 14, or positions 10, 12, 14, 16, 18, and 20, or positions 2, 14, and 16 are fluorinated nucleotides, and the nucleotide groups at the remaining positions in the antisense strand are each non-fluorinated nucleotide groups; Each of the non-fluorinated modified nucleotide groups is independently selected from one of the nucleotide groups or nucleotide analog groups formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group; the non-fluorinated modified nucleotide is a methoxy-modified nucleotide, a 2′-deoxynucleotide, a glycol nucleic acid, an inosine nucleotide, and a 2′-O-alkyl-modified nucleotide. Optionally, the 2'-O-alkyl-modified nucleotide is a 2'-O-C22-modified nucleotide or a 2'-O-hexadecyl-modified nucleotide.

[0010] Furthermore, in the direction from the 5' end to the 3' end, at least one nucleotide in the nucleotides at positions 2 to 18 of the antisense strand is a thermally unstable modified nucleotide.

[0011] Furthermore, in the direction from the 5' end to the 3' end, at least one nucleotide in the nucleotides at positions 2, 5, 7, 10, 11, 12, 13, and 16 of the antisense strand is a thermally unstable modified nucleotide.

[0012] Furthermore, in the direction from the 5' end to the 3' end, at least two nucleotides in the second to eighth of the antisense strand are thermally unstable modified nucleotides.

[0013] Furthermore, the 5th and 7th positions of the antisense strand are nucleotides modified to be thermally unstable.

[0014] Furthermore, the thermally unstable modified nucleotide is ethylene glycol nucleic acid (GNA) and / or 2′-deoxynucleotide.

[0015] Furthermore, the 2'-O-C22 modified nucleotide is located in the sense strand and / or antisense strand, following the direction from the 5' end to the 3' end; Optionally, the nucleotide modified by 2'-O-C22 is located at the 1st, 6th and / or 1st to the end of the positive strand.

[0016] Furthermore, the 5' and / or 3' ends of the sense strand and / or antisense strand are also connected to a 2'-O-C22 modified nucleotide; Optionally, the 2'-O-C22 modified nucleotide is linked to the 3' end of the sense strand and / or antisense strand via a thiophosphate group.

[0017] Furthermore, at least one of the phosphate ester groups in the phosphate-sugar backbone of at least one single chain of the sense chain and the antisense chain is a phosphate ester group with a modifying group; Optionally, the phosphate ester group having the modifying group includes a thiophosphate ester group; Optionally, the nucleotides at least the first and second, the second and third, and the penultimate and penultimate positions of the antisense strand are linked by phosphate thioester groups in the direction from the 5' end to the 3' end. Optionally, in the direction from the 5' end to the 3' end, at least the nucleotides at the 1st and 2nd, 2nd and 3rd, penultimate and penultimate, and penultimate and penultimate positions of the antisense strand are linked by phosphate thioester groups. Optionally, the nucleotides at least the first and second positions, and the second and third positions of the positive strand are linked by thiophosphate groups in the direction from the 5' end to the 3' end.

[0018] Furthermore, the antisense strand contains at least two thermally unstable modified nucleotides in the direction from the 5' end to the 3' end; more examples of thermally unstable modified nucleotides are described in detail in WO2018098328A1 (the entire contents of which are incorporated herein by reference), and thermally unstable modification in this invention is equivalent to thermal destabilization modification in WO2018098328A1; In the direction from the 5' end to the 3' end, at least two nucleotides in the seed region of the antisense strand are thermally unstable modified nucleotides; In the direction from the 5' end to the 3' end, at least the nucleotides at positions 5 and 7 of the antisense strand are thermally unstable modified nucleotides; The thermally unstable modified nucleotide is ethylene glycol nucleic acid (GNA) and / or 2′-deoxynucleotide, wherein the 2′-deoxynucleotide is located in the antisense strand of the nucleotide sequence.

[0019] Furthermore, the 5' end and / or 3' end of the siRNA sense strand or antisense strand contains at least one reverse abase-free nucleotide; Furthermore, the 5' end and / or 3' end of the siRNA's positive strand contains at least one reverse abase-free nucleotide; Optionally, the reverse abase-free nucleotide is connected to the 5' and / or 3' ends of the sense and / or antisense strands via thiophosphate groups.

[0020] Furthermore, the first nucleotide at the 5' and / or 3' end of the siRNA's sense and / or antisense strands is a vinyl phosphate-modified nucleotide.

[0021] Furthermore, the 5' end / 3' end of the sense strand may also be connected to two nucleotides complementary to the 3' end / 5' end of the antisense strand; and the 3' end / 5' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region.

[0022] Furthermore, the 5' and / or 3' ends of the sense strand and / or antisense strand are also connected to a 2'-O-C22 modified nucleotide; Optionally, the 2'-O-hexadecyl modified nucleotide is linked to the 3' end of the sense and / or antisense strands via a thiophosphate group.

[0023] Furthermore, one or more lipophilic portions are coupled or conjugated at one or more internal or end positions on at least one of the sense or antisense strands of the siRNA.

[0024] Furthermore, the siRNA has a ligand coupled to the end of either the sense or antisense strand.

[0025] The present invention also provides a product for inhibiting ACVR1C, the product comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is siRNA as described in any of the preceding claims.

[0026] The present invention also provides the use of siRNA or the above-described products according to any one of the preceding claims in the preparation of products for the prevention, diagnosis and / or treatment of pathological conditions or diseases caused by ACVR1C.

[0027] In one embodiment of the present invention, taking the positive and antisense strands shown in SEQ ID NO.1 and SEQ ID NO.160 as examples, the positive strand comprises: a nucleotide sequence as shown in SEQ ID NO.1 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO.1 and retaining the biological function of its derived sequence; the antisense strand comprises: a nucleotide sequence as shown in SEQ ID NO.160 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO.160 and retaining the biological function of its derived sequence. The remaining positive and antisense strands each have nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity and retaining the biological function of their derived sequences.

[0028] In one embodiment of the present invention, the 5' end of the siRNA sense strand may also be connected to 1-3 nucleotides complementary to the 3' end of the antisense strand; Alternatively, the 3' end of the siRNA sense strand may also be linked to 1-3 nucleotides complementary to the 5' end of the antisense strand; Alternatively, the 5' end of the siRNA antisense strand may also be linked to 1-3 nucleotides complementary to the 3' end of the sense strand; Alternatively, the 3' end of the siRNA antisense strand may be linked to 1-3 nucleotides complementary to the 5' end of the sense strand.

[0029] In one embodiment of the present invention, the 5' end of the siRNA sense strand may also be connected to two nucleotides complementary to the 3' end of the antisense strand; Alternatively, the 3' end of the siRNA sense strand may also be linked to two nucleotides complementary to the 5' end of the antisense strand; Alternatively, the 5' end of the siRNA antisense strand may also be linked to two nucleotides complementary to the 3' end of the sense strand; Alternatively, the 3' end of the siRNA antisense strand may also be linked to two nucleotides complementary to the 5' end of the sense strand.

[0030] In one embodiment of the present invention, the sense strand and antisense strand of the double-stranded RNA molecule are complementary to form the double-stranded region of the siRNA, the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region. Alternatively, the sense and antisense strands of the double-stranded RNA molecule are complementary to form the double-stranded region of the siRNA, the 5' end of the sense strand forms a blunt end, and the 5' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region; Alternatively, the sense and antisense strands of the double-stranded RNA molecule are complementary to form the double-stranded region of the siRNA, with the 3' end of the sense strand forming a blunt end and the 3' end of the antisense strand forming a blunt end; Alternatively, the sense and antisense strands of the double-stranded RNA molecule are complementary to form the double-stranded region of the siRNA, and the 3' ends of the sense and antisense strands have 1-3 protruding nucleotides extending out of the double-stranded region, or the 5' ends of the sense and antisense strands have 1-3 protruding nucleotides extending out of the double-stranded region.

[0031] In one embodiment of the present invention, the siRNA is prepared by solid-phase synthesis or liquid-phase synthesis.

[0032] In one embodiment of the present invention, the nucleotides in the siRNA are each independently modified or unmodified nucleotides.

[0033] In one embodiment of the present invention, each nucleotide in the siRNA is an unmodified nucleotide.

[0034] In one embodiment of the present invention, some or all of the nucleotides in 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 ACVR1C gene expression.

[0035] In one embodiment of the present invention, at least one nucleotide in the sense strand or antisense strand of the siRNA is a modified nucleotide.

[0036] 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 ACVR1C gene expression.

[0037] In one embodiment of the present invention, each nucleotide in the sense and antisense strands of the siRNA is independently a fluorinated nucleotide or a non-fluorinated nucleotide.

[0038] In one embodiment of the invention, the fluorinated nucleotide is located in both the antisense and sense strands of the nucleotide sequence, and the fluorinated nucleotide is present at least at one of the following positions: Following the direction from the 5' end to the 3' end, at least the 7th, 8th, and 9th nucleotides of the positive strand are fluorinated nucleotides; at least the 5th, 7th, 8th, and 9th nucleotides of the positive strand are fluorinated nucleotides; at least the 9th, 10th, and 11th nucleotides of the positive strand are fluorinated nucleotides; at least the 7th, 9th, 10th, and 11th nucleotides of the positive strand are fluorinated nucleotides. Following the direction from the 5' end to the 3' end, at least the 2nd, 6th, 12th, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides; at least the 2nd, 8th, 12th, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides; at least the 2nd, 9th, 12th, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides; at least the 2nd, 10th, 12th, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides; at least the 2nd, 6th, 10th, 12th, and 14th nucleotides of the antisense strand are fluorinated nucleotides; at least the 2nd, 8th, 10th, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides; at least the 2nd, 6th, 10th, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides; at least the 2nd, 6th, 10th, 14th, and 16th nucleotides of the antisense strand are nucleotides. The acid is a fluorinated nucleotide; at least the 2nd, 4th, 12th, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides; at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides; at least the 2nd, 10th, 12th, 14th, 16th, 18th, and 20th nucleotides of the antisense strand are fluorinated nucleotides; at least the 2nd, 6th, 8th, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides; at least the 14th nucleotide of the antisense strand is a fluorinated nucleotide; at least the 10th, 12th, 14th, 16th, 18th, and 20th nucleotides of the antisense strand are fluorinated nucleotides; at least the 2nd, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides. Alternatively, any combination thereof, meaning that the fluorinated nucleotides of the sense and antisense strands are selected from any combination thereof. The remaining nucleotide groups in the sense / antisense strands are each non-fluorinated nucleotide groups.

[0039] In one embodiment of the invention, each of the non-fluorinated modified nucleotide groups is independently selected from one of the nucleotide groups or nucleotide analog groups formed by replacing the hydroxyl group at the 2' position of the ribosome of the nucleotide group with a non-fluorinated group.

[0040] In one embodiment of the invention, in the direction from the 5' end to the 3' end, at least one nucleotide of the antisense strand is a thermally unstable modified nucleotide, and each of the remaining non-fluorinated modified nucleotide groups is independently selected from a nucleotide group or a nucleotide analog group formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group.

[0041] In one embodiment of the present invention, at least one nucleotide from 2 to 8 of the antisense strand is a thermally unstable modified nucleotide, in the direction from the 5' end to the 3' end.

[0042] In one embodiment of the present invention, at least two nucleotides in the antisense strand 2 to 8 are thermally unstable modified nucleotides, arranged from the 5' end to the 3' end.

[0043] In one embodiment of the invention, the nucleotides at least the 5th and 7th positions of the antisense strand are thermally unstable modified nucleotides, following the direction from the 5' end to the 3' end.

[0044] In one embodiment of the present invention, a "fluorinated nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group with fluorine, having the structure shown in formula (1). The non-fluorinated nucleotide is independently selected from nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group.

[0045] In one embodiment of the present invention, the nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with a non-fluorinated group is well known to those skilled in the art, and these nucleotides may be selected from one of 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxynucleotides.

[0046] In one embodiment of the present invention, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe) modified nucleotide, as shown in formula (2), i.e., methoxy modified; the 2'-substituted alkoxy modified nucleotide may be, for example, a 2'-O-methoxyethyl (2'-MOE) modified nucleotide, as shown in formula (3), a 2'-amino (2'-NH2) modified nucleotide as shown in formula (4), and a 2'-deoxynucleotide (DNA) as shown in formula (5), wherein Base refers to the modified or unmodified nucleotide base A, U, G, C, T or other nucleotide bases:

[0047] Equation (1) Equation (2) Equation (3) Equation (4) Equation (5) In one embodiment of the present invention, the nucleotide analogue refers to a group that can replace a nucleotide in nucleic acids, but whose structure is different from that of adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide.

[0048] In one embodiment of the present invention, the nucleotide analog may be a heteronucleotide, a bridged nucleotide, or an acyclic nucleotide.

[0049] In one embodiment of the invention, the bridged nucleic acid (BNA) refers to a restricted or inaccessible nucleotide. The BNA may contain a bridging structure with a "fixed" C3'-endoglucan condensation of a five-membered, six-membered, or seven-membered ring. Typically, the bridge is incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide.

[0050] In one embodiment of the present invention, the BNA may be LNA, ENA, cET BNA, etc., wherein LNA is as shown in formula (6), ENA is as shown in formula (7), and cET BNA is as shown in formula (8), wherein Base refers to modified or unmodified nucleotide bases A, U, G, C, T or other nucleotide bases:

[0051] Equation (6) Equation (7) Equation (8) In one embodiment of the present invention, the thermally unstable modification may include, but is not limited to, debasement modification; mismatch with relative nucleotides in the opposite chain; and sugar modification, such as 2'-deoxy modification or acyclic nucleotides, for example, unlocked nucleic acid (UNA) or glycol nucleic acid (GNA).

[0052] Examples of debasing modifications include, but are not limited to, the following, with structural formulas as shown in formulas (9) to (15):

[0053] Formula (9) Formula (10) Formula (11) Formula (12)

[0054] Equation (13) Equation (14) Equation (15) Where R = H, Me, Et or OMe; R' = H, Me, Et or OMe; R” = H, Me, Et or OMe.

[0055] In one embodiment of the present invention, sugar modification includes, but is not limited to, the following: 2'-deoxynucleotides, unlocked nucleic acids, and glycol-based nucleic acids.

[0056] In one embodiment of the invention, an acyclic nucleotide refers to any nucleotide having a noncyclic ribose, for example, wherein any bond between ribose carbons in the nucleotide (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') is absent and / or at least one of the ribose carbons or oxygen (e.g., C1', C2', C3', C4', or O4') is absent independently or in combination.

[0057] In one embodiment of the invention, UNA represents an unlocked acyclic nucleic acid (or open-ring nucleotide) in which any bond of the sugar is removed, forming an unlocked "sugar" residue. In one instance, UNA also encompasses monomers in which the bond between C1' and C4' has been removed (i.e., the covalent carbon-oxygen-carbon bond between C1' and C4' carbons). In another instance, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between C2' and C3' carbons) is removed (see Mikhailov et al., Tetrahedron Letters, 26(17):2059(1985); and Fluiter et al., Mol. Biosyst., 10:1039(2009), which are incorporated herein by reference in their entirety). Acyclic derivatives provide greater skeletal flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' bonds.

[0058] In one embodiment of the present invention, GNA represents ethylene glycol nucleic acid, which is a polymer similar to DNA or RNA, but with a different "backbone" composed of repeating glycerol units linked by phosphodiester bonds. The structure of A (GNA) is shown in Formula (16), the structure of G (GNA) is shown in Formula (17), the structure of C (GNA) is shown in Formula (18), the structure of U (GNA) is shown in Formula (19), and the structure of T (GNA) is shown in Formula (20).

[0059]

[0060] Equation (16) Equation (17) Equation (18)

[0061] Equation (19) Equation (20) In one embodiment of the present invention, inosine nucleotides are also thermally unstable modifications. Inosine nucleotides are represented by the following structures: dI structure as shown in formula (21), rI structure as shown in formula (22), 2'-F rI structure as shown in formula (23), and 2'OMe rI structure as shown in formula (24).

[0062]

[0063] Equation (21) Equation (22) Equation (23) Equation (24) The thermally unstable modification of the duplex can be a mismatch (i.e., a non-complementary base pair) between a thermally unstable nucleotide and a corresponding nucleotide in the opposite strand of the dsRNA duplex. Exemplary mismatched base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof. Other mismatched base pairs known in the art are also applicable to the present invention. Mismatches can occur between nucleotides, which are naturally occurring nucleotides or modified nucleotides; that is, mismatched base pairs can occur between nucleobases from the respective nucleotides, regardless of modifications to the ribose of the nucleotides. In some embodiments, the dsRNA molecule contains at least one nucleobase in the mismatched pair that is a 2'-deoxynucleobase; for example, a 2'-deoxynucleobase in the sense strand.

[0064] Further examples of thermally unstable modified nucleotides are described in detail in WO2018098328A1 (the entire contents of which are incorporated herein by reference), and thermally unstable modifications in this invention are equivalent to thermally destabilizing modifications in WO2018098328A1.

[0065] In one embodiment of the present invention, the nucleotides at least the 5th and 7th positions of the antisense strand are thermally unstable modified nucleotides, in the direction from the 5' end to the 3' end; the thermally unstable modified nucleotides are ethylene glycol nucleic acid (GNA) and / or 2′-deoxynucleotides, wherein the 2′-deoxynucleotides are located in the antisense strand of the nucleotide sequence.

[0066] In one embodiment of the present invention, at least one phosphate group in the sense or antisense strand of the siRNA is a phosphate group with a modifying group.

[0067] In one embodiment of the present invention, at least a portion of the phosphate ester groups and / or ribosomes in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA are phosphate ester groups and / or ribosomes with modifying groups.

[0068] In one embodiment of the present invention, the phosphate ester group with the modifying group is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphate diester bond of the phosphate ester group with a sulfur atom.

[0069] In one embodiment of the present invention, the phosphate group having the modifying group is a thiophosphate group having the structure shown in formula (25). In one embodiment of the present invention, the nucleotide linked to the thiophosphate group is shown in formula (26). In one embodiment of the present invention, the VP-modified nucleotide is a vinyl phosphate modified nucleotide. In one embodiment of the present invention, the nucleotide modified by VP and methoxy groups, i.e., the nucleotide modified by 5'-(E)-vinyl-2'-methoxy-modified phosphonate group (5'-(E)-VP-2'-OMe), is shown in formula (27). In one embodiment of the present invention, the nucleotide modified by VP, methoxy groups, and thiophosphate groups, i.e., the nucleotide modified by 5'-PS (i.e., the nucleotide modified by 5'-(E)-vinyl-2'-methoxy-modified thiophosphonate group), is shown in formula (28).

[0070]

[0071] Equation (25) Equation (26) Equation (27) Equation (28) In one embodiment of the invention, the thiophosphate group linkage is present at least at one of the following positions: between the first and second nucleotides at either end of the sense or antisense strand; between the second and third nucleotides at either end of the sense or antisense strand; or any combination thereof.

[0072] In one embodiment of the invention, the thiophosphate group linkage is present at all of the above-mentioned positions except for the end of the positive chain 5'.

[0073] In one embodiment of the invention, the thiophosphate group linkage is present at all of the above-mentioned positions except for the end of the positive chain 3'.

[0074] In one embodiment of the invention, the thiophosphate group linkage is present at at least one of the following positions: Between the first and second nucleotides at the 5' end of the positive strand; Between the second and third nucleotides at the 5' end of the positive strand; Between the first and second nucleotides at the 3' end of the positive strand; Between the second and third nucleotides at the 3' end of the positive strand; Between the first and second nucleotides at the 5' end of the antisense strand; Between the second and third nucleotides at the 5' end of the antisense strand; Between the first and second nucleotides at the 3' end of the antisense strand; and Between the second and third nucleotides at the 3' end of the antisense strand.

[0075] In one embodiment of the present invention, the reverse nucleotide is represented by the following structure, as shown in formulas (29) to (32).

[0076]

[0077] Equation (29) Equation (30)

[0078] Equation (31) Equation (32) In one embodiment of the present invention, invAb represents a reverse abase-free nucleotide, and the structural formula of invAb is shown in formula (33).

[0079]

[0080] Equation (33) In one embodiment of the present invention, the 5' end and / or 3' end of the siRNA sense strand or antisense strand contains at least one reverse abase-free nucleotide.

[0081] In one embodiment of the present invention, the 5' end and / or 3' end of the siRNA positive strand contains at least one reverse abase-free nucleotide.

[0082] In one embodiment of the present invention, the siRNA is coupled to a functional molecule, the coupled functional molecule including N-acetylgalactosamine (GalNAc), lipophilic molecules, peptides, small molecule drugs, antibodies, etc.

[0083] In one embodiment of the present invention, siRNA may be coupled to one or more coupling groups containing functional molecules.

[0084] In one embodiment of the present invention, the pharmaceutically acceptable targeting group in the siRNA conjugate is galactose or N-acetylgalgactosamine (GalNAc). N-acetylgalgactosamine (GalNAc) is a ligand that binds to the asialoglycoprotein receptor (ASGPR) on the liver surface. The ASGPR is an endocytic receptor specifically expressed by hepatocytes. N-acetylgalgactosamine serves as a targeting molecule to deliver small RNA to the liver.

[0085] In one embodiment of the present invention, the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent; the monovalent, divalent, trivalent, and tetravalent respectively refer to the siRNA molecule forming an siRNA conjugate with a coupling group containing a galactose or N-acetylgalactosamine molecule as a targeting group, wherein the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate is 1:1, 1:2, 1:3, or 1:4.

[0086] In one embodiment of the present invention, when siRNA is coupled to a coupling group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent.

[0087] In one embodiment of the present invention, when siRNA is coupled to a coupling group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0088] In one embodiment of the present invention, siRNA may be coupled to one or more coupling groups containing N-acetylgalactosamine.

[0089] In one embodiment of the present invention, the targeting group can be linked to the siRNA molecule via a suitable adapter, and those skilled in the art can select a suitable adapter according to the specific type of the targeting group.

[0090] The types of adapters, targeting groups, and the methods of ligation with siRNA are described in detail in WO2015006740A2 (the entire contents of which are incorporated herein by reference).

[0091] In one embodiment of the present invention, the 5' and / or 3' ends of the sense or antisense strand of the siRNA are coupled with a ligand, which may be a lipophilic moiety or GalNAc.

[0092] In one embodiment of the present invention, the siRNA has a ligand coupled to the 3' end of the positive strand, the ligand being GalNAc.

[0093] In one embodiment of the present invention, the 3' end of the positive strand of the siRNA is connected to the ligand via a thiophosphate group.

[0094] In one embodiment of the present invention, the siRNA conjugate formed by GalNAc and siRNA molecules has the structure shown in formula (34) below:

[0095] Equation (34) In one embodiment of the invention, the functional molecule is a lipophilic molecule (or a lipophilic or lipophilic moiety). The siRNA comprises one or more lipophilic moieties coupled or conjugated to one or more nucleotides on at least one chain via a linker or vector. The lipophilic moieties include, but are not limited to, one or more of saturated alkanes, unsaturated alkanes, saturated fatty acids, unsaturated fatty acids, and cholesterol of varying chain lengths.

[0096] The lipophilic portion of the example includes, but is not limited to, the following:

[0097] The lipophilic portion is described in more detail in WO2025228348A1 (the entire contents of which are incorporated herein by reference).

[0098] In one embodiment of the invention, the lipophilic moiety is aliphatic, cyclic (e.g., alicyclic), or polycyclic, such as alicyclic compounds, such as steroids (e.g., sterols), or straight-chain or branched aliphatic hydrocarbons. Exemplary lipophilic moieties include lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geraniol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytriphenylmethyl, or phenoxazine.

[0099] In one embodiment of the invention, the lipophilic portion also includes saturated or unsaturated C4-C. 30 Hydrocarbon chains (e.g., C4-C) 30 A lipophilic moiety comprising an alkyl or alkenyl group and optional functional groups selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. These functional groups can be used to attach the lipophilic moiety to an iRNA agent. In some embodiments, the lipophilic moiety contains saturated or unsaturated C6-C6 groups. 18 Hydrocarbon chains (e.g., straight-chain C6-C) 18(alkyl or alkenyl). In one embodiment, the lipophilic moiety contains saturated or unsaturated C. 16 Hydrocarbon chains (e.g., straight-chain C) 16 (alkyl or alkenyl).

[0100] In one embodiment of the present invention, the lipophilic portion is C6-C. 30 Acids (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenoic acid, etc.) or C6-C 30 Alcohols (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linolenic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexaenoic acid, retinol, vitamin E, cholesterol, etc.).

[0101] In one embodiment of the invention, the lipophilic portion may conjugate to the iRNA agent via direct attachment to the ribose of the iRNA agent. Alternatively, the lipophilic portion may conjugate to the iRNA agent via a linker or a vector.

[0102] In one embodiment of the invention, the lipophilic portion is conjugated to an iRNA agent via one or more linkers.

[0103] In one embodiment of the invention, the lipophilic portion is conjugated to a double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphate diester, sulfonamide bond, product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or carbamate.

[0104] In one embodiment of the invention, at least one connector is a redox-cleavable connector (such as a reductant connector, e.g., a disulfide group), an acid-cleavable connector (e.g., an hydrazone group, an ester group, an acetal group, or a ketal group), an esterase-cleavable connector (e.g., an ester group), a phosphatase-cleavable connector (e.g., a phosphate ester), or a peptidase-cleavable connector (e.g., a peptide bond).

[0105] In one embodiment of the invention, at least one adapter is a biolytic adapter selected from the group consisting of: DNA, RNA, disulfides, amides, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, or combinations thereof.

[0106] In one embodiment of the invention, the lipophilic portion is conjugated to a double-stranded iRNA agent via a carrier that replaces one or more nucleotides. The carrier can be a cyclic group or an acyclic group. In one embodiment, the cyclic group is selected from the group consisting of: pyrrolidinyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolinyl, isoxazolinyl, morpholinyl, thiazolinyl, isothiazolinyl, quinoxalinyl, pyridazinoneyl, tetrahydrofuranyl, and decahydronaphthalene. In one embodiment, the acyclic group is a portion based on a serine or diethanolamine backbone.

[0107] In one embodiment of the present invention, the lipophilic portion is C16, where C16 represents 2'-O-hexadecyl, a short lipid chain attached to siRNA, which is lipophilic and can interact with cell membranes or membrane proteins. The structural formula of the 2'-O-hexadecyl modified nucleotide is shown in formula (36).

[0108]

[0109] Equation (36) In one embodiment of the present invention, the C16 may be attached to any nucleotide of the positive strand.

[0110] In one embodiment of the present invention, the structural formula of the 2'-O-C22 modified nucleotide is shown in formula (37).

[0111]

[0112] Equation (37) The lipophilic portion is described in more detail in WO2019217459A1 and WO2024216155A1 (the entire contents of which are incorporated herein by reference).

[0113] In one embodiment of the present invention, the siRNA introduces modified nucleotides by using nucleotide monomers with corresponding modifications.

[0114] The present invention also provides a product for inhibiting ACVR1C, the product comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the above-mentioned siRNA or the above-mentioned modified siRNA.

[0115] In one embodiment of the present invention, the product is a pharmaceutical composition or a kit.

[0116] In one embodiment of the present invention, the product is a pharmaceutical composition, and the pharmaceutically acceptable carrier can be a carrier conventionally used in the field of siRNA delivery, such as, but not limited to, magnetic nanoparticles (e.g., 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 the following: phosphate), PPEEA, and poly(2-dimethylaminoethylmethacrylate), PDMAEMA, and their derivatives.

[0117] In one embodiment of the present invention, there are no special requirements for the content of siRNA and pharmaceutically acceptable carrier; the content of each component can be the conventional content.

[0118] In one embodiment of the present invention, the weight ratio of the active ingredient to the pharmaceutically acceptable carrier in the pharmaceutical composition is 1:(1~500).

[0119] In one embodiment of the present invention, the weight ratio of the active ingredient to the pharmaceutically acceptable carrier in the pharmaceutical composition is 1:(1~50).

[0120] In one embodiment of the invention, the pharmaceutical composition may further contain other pharmaceutically acceptable excipients, which may be one or more of various formulations or compounds conventionally used in the art.

[0121] In one embodiment of the invention, the other pharmaceutically acceptable excipients may include at least one of pH buffers, protectants, and osmotic pressure regulators.

[0122] In one embodiment of the present invention, the pH buffer may be 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.

[0123] In one embodiment of the present invention, the protective agent may be at least one selected from inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose.

[0124] In one embodiment of the present invention, the content of the protective agent may be 0.01 to 30% by weight, based on the total weight of the pharmaceutical composition.

[0125] In one embodiment of the present invention, the osmotic pressure regulator may be sodium chloride and / or potassium chloride.

[0126] In one embodiment of the present invention, the content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200 to 700 mOSM / L. The content of the osmotic pressure regulator can be easily determined by those skilled in the art based on the required osmotic pressure.

[0127] In one embodiment of the invention, the pharmaceutical composition can be administered by any method known in the art, including but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrasheathal), intravenous, intramuscular, intravitreal, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and local (including oral and sublingual) administration.

[0128] In one embodiment of the present invention, the pharmaceutical composition may be a liquid formulation, such as an injection; or it may be a lyophilized powder for injection, which is mixed with liquid excipients to form a liquid formulation when administered.

[0129] In one embodiment of the invention, the liquid formulation may be administered, but is not limited to, subcutaneous, intramuscular, intradermal, or intravenous injection; it may also be administered, but is not limited to, via spray to the lungs, or via spray to other organs or tissues (such as the liver); it may also be administered, but is not limited to, via intradermal injection, application, or spray to the skin; or it may be, but is not limited to, via intrathecal or intravitreal injection, optionally infused into the brain (e.g., the striatum), for example, via a continuous pump infusion.

[0130] In one embodiment of the present invention, the pharmaceutical composition may be in the form of a liposome formulation.

[0131] In one embodiment of the invention, the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), an auxiliary lipid, and / or a polyethylene glycol-modified lipid.

[0132] In one embodiment of the present invention, the organic amine, the auxiliary lipid, and the polyethylene glycol-modified lipid may be selected from one or more of the amine-containing transfection compounds or their pharmaceutically acceptable salts or derivatives, auxiliary lipids, and polyethylene glycol-modified lipids described in CN108220295B (which is incorporated herein by reference in its entirety).

[0133] In one embodiment of the invention, the kit further comprises a pharmaceutically acceptable carrier and / or excipients.

[0134] In one embodiment of the invention, the siRNA, pharmaceutically acceptable carrier, and / or excipients in the kit may be present individually, in a mixture of two or more of them, or in the form of a final pharmaceutical composition.

[0135] In one embodiment of the present invention, the pharmaceutically acceptable carrier in the kit is an amine-containing compound, an auxiliary lipid, or a polyethylene glycol-modified lipid.

[0136] In one embodiment of the invention, the pharmaceutically acceptable carrier in the kit is either a mixture or exists independently.

[0137] In one embodiment of the invention, the siRNA, pharmaceutically acceptable carrier, and / or excipients in the kit are provided in liquid, dry, or lyophilized form.

[0138] In one embodiment of the invention, the siRNA, pharmaceutically acceptable carriers, and / or excipients in the kit are substantially pure and / or sterile.

[0139] In one embodiment of the invention, the kit includes a container for providing siRNA, one or more containers for providing amine-containing compounds, auxiliary lipids, and polyethylene glycol-modified lipids, and optionally, a container for providing excipients.

[0140] In one embodiment of the invention, the kit further comprises one or more components necessary or beneficial for a particular application, the components being selected from: One or more components for achieving the desired cell transfection; One or more components used to diagnose, treat, or prevent a specific disease or physical disorder; One or more buffers; Positive or negative control samples; Excipients, stabilizers, or preservatives.

[0141] In one embodiment of the invention, the one or more components for diagnosing, treating or preventing a specific disease or physical disorder are one or more additional therapeutic compounds or compositions, or one or more diagnostic reagents.

[0142] In one embodiment of the present invention, the kit further comprises one or more of sterile water, physiological saline, and PBS.

[0143] The present invention also provides the use of the above-mentioned siRNA or the above-mentioned product in the preparation of products for the prevention, diagnosis and / or treatment of pathological conditions or diseases caused by ACVR1C.

[0144] The present invention also provides the use of the above-mentioned siRNA or the above-mentioned product in the preparation of products for the prevention, diagnosis and / or treatment of pathological conditions or diseases caused by ACVR1C.

[0145] In one embodiment of the present invention, the ACVR1C-related disease is obesity, tumors, or other diseases related to ACVR1C signal transduction.

[0146] The technical solution of this invention has the following advantages: This invention provides a modified siRNA for inhibiting ACVR1C, wherein the modifications include methoxy modification, fluorination modification, thiophosphate linkage, and thermally unstable modification. The siRNA exhibits high inhibitory activity against ACVR1C when its activity is detected in vitro. Detailed Implementation

[0147] 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.

[0148] In the following embodiments, ACVR1C mRNA refers to mRNA having the sequences shown in GenBank accessions XM_005573224.4, XM_005573230.4, NM_145259.3, NM_001111031.2, NM_001111032.2, NM_001033369.3, NM_001111030.1, and NM_139090.2. Further, unless otherwise specified, the term "target gene" as used in this disclosure refers to a gene capable of transcribing the aforementioned ACVR1C mRNA, and the term "target mRNA" refers to the aforementioned ACVR1C mRNA.

[0149] 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.

[0150] In the following examples, 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; and lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are modified with a thiophosphate group.

[0151] In the following examples, "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2'-hydroxyl group of the ribosyl group with another group, or a nucleotide whose bases are modified bases. "Fluorinated nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with fluorine, and "non-fluorinated nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2'-hydroxyl group of the ribosyl group with a non-fluorinated group. "Nucleotide analog" refers to a group that can replace a nucleotide in nucleic acids but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Examples include isonucleotides, bridged nucleic acids (BNA), or acyclic nucleotides. "Methoxylated nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with a methoxy group.

[0152] In the following examples, the terms "complementary" or "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired with the bases of the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the strand can be inferred from the sequence of its complementary strand.

[0153] In the following examples, particularly in describing methods for preparing siRNA, pharmaceutical compositions, or siRNA conjugates of this disclosure, unless otherwise specified, a nucleoside monomer refers to a modified or unmodified RNA phosphoramidites (sometimes also called nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis, depending on the type and sequence of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphoramidite solid-phase synthesis is a method known to those skilled in the art for RNA synthesis. All nucleoside monomers used in this disclosure are commercially available.

[0154] In the following embodiments, "coupling" refers to the covalent connection between two or more chemical parts, each with a specific function; correspondingly, "coupling" refers to a compound formed by the covalent connection between these chemical parts. Further, "siRNA conjugate" refers to a compound formed by the covalent attachment of one or more chemical parts with specific functions to siRNA. The term "siRNA conjugate" should be understood, depending on the context, as a collective term for multiple siRNA conjugates or a siRNA conjugate represented by a specific chemical formula. In the context of this disclosure, "coupling molecule" should be understood as a specific compound that can be reactively coupled to siRNA to ultimately form the siRNA conjugate of this disclosure.

[0155] In the following examples, "optional" or "optionally" means that the event or condition described thereafter may or may not occur, and the description includes both the possibility that the event or condition occurs and the possibility that it does not occur. For example, "optionally substituted" "alkyl" includes "alkyl" and "substituted alkyl" as defined below. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is spatially impractical, synthetically infeasible, and / or inherently unstable.

[0156] In the following embodiments, the terms “treatment,” “relief,” or “improvement” may be used interchangeably herein. These terms refer to methods of achieving beneficial or desired outcomes, including but not limited to therapeutic benefits. A “therapeutic benefit” means the eradication or improvement of the underlying disorder being treated. Furthermore, a therapeutic benefit is achieved by eradicating or improving one or more physical symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be suffering from the underlying disorder.

[0157] In the following examples, "prevention" and "protection" are used interchangeably. These terms refer to methods of obtaining a beneficial or desired outcome, including but not limited to preventive benefits. To obtain a "preventive benefit," the composition may be given to a subject at risk of developing a specific disease, or to a subject who reports one or more pathological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0158] Unless otherwise specified, the reagents and culture media used in the following examples are commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are performed in accordance with the methods described in Molecular Biology (4th Edition) (Alexander McLennan et al., 2019).

[0159] The siRNAs involved in the following examples are siRNA sequences synthesized via phosphoramide solid-phase synthesis.

[0160] In the examples described below, when transfecting cells with siRNA, siRNA conjugates, or siRNA or siRNA conjugates targeting the ACVR1C gene, Lipo3000 or RNAiMAX (purchased from Invitrogen) were used as transfection reagents. Specific procedures were followed 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. Specific procedures were followed according to the manufacturer's instructions.

[0161] Unless otherwise specified, all reagent ratios provided below are calculated on a volume ratio (v / v).

[0162] Example 1: A siRNA for inhibiting ACVR1C This embodiment provides an siRNA for inhibiting ACVR1C. The nucleotide sequence of the siRNA is designed based on the target mRNA, as shown in Table 1. The siRNA molecule with the following sequence was synthesized by Suzhou GeneGene Co., Ltd.

[0163] Table 1. Nucleotide sequences of siRNAs that inhibit ACVR1C

[0164] Example 2: A modified siRNA for inhibiting ACVR1C This embodiment provides a modified siRNA for inhibiting ACVR1C. The siRNA is modified based on Example 1, and the modified sequence is shown in Tables 2, 3-1 and 3-2.

[0165] It should be noted that the 5' end / 3' end of the sense strand can also be connected to two nucleotides complementary to the 3' end / 5' end of the antisense strand; and the 3' end / 5' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region.

[0166] For example, ACVR1C-1M1 is a modified version of the sequence ACVR1C-1. Similarly, ACVR1C-113M1, ACVR1C-113AM1, ACVR1C-113AM3, ACVR1C-113AM4, and ACVR1C-113AM6 are modified versions of the sequence ACVR1C-113. Finally, ACVR1C-459AM7 is a modified version of the sequence ACVR1C-459.

[0167] In Tables 2, 3-1, and 3-2, 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 string is replaced by a 2'-deoxyribonucleotide (for example, when its ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide is used instead of uracil ribonucleotide). The nucleotides replaced by 2'-deoxyribonucleotides include four types: A(d), C(d), G(d), and T(d), where A(d) represents 2'-deoxyadenosine-3'-phosphate, C(d) represents 2'-deoxyadenosine-3'-phosphate, and T(d) represents 2'-deoxyadenosine-3'-phosphate. '-Deoxycytidine-3'-phosphate, G(d) represents 2'-deoxyguanosine-3'-phosphate, T(d) represents 2'-deoxythymidine-3'-phosphate; string (GNA) indicates that the nucleotide adjacent to the left of this string is a diol-modified nucleotide, A(GNA) represents adenosine-diol nucleotide (GNA), C(GNA) represents cytidine-diol nucleotide (GNA), G(GNA) represents guanosine-diol nucleotide (GNA), T(GNA) represents thymidine-diol nucleotide (GNA), U(GNA) represents uridine-diol nucleotide (GNA); string (invAb) indicates that the nucleotide adjacent to the left or right of this string is linked to an inverse abase-free nucleotide (invAb group); string VP indicates that the nucleotide adjacent to the right of this string is a vinyl phosphate-modified nucleotide.

[0168] Table 2. Nucleotide sequences of modified siRNAs that inhibit ACVR1C

[0169] Table 3-1. Nucleotide sequences of modified siRNAs that inhibit ACVR1C

[0170] Table 3-2. Nucleotide sequences of modified siRNAs that inhibit ACVR1C

[0171] Experimental Example 1: Detection of the on-target activity of modified siRNA used to inhibit ACVR1C This experimental example provides an assay for detecting the on-target activity of modified siRNA used to inhibit ACVR1C. A plasmid vector, psiCHECK2, was constructed for the assay. psiCHECK2 is a plasmid vector that monitors changes in the expression of a target gene fused with a reporter gene. This vector uses Renal luciferase as the primary reporter gene. The target fragment is cloned into the multiple cloning site downstream of the translation stop codon of Renal luciferase. The synthesized siRNA triggers an RNAi process targeting the target gene, leading to the cleavage and subsequent degradation of the fusion mRNA. By detecting changes in Renal luciferase activity, the targeting relationship between the siRNA and the target gene fragment can be determined. The experimental procedure is as follows: Step 1: Construct the detection plasmid ACVR1C-psiCHECK2 Using psiCHECK TM -2(Promega TMThe plasmid was constructed to detect the insertion sequence, which contained the sequence shown in SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321, SEQ ID NO: 322, SEQ ID NO: 323, or SEQ ID NO: 324 (see Table 4 for the sequences, which were purchased from Hongxun Biotechnology Co., Ltd.). The insertion sequence was obtained by splicing the target sequence, which was completely complementary to all nucleotide sequences of the antisense strand of the siRNA shown in Table 1. A single copy of the spliced ​​sequence was cloned into psiCHECK. TM -2 Xho I / Not I sites of plasmid were used to obtain detection plasmids ACVR1C-psiCHECK2-1~ACVR1C-psiCHECK2-6; Table 4 Insertion Sequence

[0172] Step 2: Cell Culture and Transfection Add 5 μL of the siRNA listed in Table 2 to each well of a 96-well plate. Add 12.5 μL of Opti-MEM containing 20 ng of hACVR1C-psiCHECK2-1 (or hACVR1C-psiCHECK2-2, hACVR1C-psiCHECK2-3, hACVR1C-psiCHECK2-4, hACVR1C-psiCHECK2-5, or hACVR1C-psiCHECK2-6) of the detection plasmid to each well. Add 32.5 μL of Opti-MEM (Gibco) to each well. Add 0.3 μL of Lipofectamine 2000 (purchased from Invitrogen, catalog number 11668-019) to each well. Incubate at room temperature (22°C) for 15 minutes to obtain a mixture. Add 50 μL of 1×10⁻⁶ siRNA to each well of the above mixture. 4 Two 293T cells were cultured in DMEM complete medium (purchased from Transgen Biotech, catalog number FI101-01) at 37°C for 24 h for subsequent dual-luciferase assays. Experiments were performed at siRNA final concentrations of 10 nM and 1 nM.

[0173] Specifically, siRNAs corresponding to ACVR1C-1M1~ACVR1C-23M1 were used with Opti-MEM containing the hACVR1C-psiCHECK2-1 detection plasmid; siRNAs corresponding to ACVR1C-24M1 were used with Opti-MEM containing the hACVR1C-psiCHECK2-2 detection plasmid; siRNAs corresponding to ACVR1C-25M1~ACVR1C-31M1 were used with Opti-MEM containing the hACVR1C-psiCHECK2-3 detection plasmid; siRNAs corresponding to ACVR1C-32M1~ACVR1C-97M1 were used with Opti-MEM containing the hACVR1C-psiCHECK2-4 detection plasmid; and siRNAs corresponding to ACVR1C-98M1~ACVR1C-129M1 were used with Opti-MEM containing the hACVR1C-psiCHECK2-6 detection plasmid.

[0174] Step 3: Dual-luciferase assay Dilute the 5× lysis buffer in the Dual Luciferase Assay Kit (purchased from Promega, catalog number E2940) with water to make 1× lysis buffer. Take the cells obtained from step two, discard the supernatant, dilute each well with PBS buffer (purchased from Hyclone, catalog number SH30256.01) and wash twice. Add 50 μL of 1× lysis buffer to each well of each cell plate and lyse at room temperature (22℃) for 20 min to obtain lysed cell plates. Take 30 μL / well of lysis buffer from each lysed cell plate and add it to an opaque 96-well detection plate. Take the dual-luciferase assay kit, prepare substrate 1 and substrate 2 according to the instructions, and add 30 μL of each substrate to each well of the opaque 96-well detection plate. After each addition of substrate, use a multi-mode microplate reader to detect the values ​​of firefly luciferase and Renilla luciferase.

[0175] The luminescence ratio of each well in the ELISA plate was calculated as Renilla / Firefly. The luminescence ratio of each test group or control group was the average of the luminescence ratios of the three culture wells. Using the luminescence ratio of the control group as a baseline, the luminescence ratios of each test group were normalized to obtain the ratio R of luminescence ratio (test) / luminescence ratio (control), which represents the expression level of the Renilla reporter gene, i.e., its relative residual activity. The inhibition rate of siRNA was (1-R)×100%.

[0176] The results of the on-target activity of siRNA inhibiting ACVR1C are shown in Table 5.

[0177] Table 5. Target activity of siRNA

[0178] Experimental Example 2: Detection of the on-target activity of modified siRNA used to inhibit ACVR1C The on-target activity of the modified siRNA of the preferred sequence in Experiment 1 was detected. The experimental procedure was the same as in Experiment 1, but the final concentration of siRNA was set to 0.1 nM.

[0179] The experimental results are shown in Table 6.

[0180] Table 6. Target activity of siRNA

[0181] Experimental Example 3: Detection of the on-target activity of modified siRNA used to inhibit ACVR1C Based on the results in Experiment 2, the on-target activity of the modified siRNA in the sequence in Table 3-1 of Example 2 was detected. The experimental steps were the same as in Experiment 1, but the detection plasmid used was shown in Table 7, and the final concentration of siRNA was set to 0.1 nM.

[0182] Table 7. Sequence-to-Vector Table

[0183] The experimental results are shown in Table 8.

[0184] Table 8. Target activity of siRNA

[0185] Experiment Example 4: Detection of the on-target activity of modified siRNA used to inhibit ACVR1C The on-target activity of the modified siRNA sequences in Table 3-2 of Example 2 was detected. The experimental steps were the same as in Example 1, but the detection plasmid used was shown in Table 9, and the final concentration of siRNA was set to 0.1 nM.

[0186] Table 9. Sequence-to-Vector Table

[0187] The experimental results are shown in Table 10.

[0188] Table 10. Target activity of siRNA

[0189] Experimental Example 5: Activity assay of modified siRNA for inhibiting ACVR1C in SK-MEL-28 cells This embodiment provides a method for determining the relative inhibitory level of the siRNA compound of the present invention on ACVR1C mRNA in SK-MEL-28 cells using quantitative real-time PCR (qPCR). The experiment for detecting the activity of the ACVR1C-modified siRNA in SK-MEL-28 cells is as follows: 1. SK-MEL-28 cells (human malignant melanoma cells, purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) were seeded into MEM medium (purchased from Gibco, catalog number 11095-080) containing 10% (v / v) fetal bovine serum (FBS, purchased from Hyclone), 1% (v / v) NEAA (MEM non-essential amino acid solution, purchased from Gibco), 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), and cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 48 days. h; After culture, SK-MEL-28 cells were digested with trypsin (purchased from GIBCO, catalog number 25200-072); after digestion, the cells were first rinsed with PBS buffer, and then resuspended in MEM medium to obtain a cell concentration of 3×10⁻⁶. 5 Cell suspension of cells / mL; 2. Dilute the different siRNAs listed in Tables 3-1 and 3-2 using opti-MEM (Gibco, catalog number 31985-070) to obtain siRNA dilutions containing different siRNAs; mix 25 μL opti-MEM with 0.25 μL Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, catalog number 11668-019) to obtain transfection reagent dilutions; mix 25 μL of each siRNA dilution with the transfection reagent dilutions and incubate at room temperature (25℃) for 15 min to obtain transfection solutions containing different siRNAs; 3. After seeding the cell suspension into 96-well plates at a seeding rate of 50 μL / well, set up MOCK group (MOCK group is the transfection reagent control group), ACVR1C-11AM1 group to ACVR1C-113AM6 group in 96-well plates, with 3 replicates in each group. 4. After setting up, add 50 μL of transfection buffer containing different siRNAs to the wells of each experimental group (ACVR1C-11AM1 experimental group was given transfection buffer containing ACVR1C-11AM1, ACVR1C-113AM6 experimental group was given transfection buffer containing ACVR1C-113AM6, and so on, with the final concentration of siRNA in the wells being 1 nM or 0.1 nM), and culture in a 5% (v / v) CO2, 37℃ cell culture incubator; after 48 h of transfection, discard the cell supernatant, wash the cells with PBS, and then proceed with subsequent treatments.

[0190] 5. Cell sample processing: Prepare the lysis buffer according to Table 11: Table 11. Cell lysis reaction system

[0191] Add 50 μL of the prepared lysis buffer to each well of the cell culture plate, mix thoroughly, and let stand at room temperature for 5 min to lyse the cells.

[0192] After lysis, add 5 μL of stop solution to each well of the cell culture plate, mix well, and let stand at room temperature for 2 min to terminate the reaction.

[0193] 6. Preparation of qPCR reaction system For the qPCR reaction system, 50 μL of the cell lysis buffer was diluted with 150 μL of DEPC H2O to prepare RNA templates. 13 μL of qPCR reaction system was prepared according to Table 12. The primer and probe sequences are shown in Table 13. Detection: Real-time quantitative PCR reaction was performed on an LC480. The program is shown in Table 14. The fluorescence signal values ​​of the target gene ACVR1C and the internal reference gene GAPDH in the product were collected by the real-time quantitative PCR instrument to obtain the Ct values ​​of the target gene ACVR1C and the internal reference gene GAPDH.

[0194] Table 12. One-step RT-qPCR probe method reaction system

[0195] Table 13. Primer Information

[0196] Table 14. RT-qPCR reaction procedure

[0197] The relative quantification of the target gene ACVR1C in each test group was performed using the comparison ΔCt 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.

[0198] Using the control group as a baseline, the expression level of ACVR1C mRNA in the test group was normalized, and the expression level of ACVR1C mRNA in the control group was defined as 100%.

[0199] The relative expression level of ACVR1C mRNA in the test group was 2. -ΔΔCt(测试组) ×100% The inhibition rate of ACVR1C mRNA in the test group = 1 - the relative expression level of ACVR1C mRNA in the test group.

[0200] The ACVR1C mRNA level was compared with the internal reference gene GAPDH, and the value was normalized to the mean of the control group. The data were expressed as a percentage relative to the control group and presented as the mean plus the standard deviation.

[0201] The experimental results are shown in Table 15.

[0202] Table 15. Inhibitory levels of siRNA in SK-MEL-28 cells at 1 nM / 0.1 nM mRNA levels.

[0203] Example 3: A modified siRNA for inhibiting ACVR1C This embodiment provides a modified siRNA for inhibiting ACVR1C. The siRNA is modified based on Example 1, and the modified sequence is shown in Table 16.

[0204] It should be noted that the 5' end / 3' end of the sense strand can also be connected to two nucleotides complementary to the 3' end / 5' end of the antisense strand; and the 3' end / 5' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region.

[0205] Taking ACVR1C-108L23M2C22VP, ACVR1C-108L23M2VP, ACVR1C-108L23M2VP-C22-2, ACVR1C-108L23M2VP-C22-3, and ACVR1C-108L23M2VP-C22-4 as examples, they are modifications based on the sequence ACVR1C-108.

[0206] In Table 16, 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 methoxylated 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 string is replaced by a 2'-deoxyribonucleotide (for example, when its ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide is used instead of uracil ribonucleotide). The nucleotides replaced by 2'-deoxyribonucleotides include four types: A(d), C(d), G(d), and T(d), where A(d) represents 2'-deoxyadenosine-3'-phosphate, C(d) represents 2'-deoxycytidine-3'-phosphate, and G(d) represents 2'- Deoxyguanosine-3'-phosphate, T(d) represents 2'-deoxythymidine-3'-phosphate; the string (GNA) indicates that the nucleotide adjacent to the left of this string is a diol-modified nucleotide, A(GNA) represents adenosine-diol nucleotide (GNA), C(GNA) represents cytidine-diol nucleotide (GNA), G(GNA) represents guanosine-diol nucleotide (GNA), T(GNA) represents thymidine-diol nucleotide (GNA), U(GNA) represents uridine-diol nucleotide (GNA); the string (invAb) indicates that the nucleotide adjacent to the left or right of this string is linked to an inverse abase-free nucleotide (invAb group); the string VP indicates that the nucleotide adjacent to the right of this string is a vinyl phosphate-modified nucleotide; the string mC22 indicates that the nucleotide adjacent to the left of this string is a 2'-O-C22-modified nucleotide.

[0207] Table 16 Nucleotide sequences of modified siRNAs that inhibit ACVR1C

[0208] Experimental Example 6: Activity assay of modified siRNA for inhibiting ACVR1C in SK-MEL-28 cells This experimental example provides a determination of the relative inhibitory level of the siRNA compound of the present invention on ACVR1C mRNA in SK-MEL-28 cells using quantitative real-time PCR (qPCR).

[0209] For the activity detection experiment of the sequences in Table 16 of Example 3 in SK-MEL-28 cells, the experimental procedure was the same as in Example 5, but the final concentrations of siRNA were set to 1 nM and 0.1 nM, respectively.

[0210] The experimental results are shown in Table 17.

[0211] Table 17. Inhibitory levels of siRNA in SK-MEL-28 cells at 1 nM / 0.1 nM mRNA levels.

[0212] Experimental Example 7: The activity of modified siRNA used to inhibit ACVR1C in animals This experimental example provides a determination of the relative inhibitory level of the siRNA compound of the present invention on ACVR1C mRNA in animals by real-time quantitative PCR.

[0213] Twenty-four male C57BL / 6J mice were selected, with three mice in each group. Each group of mice was given a single dose of 5 mg / kg of siRNA conjugate (the siRNA conjugates measured are shown in Table 16) or physiological saline (physiological saline was the control). On the 14th day after administration, the mice were sacrificed, and samples of white adipose tissue around the gonads were collected to detect the ACVR1C mRNA expression level, as shown in Table 18.

[0214] Table 18. Relative inhibitory levels of siRNA on ACVR1C mRNA in animals.

[0215] The specific testing steps are as follows: Step 1: RNA extraction 1) Take 20 mg of mouse tissue, place it in RNA protection solution, incubate overnight at 4°C, discard the RNA protection solution, add 1 mL of Trizol Lysis Buffer (purchased from Life Technology, catalog number 410701), and grind the tissue at low temperature to lyse it. After thorough grinding, transfer it to an RNase-free 1.5 ml centrifuge tube; shake vigorously for 15 s to fully lyse the tissue cells, and let it stand at room temperature for 5 min. 2) Add 200 μL of chloroform (purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., catalog number: 20140925); shake vigorously for 20 seconds, let stand at room temperature for 3 minutes; centrifuge at 12000×g for 20 minutes at 4℃. 3) After centrifugation, carefully remove the centrifuge tubes to the centrifuge tube rack, transfer the supernatant to a new 2.0 mL centrifuge tube, add 1.5 times the volume of anhydrous ethanol (purchased from Jiangsu Qiangsheng Functional Chemical Co., Ltd., product number: 20210802) to the supernatant, and mix by inverting. 4) Take a purification column with a collection tube (purchased from VWI, catalog number 11822AG0627), add 700 μL of the mixture from step 3), let stand for 2 min; centrifuge at 10000×g for 1 min at 4℃, discard the filtrate; repeat the above steps with the remaining mixture. 5) Add 700 μL of 80% ethanol to the purification column, centrifuge at 10000×g for 1 min at 4℃, and discard the filtrate; 6) Add 700 μL of 80% ethanol to the purification column, centrifuge at 10000×g for 1 min at 4℃, and discard the filtrate; 7) Centrifuge the purification column at 4℃, 10000×g for 2 min (empty). 8) After centrifugation, remove the purification column with the collection tube, discard the collection tube, put the purification column into a new 1.5 mL centrifuge tube, add 100 μL of DEPC water to the purification column, let it stand at room temperature for 2 min; centrifuge at 4℃, 10000×g for 1 min to obtain RNA.

[0216] Step 2: RNA reverse transcription The experimental procedure was performed using HiScript III RT SuperMix for qPCR (purchased from Novizan, catalog number: R323-01) following 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. Step 3: Preparation of qPCR reaction system For each reverse transcription reaction system, 4 μL of the above-mentioned cDNA-containing solution was used as a template. Using the reagents provided by 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 19. Primer1 and Primer2 are the PCR primer sequences for amplifying the target gene ACVR1C and the internal reference gene GAPDH, respectively (as shown in Table 20). Each qPCR reaction system was placed on an LC480 instrument (Roche) and amplified using a three-step method. The reaction program is shown in Table 21. Product W containing amplified target gene ACVR1C and internal reference gene GAPDH was obtained. Product W was then incubated sequentially at 95℃ for 10 s, 60℃ for 1 min, and 95℃ for 15 s. The melting curves of target gene ACVR1C and internal reference gene GAPDH in product W were collected by a real-time fluorescence quantitative PCR instrument to obtain the Ct values ​​of target gene ACVR1C and internal reference gene GAPDH.

[0217] Table 19 RNA Amplification Reaction System

[0218] Table 20. Primer Information

[0219] Table 21. RT-qPCR reaction procedure

[0220] The relative quantification of the target gene ACVR1C in each test group was performed using the comparison ΔΔCt method, and the results are shown in Table 22 below.

[0221] Table 22 Relative expression levels of ACVR1C mRNA in perigonadal white adipose tissue

[0222] 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 ACVR1C, characterized in that, The siRNA contains a sense strand and an antisense strand, which are at least partially anticomplementary to form a double-stranded region; the sense strand of the siRNA contains at least 17 consecutive nucleotides that differ from any one of the nucleic acid sequences shown in any of SEQ ID NO. 1 to 159 by no more than 3 nucleotides; the antisense strand of the siRNA contains at least 17 consecutive nucleotides that differ from any one of the nucleic acid sequences shown in any of SEQ ID NO. 160 to 318 by no more than 3 nucleotides.

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

3. The siRNA as described in claim 2, characterized in that, The modified nucleotide is a compound formed by modifying the ribose of a nucleotide, or a compound formed by modifying the backbone between nucleotides, or a compound formed by modifying the bases on a nucleotide, or a compound formed by modifying the ends of siRNA.

4. The siRNA as described in claim 2 or 3, characterized in that, The modified nucleotide is selected from at least one of the following: 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, phosphate thioester modified nucleotides, deoxynucleotides, 2'-deoxynucleotides, 3'-terminal deoxy-thymidine nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, baseless nucleotides, reverse nucleotides, reverse baseless nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy - Modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl- modified nucleotides, morpholino nucleotides, aminophosphates, nucleotides containing non-natural bases, tetrahydropyran modified nucleotides, 1,5-dehydrohexyl modified nucleotides, cyclohexenyl modified nucleotides, glycol nucleic acids, open-ring nucleotides, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimics, terminal nucleotides linked to cholesterol derivatives, or terminal nucleotides linked to dodecanoic acid bisdecamide groups.

5. The siRNA according to any one of claims 1 to 4, characterized in that, Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence. Specifically, in the sense strand, positions 7, 8, and 9, or positions 5, 7, 8, and 9, or positions 9, 10, and 11, or positions 7, 9, 10, and 11, are fluorinated nucleotides, while the remaining nucleotide groups in the sense strand are non-fluorinated nucleotides. In the antisense strand, positions 2, 6, 12, 14, and 16, or positions 2, 8, 12, 14, and 16, or positions 2, 9, 12, 14, and 16, or positions 2, 10, 12, and 14, are fluorinated nucleotides. The nucleotides at positions 16, 2, 6, 10, 12, 14, 2, 8, 10, 14, 16, 2, 6, 10, 14, 16, 2, 4, 12, 14, 16, 2, 6, 14, 16, 2, 10, 12, 14, 16, 18, 20, 2, 6, 8, 14, 16, 14, 10, 12, 14, 16, 18, 20, 2, 14, 16 are fluorinated nucleotides, and the nucleotide groups at the remaining positions in the antisense strand are each non-fluorinated nucleotide groups. The non-fluorinated modified nucleotide group is independently selected from one of the nucleotide groups or nucleotide analog groups formed by replacing the hydroxyl group at the 2' position of the ribosome of the nucleotide group with a non-fluorinated group; the non-fluorinated modified nucleotide is a methoxy-modified nucleotide, a 2′-deoxynucleotide, a glycol nucleic acid, an inosine nucleotide, and a 2′-O-alkyl-modified nucleotide. Optionally, the 2'-O-alkyl-modified nucleotide is a 2'-O-C22-modified nucleotide or a 2'-O-hexadecyl-modified nucleotide.

6. The siRNA according to any one of claims 1 to 5, characterized in that, In the direction from the 5' end to the 3' end, at least one nucleotide in the nucleotides at positions 2 to 18 of the antisense strand is a thermally unstable modified nucleotide; Optionally, at least one nucleotide in the nucleotides at positions 2, 5, 7, 10, 11, 12, 13, and 16 of the antisense strand is a thermally unstable modified nucleotide.

7. The siRNA according to any one of claims 1 to 6, characterized in that, In the direction from the 5' end to the 3' end, at least two nucleotides in the 2nd to 8th of the antisense strand are thermally unstable modified nucleotides; Optionally, the 5th and 7th positions of the antisense strand are nucleotides modified to be thermally unstable.

8. The siRNA according to any one of claims 1 to 7, characterized in that, The thermally unstable modified nucleotides are ethylene glycol nucleic acids and / or 2′-deoxynucleotides.

9. The siRNA according to any one of claims 1 to 8, characterized in that, The 2'-O-C22 modified nucleotides are located in the sense strand and / or antisense strand, with the direction from the 5' end to the 3' end. Optionally, the nucleotide modified by 2'-O-C22 is located at the 1st, 6th and / or 1st to the end of the positive strand.

10. The siRNA according to any one of claims 1 to 9, characterized in that, The 5' and / or 3' ends of the sense strand and / or antisense strand are also connected to a 2'-O-C22 modified nucleotide; Optionally, the 2'-O-C22 modified nucleotide is linked to the 3' end of the sense strand and / or antisense strand via a thiophosphate group.

11. The siRNA according to any one of claims 1 to 10, characterized in that, At least one of the phosphate ester groups in the phosphate-sugar backbone of at least one single chain of the sense chain and the antisense chain is a phosphate ester group with a modifying group; Optionally, the phosphate ester group having the modifying group includes a thiophosphate ester group; Optionally, the nucleotides at least the first and second, the second and third, and the penultimate and penultimate positions of the antisense strand are linked by phosphate thioester groups in the direction from the 5' end to the 3' end. Optionally, in the direction from the 5' end to the 3' end, at least the nucleotides at the 1st and 2nd, 2nd and 3rd, penultimate and penultimate, and penultimate and penultimate positions of the antisense strand are linked by phosphate thioester groups. Optionally, the nucleotides at least the first and second positions, and the second and third positions of the positive strand are linked by thiophosphate groups in the direction from the 5' end to the 3' end.

12. The siRNA according to any one of claims 1 to 11, characterized in that, The 5' and / or 3' ends of the siRNA's sense and / or antisense strands contain at least one reverse-abase-free nucleotide. Optionally, the reverse abase-free nucleotide is connected to the 5' and / or 3' ends of the sense and / or antisense strands via thiophosphate groups.

13. The siRNA according to any one of claims 1 to 12, characterized in that, The first nucleotide at the 5' and / or 3' end of the siRNA's sense and / or antisense strands is a vinyl phosphate-modified nucleotide.

14. The siRNA according to any one of claims 1 to 13, characterized in that, The 5' end / 3' end of the sense strand may also be connected to two nucleotides complementary to the 3' end / 5' end of the antisense strand; and the 3' end / 5' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region.

15. The siRNA according to any one of claims 1 to 14, characterized in that, One or more lipophilic portions coupled or conjugated at one or more internal or end positions on at least one strand of the sense or antisense strand of the siRNA.

16. The siRNA according to any one of claims 1 to 15, characterized in that, The siRNA has a ligand coupled to the end of either the sense or antisense strand.

17. A product for suppressing ACVR1C, characterized in that, The product comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is siRNA as described in any one of claims 1 to 16.

18. The use of the siRNA of any one of claims 1 to 16 or the product of claim 17 in the preparation of products for the prevention, diagnosis and / or treatment of pathological conditions or diseases caused by ACVR1C.

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