Sirna for inhibiting inhbe, and conjugate thereof and use thereof

Abstract

The present invention belongs to the field of biotechnology. Provided is an siRNA for inhibiting INHBE, and a conjugate thereof and the use thereof. Provided is an siRNA for inhibiting INHBE. Experimental results verify that the siRNA exhibits a high INHBE inhibitory activity. Further provided is a modified siRNA for inhibiting INHBE. The modification comprises methoxy modification, fluoro modification, phosphorothioate linkage, inverted abasic nucleotide modification, vinyl phosphate modification and deoxyribonucleotide substitution. Experimental results verify that the modified siRNA exhibits good INHBE inhibitory effects at concentrations ranging from 0.1 nM to 1 nM.

Description

siRNA and its conjugates for inhibiting INHBE and their applications

[0001] Cross-reference of related applications

[0002] This application claims priority to Chinese Patent Application No. 202510093642.9, filed on January 21, 2025, entitled "siRNA for inhibiting INHBE 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. 202511794316.7, filed on December 1, 2025, entitled "siRNA for inhibiting INHBE and its conjugates and applications," the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to siRNAs and their conjugates for inhibiting INHBE and their applications, and belongs to the field of biotechnology. Background Technology

[0004] Inhibin subunit βE (INHBE), encoded by the INHBE gene, is a member of the TGF-β (transforming growth factor β) protein superfamily and is involved in the regulation of various cellular processes, including cell proliferation, apoptosis, immune response, and hormone secretion. INHBE is mainly expressed in liver tissue, and studies have shown that INHBE expression is related to lipid distribution.

[0005] Building on this, both epidemiological and genetic studies have demonstrated the role of adipose tissue distribution in metabolic health and cardiovascular disease risk. Furthermore, abdominal obesity, characterized by an increased waist-to-hip ratio, is a risk marker for cardiovascular disease. Simultaneously, multiple genome-wide association studies have indicated that genetic variations associated with abdominal fat accumulation are linked to hepatic steatosis, a driver of metabolic diseases such as insulin resistance, dyslipidemia, and non-alcoholic steatohepatitis. Therefore, blocking INHBE expression may be beneficial for controlling abdominal obesity, and consequently, for the treatment of metabolic diseases and cardiovascular diseases related to fat distribution.

[0006] RNA interference (RNAi) is a highly conserved evolutionary phenomenon characterized by the efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA). Small interfering RNA (siRNA) is a 20-25 nucleotide long double-stranded RNA that mediates the silencing of specific genes through RNA interference (RNAi). In the RNAi pathway, siRNA interferes with gene expression by hybridizing with complementary mRNA molecules. This interference triggers mRNA degradation, thereby inhibiting the expression of specific genes. This specific regulation of gene expression allows siRNA to serve as a targeted therapeutic agent, specifically regulating the expression of disease-related genes to achieve therapeutic goals. Developing siRNA that can effectively inhibit INHBE expression would be a more effective and targeted treatment for metabolic diseases related to fat distribution and cardiovascular diseases. Summary of the Invention

[0007] To address the aforementioned problems, this application provides an siRNA for inhibiting INHBE 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 to SEQ ID NO.332 or SEQ ID NO.726 to SEQ ID NO.732 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.333 to SEQ ID NO.664 or SEQ ID NO.733 to SEQ ID NO.739 differing by no more than 3 nucleotides.

[0008] In one embodiment of this application, the nucleotide sequence of the sense strand of the siRNA is shown in any one of SEQ ID NO.1 to SEQ ID NO.332 or SEQ ID NO.726 to SEQ ID NO.732; the nucleotide sequence of the antisense strand of the siRNA is shown in any one of SEQ ID NO.333 to SEQ ID NO.664 or SEQ ID NO.733 to SEQ ID NO.739.

[0009] In one embodiment of this application, at least one nucleotide in the sense strand and / or antisense strand of the siRNA is a modified nucleotide.

[0010] In one embodiment of this application, all nucleotides in the sense and / or antisense strands 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 INHBE gene expression.

[0011] In one embodiment of this application, the modified nucleotide includes compounds formed by replacing the 2' hydroxyl group of the ribosyl group of a nucleotide with other groups, compounds formed by modifying the nucleotide with a thiophosphate group, and / or compounds formed by modifying the bases on the nucleotide.

[0012] In one embodiment of this application, the modified nucleotide includes fluorinated nucleotides, methoxylated nucleotides, phosphate-thioester linked nucleotides, glycolic acid (GNA) modified nucleotides, inverse baseless nucleotides, vinyl phosphate modified nucleotides, deoxyribonucleotide-substituted nucleotides, 2'-O-methoxyethyl (MOE) modified nucleotides, 2'-deoxy-nucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, 3'-deoxy-nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, baseless nucleotides, 2'-amino-modified nucleotides, and 2'-O-allyl-modified nucleotides. Nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinonucleotide-modified nucleotides, aminophosphate-modified nucleotides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl alcohol-modified nucleotides, cyclohexenyl-modified nucleotides, open-ring nucleotides (UNA)-modified nucleotides, nucleotides containing methyl phosphate groups, nucleotides containing 5'-phosphate groups, and / or nucleotides containing 5'-phosphate mimics.

[0013] In one embodiment of this application, the fluorinated nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with fluorine, and has the following structure (where Base represents a base):

[0014] In one embodiment of this application, the methoxy-modified nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with a methoxy group, having the following structure (where Base represents a base):

[0015] In one embodiment of this application, the nucleotides linked by the thiophosphate group refer to two adjacent nucleotides linked by a thiophosphate group.

[0016] The thiophosphate group has the following structure:

[0017] The nucleotide linked to the thiophosphate group has the following structure (where Base represents a base):

[0018] In one embodiment of this application, the glycol nucleic acid (GNA) modified nucleotide is a polymer similar to DNA or RNA, but its "backbone" is different, consisting 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);

[0019] The A(GNA) has the following structure:

[0020] The G(GNA) has the following structure:

[0021] The C(GNA) has the following structure:

[0022] The U(GNA) has the following structure:

[0023] The T(GNA) has the following structure:

[0024] In one embodiment of this application, the nucleotide modified with the inverse abase-free nucleotide refers to a nucleotide coupled with an inverse abase-free nucleotide (invAb), wherein the inverse abase-free nucleotide has the following structure:

[0025] In one embodiment of this application, 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);

[0026] The VPAms have the following structure:

[0027] The VPUms have the following structure:

[0028] The VPGms has the following structure:

[0029] The VPCms has the following structure:

[0030] In one embodiment of this application, 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):

[0031] In one embodiment of this application, the nucleotide modified with 2'-O-methoxyethyl (MOE) refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with 2'-O-methoxyethyl (MOE), which has the following structure (where Base represents a base):

[0032] In one embodiment of this application, 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, at least the 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; or,

[0033] The 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 5, 7, 8, and 9 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,

[0034] The 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, 10, 12, 14, 16, 18, and 20 of the antisense strand are fluorinated nucleotides; or,

[0035] The 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,

[0036] The 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, 14, and 16 of the antisense strand are fluorinated nucleotides; or,

[0037] The 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 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.

[0038] In one embodiment of this application, 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 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,

[0039] 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 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; or,

[0040] 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, 10, 12, 14, 16, 18, and 20 of the antisense strand are fluorinated nucleotides; or,

[0041] 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, 10, 14, and 16 of the antisense strand are fluorinated nucleotides; or,

[0042] 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, 14, and 16 of the antisense strand are fluorinated nucleotides; or,

[0043] The 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 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.

[0044] In one embodiment of this application, the methoxylated nucleotides are located in the antisense and sense strands of the nucleotide sequence. In the sense strand, the nucleotides that are not fluorinated, not glycolic acid (GNA) modified, and not deoxyribonucleotide substituted are all methoxylated nucleotides. In the antisense strand, the nucleotides that are not fluorinated, not glycolic acid modified, and not deoxyribonucleotide substituted are all methoxylated nucleotides.

[0045] In one embodiment of this application, 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 of the positive strand is attached to an inverse abase-free nucleotide (invAb group); and / or,

[0046] 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).

[0047] In one embodiment of this application, 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).

[0048] In one embodiment of this application, 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 trans vinyl phosphate group (VP group).

[0049] In one embodiment of this application, 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 trans vinyl phosphate group (VP group).

[0050] In one embodiment of this application, 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 nucleotides at positions 5 and 7 of the antisense strand are deoxyribonucleotide-substituted nucleotides; or,

[0051] 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.

[0052] In one embodiment of this application, the deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 5 and 7 of the antisense strand are deoxyribonucleotide-substituted nucleotides; or,

[0053] The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and the nucleotide at position 12 of the antisense strand is the deoxyribonucleotide-substituted nucleotide in the direction from the 5' end to the 3' end.

[0054] In one embodiment of this application, 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, and positions 2 to 1 ...

[0055] The phosphate-thioester linked 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 nucleotide at position 1 of the sense strand is linked to the inverted abasic nucleotide by a phosphate-thioester group, and the nucleotide at position 1-1-3 of the sense strand is linked to the inverted abasic nucleotide by a phosphate-thioester group; at least the nucleotides at positions 1-2, 2-3, 1-1-2-3, and 1-1-2-2-3 of the antisense strand are linked by a phosphate-thioester group; or,

[0056] The nucleotides linked by the thiophosphate group are located in the antisense strand 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, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by the thiophosphate group.

[0057] In one embodiment of this application, 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 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, and positions 2 to 1 ...

[0058] The phosphate-thioester linked 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 nucleotide at position 1 of the sense strand is linked to the inverted abasic nucleotide via a phosphate-thioester group, and the nucleotide at position 1-3 of the sense strand is linked to the inverted abasic nucleotide via a phosphate-thioester group; the nucleotides at positions 1-2, 2-3, 1-1-2, and 1-1-2 of the antisense strand are linked via phosphate-thioester groups; or,

[0059] The nucleotides linked by the thiophosphate group are located in the antisense strand of the nucleotide sequence, and the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate of the antisense strand are linked by the thiophosphate group in the direction from the 5' end to the 3' end.

[0060] This application also provides an siRNA conjugate for inhibiting INHBE, the siRNA conjugate containing the above-mentioned siRNA and a conjugating group conjugated to the siRNA.

[0061] In one embodiment of this application, the conjugation group comprises a ligand formed from a targeting molecule or a derivative thereof; the targeting molecule comprises galactose and / or N-acetylgalactosamine (GalNAc).

[0062] In one embodiment of this application, the conjugating group is N-glucose amino acid (N-Acetylgalactosamine, GalNAc).

[0063] In one embodiment of this application, 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.

[0064] In one embodiment of this application, the conjugation site of the siRNA and the conjugating group is located at the 3' end of the siRNA's positive strand.

[0065] In one embodiment of this application, the conjugating group is conjugated to the 3' end of the siRNA positive strand via a phosphodiester bond.

[0066] In one embodiment of this application, the siRNA conjugate has the following structure:

[0067] This application also provides an antisense oligonucleotide (ASO) for suppressing INHBE, said antisense oligonucleotide comprising a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO. 675 to SEQ ID NO. 725.

[0068] In one embodiment of this application, the nucleotide sequence of the antisense oligonucleotide is shown in any one of SEQ ID NO.675 to SEQ ID NO.725.

[0069] In one embodiment of this application, at least one nucleotide of the antisense oligonucleotide is a modified nucleotide.

[0070] In one embodiment of this application, all nucleotides in the antisense oligonucleotide are modified nucleotides, and these modifications on the nucleotide groups do not cause the function of the antisense oligonucleotide of this disclosure in inhibiting INHBE gene expression to be significantly weakened or lost.

[0071] In one embodiment of this application, the modified nucleotide includes compounds formed by replacing the 2' hydroxyl group of the ribosyl group of a nucleotide with other groups, compounds formed by modifying the nucleotide with a thiophosphate group, and / or compounds formed by modifying the bases on the nucleotide.

[0072] In one embodiment of this application, the modified nucleotide includes nucleotides linked by thiophosphate groups, nucleotides substituted with deoxyribonucleotides, and / or nucleotides modified with 2'-O-methoxyethyl (MOE).

[0073] In one embodiment of this application, the antisense oligonucleotide has at least positions 1 to 5 and positions 16 to 20 modified with 2'-O-methoxyethyl (MOE) in the direction from the 5' end to the 3' end.

[0074] In one embodiment of this application, the antisense oligonucleotide has nucleotides modified with 2'-O-methoxyethyl (MOE) at positions 1-5 and 16-20, in the direction from the 5' end to the 3' end.

[0075] In one embodiment of this application, the antisense oligonucleotide has at least positions 6 to 15 substituted with deoxyribonucleotides in the direction from the 5' end to the 3' end.

[0076] In one embodiment of this application, the antisense oligonucleotide has its 6th to 15th positions substituted with deoxyribonucleotides in the direction from the 5' end to the 3' end.

[0077] In one embodiment of this application, the antisense oligonucleotide is connected to adjacent nucleotides via phosphate thioester groups.

[0078] This application also provides a recombinant plasmid expressing the above-mentioned siRNA; or, the recombinant plasmid expressing the above-mentioned antisense oligonucleotide.

[0079] In one embodiment of this application, 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.

[0080] In one embodiment of this application, the recombinant plasmid is prepared by: designing shRNA based on siRNA; and ligating the shRNA with a linearized vector to obtain the recombinant plasmid.

[0081] This application also provides a host cell whose genome integrates the above-mentioned siRNA; or, whose genome integrates the above-mentioned antisense oligonucleotide; or, whose host cell carries the above-mentioned recombinant plasmid.

[0082] In one embodiment of this application, the host cell includes fungi, bacteria, plant cells, and / or animal cells.

[0083] This application also provides the use of the above-mentioned siRNA, the above-mentioned siRNA conjugate, the above-mentioned antisense oligonucleotide, the above-mentioned recombinant plasmid or the above-mentioned host cell in the preparation of a drug for the prevention and / or treatment of a disease, wherein the disease is a disease related to INHBE gene expression.

[0084] In one embodiment of this application, the diseases associated with INHBE gene expression include obesity, metabolic diseases associated with fat distribution, and / or cardiovascular diseases associated with fat distribution.

[0085] This application also provides a medicament for the prevention and / or treatment of a disease related to INHBE gene expression; the medicament comprises the above-mentioned siRNA, the above-mentioned siRNA conjugate, the above-mentioned antisense oligonucleotide, the above-mentioned recombinant plasmid, or the above-mentioned host cell.

[0086] In one embodiment of this application, the diseases associated with INHBE gene expression include obesity, metabolic diseases associated with fat distribution, and / or cardiovascular diseases associated with fat distribution.

[0087] In one embodiment of this application, the drug composition further includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include drug carriers, pH buffers, lyophilization protectants, osmotic pressure regulators, excipients, stabilizers and / or preservatives.

[0088] In one embodiment of this application, 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.

[0089] In one embodiment of this application, 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.

[0090] In one embodiment of this application, the protective agent includes one or more of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose.

[0091] In one embodiment of this application, the osmotic pressure regulator includes sodium chloride and / or potassium chloride.

[0092] This application also provides a method for preventing or treating diseases associated with INHBE gene expression, comprising administering the siRNA, the siRNA conjugate, the antisense oligonucleotide, the recombinant plasmid, the host cell, or the drug to a subject in need.

[0093] In one embodiment of this application, diseases associated with INHBE gene expression include obesity, metabolic diseases associated with fat distribution, and / or cardiovascular diseases associated with fat distribution.

[0094] In one embodiment of this application, when the subject of the application is a human, the dosage of the siRNA, siRNA conjugate, antisense oligonucleotide, recombinant plasmid, host cell, or drug is 0.1-10 mg / kg.

[0095] In one embodiment of this application, when the subject of the application is a human, the dosage of the siRNA, siRNA conjugate, antisense oligonucleotide, recombinant plasmid, host cell, or drug is 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0096] The technical solution of this application has the following advantages:

[0097] 1. This application provides an siRNA for inhibiting INHBE, 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 to SEQ ID NO.332 or SEQ ID NO.726 to SEQ ID NO.732; the antisense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO.333 to SEQ ID NO.664 or SEQ ID NO.733 to SEQ ID NO.739. Experiments have shown that the siRNA has high inhibitory activity against INHBE; therefore, the siRNA has great application potential in the preparation of drugs for the prevention and / or treatment of diseases related to INHBE expression (e.g., obesity, metabolic diseases related to fat distribution, and cardiovascular diseases related to fat distribution).

[0098] Furthermore, at least one nucleotide in the sense and / or antisense strands of the siRNA is a modified nucleotide; the modification includes methoxy modification, fluorination modification, thiophosphate linkage, reverse abasic nucleotide modification, vinyl phosphate modification, and deoxyribonucleotide substitution. Experiments have shown that the modified siRNA exhibits good INHBE inhibition at concentrations ranging from 0.1 nM to 1 nM. Therefore, the modified siRNA shows great promise in the preparation of drugs for the prevention and / or treatment of diseases related to INHBE expression (e.g., obesity, metabolic diseases related to fat distribution, and cardiovascular diseases related to fat distribution).

[0099] 2. This application provides an antisense oligonucleotide (ASO) for inhibiting INHBE, wherein the antisense oligonucleotide comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO. 675 to SEQ ID NO. 725. Experiments have demonstrated that the antisense oligonucleotides all possess high inhibitory activity against INHBE. Therefore, the antisense oligonucleotides show great promise in the preparation of drugs for the prevention and / or treatment of diseases related to INHBE expression (e.g., obesity, metabolic diseases related to fat distribution, and cardiovascular diseases related to fat distribution).

[0100] Furthermore, at least one nucleotide in the sense strand and / or antisense strand of the antisense oligonucleotide is a modified nucleotide; the modification includes phosphate thioester linkage, 2'-O-methoxyethyl modification, and deoxyribonucleotide substitution. Experiments have shown that the modified antisense oligonucleotides exhibit good INHBE inhibition at a concentration of 50 nM. Therefore, the modified antisense oligonucleotides show great promise in the preparation of drugs for the prevention and / or treatment of diseases related to INHBE expression (e.g., obesity, metabolic diseases related to fat distribution, and cardiovascular diseases related to fat distribution). Detailed Implementation

[0101] The following embodiments are provided to better understand this application and are not limited to the preferred embodiments described herein. They do not constitute a limitation on the content and scope of protection of this application. Any product that is the same as or similar to this application, derived by anyone under the guidance of this application or by combining features of this application with other prior art, falls within the scope of protection of this application.

[0102] 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.

[0103] In the following embodiments, INHBE mRNA refers to mRNA having the sequence shown in GeneBank registration numbers NM_031479.5, NM_008382.3, NM_031815.3, or XM_005571319.3. Further, unless otherwise specified, the term "target gene" as used in this disclosure refers to the gene that transcribes the aforementioned INHBE mRNA, and the term "target mRNA" refers to the aforementioned INHBE mRNA.

[0104] 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).

[0105] The experimental cells used in the following examples are Hep3B cells, purchased from the Chinese Academy of Sciences Cell Bank.

[0106] The experimental animals were C57 / 6J mice (hINHBE heterozygous strain), male, 4–6 weeks old, housed in the SPF-grade animal facility of Suzhou Gemma Gene Co., Ltd. Animals were kept under a 12-hour light-dark cycle, with free access to food and water. Experiments began after one week of acclimatization. The use and handling of experimental animals complied with the requirements of the Animal Management Committee of Suzhou Gemma Gene Co., Ltd. regarding experimental animals and animal welfare.

[0107] The siRNAs involved in the following examples are siRNA sequences synthesized via phosphoramide solid-phase synthesis.

[0108] In the following examples, when transfecting cells with siRNA targeting INHBE, siRNA conjugates, or siRNA or siRNA conjugates used as negative controls, Lipofectamine 2000 (purchased from Invitrogen) was used as the transfection reagent, and the specific operation was 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 operation was performed according to the manufacturer's instructions.

[0109] Example 1: A siRNA for inhibiting INHBE

[0110] This embodiment provides an siRNA for inhibiting INHBE, the nucleotide sequence of which is designed based on the target mRNA, as shown in Table 1.

[0111] Table 1. siRNAs and their sequences that inhibit INHBE gene expression

[0112] Example 2: A modified siRNA and siRNA conjugate for inhibiting INHBE

[0113] This embodiment provides a modified siRNA and siRNA conjugate for inhibiting INHBE. The modified siRNA is based on the siRNA of Example 1, with the 5th, 7th, 8th, and 9th nucleotides of the sense strand having a 2' fluorination, and the other nucleotides having a 2' methoxylation. A thiophosphate bond connects the 1st and 2nd nucleotides, and the 2nd and 3rd nucleotides. Similarly, the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand have a 2' fluorination, and the other nucleotides have a 2' methoxylation. A thiophosphate bond connects the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the penultimate and penultimate nucleotides, and the penultimate and penultimate nucleotides. Alternatively,

[0114] The modified siRNA is based on the siRNA of Example 1, with the 7th, 8th, and 9th nucleotides of the sense strand having a 2' fluorination, and the other nucleotides having a 2' methoxylation. A thiophosphate bond connects the 1st and 2nd nucleotides, and the 2nd and 3rd nucleotides. Similarly, the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand have a 2' fluorination, and the other nucleotides having a 2' methoxylation. A thiophosphate bond connects the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the penultimate and penultimate nucleotides, and the penultimate and penultimate nucleotides. Alternatively,

[0115] 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. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the first nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the last nucleotide. The first nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group, and the last nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group. The second, third, and fourth positions of the antisense strand are also modified. Nucleotides at positions 10, 12, 14, 16, 18, and 20 are fluorinated at the 2' position; nucleotides at positions 5 and 7 are deoxyribonucleotides (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide is used instead of uracil ribonucleotide, and so on); 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; nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by thiophosphate bonds; or,

[0116] 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 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 phosphate thioester group. The penultimate nucleotide... The antisense strand is linked to the non-base nucleotide via a thiophosphate 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, 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 nucleotide at position 1. Phosphophosphate bonds connect the nucleotides at positions 1 and 2, 2 and 3, the penultimate and penultimate, and the penultimate and penultimate.

[0117] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, 10, and 11 of the positive strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the first nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The first nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group. The penultimate nucleoside... The acid and the reverse abasic nucleotide are linked by a thiophosphate group. The nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated at the 2' position, the nucleotide at position 12 is substituted with a deoxyribonucleotide, 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 nucleotide at position 1. The nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by thiophosphate bonds; or...

[0118] 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. The 3' end of the 1st nucleotide is connected to an inverse abasic nucleotide (invAb group), and the 5' end of the penultimate nucleotide is connected to an inverse abasic nucleotide (invAb group). The 1st nucleotide and the inverse abasic nucleotide are linked by a thiophosphate group, and the penultimate nucleotide and the inverse 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. The 5' end of the 1st nucleotide is connected to a trans-vinyl phosphate group (VP group), and the 1st and 2nd, 2nd and 3rd, penultimate and penultimate, and penultimate and penultimate nucleotides are linked by thiophosphate bonds.

[0119] The siRNA conjugate consists of modified siRNA and a GalNAc group conjugated to the modified siRNA; the siRNA conjugate has the following structure (double helix structure representing siRNA, and linker attached to the 3' end of the siRNA's positive strand):

[0120] Modified siRNAs and siRNA conjugates are shown in Tables 2 and 3.

[0121] Table 2. Modified siRNAs and their sequences that inhibit INHBE gene expression

[0122] In Table 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; and lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by thiophosphate groups.

[0123] Table 3. siRNA conjugates that inhibit INHBE gene expression and their sequences

[0124] In Table 3, 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 (when its ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide is used instead of uracil ribonucleotide, and so on); string (GNA) indicates that the nucleotide adjacent to the left of the string is a glycolic acid (GNA) modified nucleotide; string (invAb) indicates that the 3' end of the nucleotide adjacent to the left or right of the string is connected to an inverse abase-free nucleotide (invAb group); string VP indicates that the nucleotide adjacent to the right of the string is a vinyl phosphate modified nucleotide.

[0125] Example 3: A modified antisense oligonucleotide (ASO) for inhibiting INHBE

[0126] This embodiment provides a modified antisense oligonucleotide (ASO) for inhibiting INHBE. The nucleotide sequence of the modified antisense oligonucleotide is designed based on the target mRNA. The first 5 and last 5 nucleotides are modified with 2'-O-methoxyethyl, the middle 10 nucleotides are substituted with deoxyribonucleotides, and adjacent nucleotides are linked by phosphate thioester groups, as shown in Table 4.

[0127] Table 4. Modified antisense oligonucleotides (ASOs) that inhibit INHBE gene expression and their sequences

[0128] In Table 4, uppercase letters C, G, T, and A represent ribonucleotides; lowercase letter s indicates that the two nucleotides adjacent to s are linked by thiophosphate groups; string (d) indicates that the nucleotide adjacent to the left of the string is replaced by a deoxyribonucleotide; string (MOE) indicates that the nucleotide adjacent to the left of the string is a nucleotide modified with 2'-O-methoxyethyl.

[0129] Experimental Example 1: Verification of the activity of siRNA used to inhibit INHBE in Hep3B cells

[0130] This experimental example provides a validation experiment for the activity of siRNA used to inhibit INHBE in Hep3B cells. The experimental procedure is as follows:

[0131] Hep3B cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into MEM medium (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 Hep3B 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 × 10⁶ cells / year. 5 Cell suspension of cells / mL;

[0132] 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°C) for 15 min to obtain transfection solutions containing different siRNAs.

[0133] After seeding the cell suspension into 96-well plates at a seeding rate of 50 μL / well, the transfection reagent control group (MOCK), siRNA experimental group, AD1708473 control group and AD1711744 control group were set up in the 96-well plates, with 3 replicates in each group.

[0134] After setup, 50 μL of transfection buffer containing different siRNAs was added to the wells of the siRNA experimental groups (hINH-1M3 experimental group was given transfection buffer containing hINH-1M3, hINH-140M3 experimental group was given transfection buffer containing hINH-140M3, and so on, with the final concentration of siRNA in the wells being 0.1 nM, 1 nM, 2.5 nM, or 10 nM, respectively, and the final concentration of siRNA was adjusted by using Opti-MEM dilution). 50 μL of transfection buffer containing the same concentration of AD1708473 or AD1711744 as the siRNA experimental groups was added to the wells of the AD1708473 control group and the AD1711744 control group, respectively. 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 48 h for transfection.

[0135] 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.

[0136] 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.

[0137] For each reverse transcription reaction system, take 4 μL of the above-mentioned cDNA-containing solution as a template. Using the reagents provided in the AceQ Universal SYBR qPCR Master Mix kit (purchased from Vazyme, catalog number Q511-02), prepare a 20 μL qPCR reaction system on an ice box according to Table 5. Primer 1 and Primer 2 are the PCR primer sequences for amplifying the target gene INHBE and the internal reference gene GAPDH, respectively (as shown in Table 6). Place each qPCR reaction system in an ABIStepOnePlus Real-Time... On a PCR instrument, a three-step amplification method was used. The amplification program was 95℃ pre-denaturation for 10 min, followed by 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 the amplified target gene INHBE and the internal reference gene GAPDH. Product W was then incubated sequentially at 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s. The melting curves of the target gene INHBE and the internal reference gene GAPDH in product W were collected by a real-time quantitative PCR instrument to obtain the Ct values ​​of the target gene INHBE and the internal reference gene GAPDH.

[0138] The relative quantification of the target gene INHBE in each test group was performed using the comparison Ct(ΔΔCt) method, as follows:

[0139] ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group);

[0140] ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group);

[0141] ΔCt(test group) = ΔCt(test group) - ΔCt(control group average);

[0142] ΔCt(control group) = ΔCt(control group) - ΔCt(control group average);

[0143] 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;

[0144] Using the control group as a baseline, the expression level of INHBE mRNA in the test group was normalized, and the expression level of INHBE mRNA in the control group was defined as 100%.

[0145] The relative expression level of INHBE mRNA in the test group was 2- ΔΔCt(测试组) ×100%;

[0146] INHBE mRNA inhibition rate in the test group = 1 - relative expression level of INHBE mRNA in the test group;

[0147] The INHBE 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 7 to 12.

[0148] siRNA concentration gradients were set at 10 nM, 1 nM, 0.33 nM, 0.11 nM, 0.037 nM, 0.0123 nM, and 0.0041 nM. Following the method described above for detecting the relative expression level of INHBE mRNA, the effects of these gradient concentrations of siRNA on the relative expression level of INHBE mRNA were examined. Then, using Grapdhpad software, IC50 curves for different siRNAs were fitted using nonlinear regression. The experimental results are shown in Table 13.

[0149] As shown in Tables 7 to 13, the modified siRNA and siRNA conjugate in Example 2 both exhibited good INHBE inhibition effects at concentrations ranging from 0.1 nM to 10 nM.

[0150] Table 5. DNA amplification reaction system

[0151] Table 6. Primer Information

[0152] Table 7. Inhibitory levels of different modified siRNAs on INHBE mRNA in Hep3B cells

[0153] Table 8. Inhibitory levels of different modified siRNAs on INHBE mRNA in Hep3B cells

[0154] Table 9. Inhibitory levels of different modified siRNAs on INHBE mRNA in Hep3B cells

[0155] Table 10. Inhibitory levels of different modified siRNAs on INHBE mRNA in Hep3B cells

[0156] Table 11. Inhibitory levels of different modified siRNAs on INHBE mRNA in Hep3B cells

[0157] Table 12. Inhibitory levels of different modified siRNAs on INHBE mRNA in Hep3B cells

[0158] Table 13. Median inhibition concentrations of different modified siRNAs on INHBE mRNA in Hep3B cells

[0159] (concentration, IC50)

[0160] Experimental Example 2: Validation of the activity of modified antisense oligonucleotides used to inhibit INHBE in Hep3B cells

[0161] This experimental example provides a validation experiment for the activity of modified antisense oligonucleotides (ASO) used to inhibit INHBE in Hep3B cells. The experimental procedure is as follows:

[0162] The activity of the modified antisense oligonucleotide (ASO) used to inhibit INHBE in Hep3B cells was verified using the same method as in Experiment Example 1, except that the final concentration of ASO was set to 50 nM. The experimental results are shown in Table 14.

[0163] As shown in Table 14, the modified antisense oligonucleotides (ASO) in Example 3 all exhibited good INHBE inhibition effects.

[0164] Table 14. Inhibitory effects of different modified ASOs on INHBE mRNA in Hep3B cells

[0165] 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 INHBE 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 to SEQ ID NO.332 or SEQ ID NO.726 to SEQ ID NO.732, 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.333 to SEQ ID NO.664 or SEQ ID NO.733 to SEQ ID NO.739, 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 replacing the 2' hydroxyl group of the ribosyl group of a nucleotide with other groups, compounds formed by modifying the thiophosphate group of a nucleotide, and / or compounds formed by modifying the bases on a nucleotide.

4. The siRNA according to any one of claims 2 to 3, characterized in that, The modified nucleotides include fluorinated nucleotides, methoxylated nucleotides, phosphate-thioester linked nucleotides, ethylene glycol-modified nucleotides, inverse abase-free nucleotides, vinyl phosphate-modified nucleotides, deoxyribonucleotide-substituted nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-deoxy-nucleotides, 3'-terminal deoxy-thymidine nucleotides, 3'-deoxy-nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, abase-free nucleotides, and 2'-amino- - Modified nucleotides, 2'-O-allyl- modified nucleotides, 2'-C-alkyl- modified nucleotides, 2'-hydroxy- modified nucleotides, 2'-O-alkyl- modified nucleotides, morpholinyl nucleotide modified nucleotides, aminophosphate-modified nucleotides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl alcohol-modified nucleotides, cyclohexenyl-modified nucleotides, open-ring nucleotide modified nucleotides, nucleotides containing methyl phosphate groups, nucleotides containing 5'-phosphate groups, and / or nucleotides containing 5'-phosphate mimics.

5. The siRNA as described in claim 4, characterized in that, The 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, 8, and 9 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 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 5, 7, 8, and 9 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 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, 10, 12, 14, 16, 18, and 20 of the antisense strand are fluorinated nucleotides; or, The 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 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, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The 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 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.

6. The siRNA as described in claim 4 or 5, characterized in that, The methoxylated nucleotides are located in the antisense and sense strands of the nucleotide sequence. In the sense strand, the nucleotides that are not fluorinated, not ethylene glycol-modified, and not substituted with deoxyribonucleotides are all methoxylated nucleotides. In the antisense strand, the nucleotides that are not fluorinated, not ethylene glycol-modified, and not substituted with deoxyribonucleotides are all methoxylated nucleotides.

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 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 trans vinyl phosphate group.

9. The siRNA according to any one of claims 4 to 8, 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 nucleotides at positions 5 and 7 of the antisense strand are deoxyribonucleotide-substituted nucleotides; or, 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.

10. The siRNA according to any one of claims 4 to 9, characterized in that, The phosphate-thioester linked 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 nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by phosphate-thioester groups; and at least the nucleotides at positions 1 and 2, positions 2 and 3, positions 1 to 1 and 2 to 1, and positions 2 to 1 ... The phosphate-thioester linked 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 nucleotide at position 1 of the sense strand is linked to the inverted abasic nucleotide by a phosphate-thioester group, and the nucleotide at position 1-1-3 of the sense strand is linked to the inverted abasic nucleotide by a phosphate-thioester group; at least the nucleotides at positions 1-2, 2-3, 1-1-2-3, and 1-1-2-2-3 of the antisense strand are linked by a phosphate-thioester group; or, The nucleotides linked by the thiophosphate group are located in the antisense strand 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, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by the thiophosphate group.

11. A siRNA conjugate for inhibiting INHBE, characterized in that, The siRNA conjugate contains the siRNA as described in any one of claims 1 to 10 and a conjugating group conjugated to the siRNA.

12. An antisense oligonucleotide for inhibiting INHBE, characterized in that, The antisense oligonucleotide comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO. 675 to SEQ ID NO.

725.

13. The antisense oligonucleotide as described in claim 12, characterized in that, At least one nucleotide of the antisense oligonucleotide is a modified nucleotide.

14. The antisense oligonucleotide as described in claim 13, characterized in that, The modified nucleotides include compounds formed by replacing the 2' hydroxyl group of the ribosyl group of a nucleotide with other groups, compounds formed by modifying the thiophosphate group of a nucleotide, and / or compounds formed by modifying the bases on a nucleotide.

15. The antisense oligonucleotide as described in claim 13 or 14, characterized in that, The modified nucleotides include nucleotides linked by thiophosphate groups, nucleotides substituted with deoxyribonucleotides, and / or nucleotides modified with 2'-O-methoxyethyl groups.

16. The antisense oligonucleotide as described in claim 15, characterized in that, The antisense oligonucleotide has at least positions 1 to 5 and positions 16 to 20 modified with 2'-O-methoxyethyl, in the direction from the 5' end to the 3' end.

17. The antisense oligonucleotide as described in claim 15 or 16, characterized in that, The antisense oligonucleotide has at least nucleotides substituted with deoxyribonucleotides at positions 6 to 15, following the direction from the 5' end to the 3' end.

18. The antisense oligonucleotide according to any one of claims 15 to 17, characterized in that, The antisense oligonucleotides are linked by thiophosphate groups between adjacent nucleotides.

19. A recombinant plasmid, characterized in that, The recombinant plasmid expresses the siRNA according to any one of claims 1 to 10; or, the recombinant plasmid expresses the antisense oligonucleotide according to any one of claims 12 to 18.

20. A host cell, characterized in that, The host cell genome is integrated with the siRNA according to any one of claims 1 to 10; or, the host cell genome is integrated with the antisense oligonucleotide according to any one of claims 12 to 18; or, the host cell carries the recombinant plasmid according to claim 19.

21. The use of the siRNA according to any one of claims 1 to 10, the siRNA conjugate according to claim 11, the antisense oligonucleotide according to any one of claims 12 to 18, the recombinant plasmid according to claim 19, or the host cell according to claim 20 in the preparation of a medicament for the prevention and / or treatment of a disease, wherein the disease is a disease related to INHBE gene expression.

22. A medicine for preventing and / or treating a disease, characterized in that, The disease is a disease related to INHBE gene expression; the drug comprises the siRNA of any one of claims 1 to 10, the siRNA conjugate of claim 11, the antisense oligonucleotide of any one of claims 12 to 18, the recombinant plasmid of claim 19, or the host cell of claim 20.

23. A method for preventing or treating diseases related to INHBE gene expression, characterized in that, This includes administering to a recipient the siRNA of any one of claims 1 to 10, the siRNA conjugate of claim 11, the antisense oligonucleotide of any one of claims 12 to 18, the recombinant plasmid of claim 19, the host cell of claim 20, or the drug of claim 22.

24. The method according to claim 23, characterized in that, Diseases associated with INHBE gene expression include obesity, metabolic diseases associated with fat distribution, and / or cardiovascular diseases associated with fat distribution.

25. The method according to claim 23, characterized in that, When applied to a human, the dosage of the siRNA, siRNA conjugate, antisense oligonucleotide, recombinant plasmid, host cell, or drug is 0.1-10 mg / kg.

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