RNA agent for inhibiting expression of inhbc gene and use thereof

Abstract

Disclosed are an RNA agent for inhibiting the expression of an INHBC gene and use thereof in the preparation of a medicament for treating diseases associated with the INHBC gene.

Description

RNA agents that inhibit INHBC gene expression and their applications

[0001] This application claims priority to Chinese Patent Application No. 2024117856643, filed on December 5, 2024, and Chinese Patent Application No. 2025100955063, filed on January 21, 2025. The full text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This invention relates to RNA agents that inhibit INHBC gene expression and their use in the preparation of treatments for diseases related to the INHBC gene. Background Technology

[0003] The inhibin subunit beta C (INHBC) gene is a member of the inhibin superfamily. Inhibins are an important class of regulatory proteins, primarily expressed and functioning in the reproductive system and other organs and tissues. Their functions include regulating growth, cell differentiation, cell death, and participating in various biological processes, such as regulating hormone levels, cell proliferation, and tissue morphogenesis. Inhibins are dimeric proteins composed of two subunits: inhibin α and inhibin βA or inhibin βB. These two subunits can form different inhibin isoforms in different combinations, such as inhibin A (composed of α and βA) and inhibin B (composed of α and βB). The βC subunit encoded by the INHBC gene can bind to the α subunit, potentially forming different inhibin isoforms that participate in the regulation of various biological processes in the body. These proteins exert their regulatory effects by binding to specific receptors.

[0004] Inhibin βC chain (INHBC) protein complexly regulates the inhibin and activin systems and balances the secretion of follicle-stimulating hormone. Its key roles encompass a variety of physiological processes, including hormone secretion, cell development, insulin release, and bone growth, depending on specific subunit composition. INHBC gene expression is positively associated with waist-to-hip ratio (WHRadjBMI) adjusted for body mass index and is positively correlated with the incidence of cardiovascular disease. WHRadjBMI, as a measure of abdominal fat, has a significant causal relationship with type 2 diabetes, coronary heart disease, glycemic characteristics, circulating lipids, and blood pressure. Therefore, inhibiting INHBC expression represents a potential therapeutic approach for controlling harmful obesity and related metabolic diseases. Summary of the Invention

[0005] The present invention provides an RNA agent for inhibiting the expression of the INHBC gene, wherein one strand of the RNA agent has at least 75% homology or complementarity with any continuous fragment of 16-35 nucleotides in length in the mRNA encoding INHBC (SEQ ID NO:729).

[0006] In this application, SEQ ID NO:729(NM_005538.4) is shown below:

[0007] In some embodiments, the aforementioned RNA agent for inhibiting INHBC gene expression comprises an antisense strand or antisense nucleic acid fragment having at least 75% complementarity with any continuous fragment of 16-35 nucleotides in length in the mRNA encoding INHBC (SEQ ID NO:729).

[0008] In some embodiments, the antisense strand or antisense nucleic acid fragment consists of 19-27 consecutive nucleotides, preferably 21-26 consecutive nucleotides, for example, 21, 22, 23, 24, 25 or 26 consecutive nucleotides.

[0009] In some schemes, the aforementioned RNA agent that inhibits INHBC gene expression, wherein the antisense strand or antisense nucleic acid fragment comprises, as in SEQ ID NO: 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 32 5. A series of consecutive nucleotide sequences with a difference of no more than 5, 4, 3, 2 or 1 nucleotide from any of the sequences shown, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363 or 364.

[0010] In some schemes, the aforementioned RNA agents that inhibit INHBC gene expression, wherein the antisense strand or antisense nucleic acid fragment comprises, for example, SEQ ID NO: 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 2 28, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 2 74, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 3 20, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, or 364.

[0011] In some embodiments, the aforementioned RNA agent for inhibiting INHBC gene expression comprises a sense strand or sense nucleic acid fragment that has at least 75% homology to any continuous fragment of 16-35 nucleotides in length in the mRNA encoding INHBC (SEQ ID NO:729).

[0012] In some embodiments, the positive chain or positive nucleic acid fragment consists of 19-27 consecutive nucleotides, preferably 21-26 consecutive nucleotides, for example, 21, 22, 23, 24, 25 or 26 consecutive nucleotides.

[0013] In some schemes, the aforementioned RNA agents that inhibit INHBC gene expression contain, for example, the sense strand or sense nucleic acid fragment of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57. 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 1 09, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 1 A series of consecutive nucleotide sequences differing by no more than 5, 4, 3, 2 or 1 nucleotide from any of the sequences shown in 50, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 or 182.

[0014] In some schemes, the aforementioned RNA agents that inhibit INHBC gene expression contain, for example, SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 5 5, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144 The sequence shown is 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, or 182.

[0015] In some embodiments, the RNA agent comprises an antisense strand or antisense nucleic acid fragment and a sense strand or sense nucleic acid fragment; the antisense strand or antisense nucleic acid fragment comprises a sequence as shown in any of SEQ ID NO: 204, 205, 208, 211, 225, 226, 240, 241, 248, 263, 271, 288, 289, 290, 293, 294, 298, 338, 339, 346, 347, or 352; the sense strand or sense nucleic acid fragment correspondingly comprises a sequence as shown in any of SEQ ID NO: 22, 23, 26, 29, 43, 44, 58, 59, 66, 81, 89, 106, 107, 108, 111, 112, 116, 156, 157, 164, 165, or 170.

[0016] In some schemes, the aforementioned RNA agent that inhibits INHBC gene expression comprises a sense strand and an antisense strand forming a reverse complementary double-stranded region, the sense strand and the antisense strand being located on two nucleic acid strands.

[0017] In some schemes, the aforementioned RNA agent for inhibiting INHBC gene expression comprises a sense nucleic acid fragment and an antisense nucleic acid fragment forming a reverse complementary double-stranded region, wherein the sense nucleic acid fragment and the antisense nucleic acid fragment are located on the same nucleic acid strand.

[0018] In some formulations, the aforementioned RNA agent for inhibiting INHBC gene expression includes, in which the reverse complementary double-stranded region comprises, for example, ds-n1, ds-n2, ds-n3, ds-n4, ds-n5, ds-n6, ds-n7, ds-n8, ds-n9, ds-n10, ds-n11, ds-n12, ds-n13, ds-n14, ds-n15, ds-n16, ds-n17, ds-n18, ds-n19, ds-n20, ds-n21, ds-n22, ds-n23, ds-n24, ds-n25, ds-n26, ds-n27, ds-n28, ds-n29, ds-n30, and ds-n31. ds-n32, ds-n33, ds-n34, ds-n35, ds-n36, ds-n37, ds-n38, ds-n39, ds-n40 , ds-n41, ds-n42, ds-n43, ds-n44, ds-n45, ds-n46, ds-n47, ds-n48, ds-n4 9. ds-n50, ds-n51, ds-n52, ds-n53, ds-n54, ds-n55, ds-n56, ds-n57, ds-n 58. ds-n59, ds-n60, ds-n61, ds-n62, ds-n63, ds-n64, ds-n65, ds-n66, ds-n 67. ds-n68, ds-n69, ds-n70, ds-n71, ds-n72, ds-n73, ds-n74, ds-n75, ds- n76, ds-n77, ds-n78, ds-n79, ds-n80, ds-n81, ds-n82, ds-n83, ds-n84, ds -n85, ds-n86, ds-n87, ds-n88, ds-n89, ds-n90, ds-n91, ds-n92, ds-n93, d s-n94, ds-n95, ds-n96, ds-n97, ds-n98, ds-n99, ds-n100, ds-n101, ds-n10 2. ds-n103, ds-n104, ds-n105, ds-n106, ds-n107, ds-n108, ds-n109, ds-n 110, ds-n111, ds-n112, ds-n113, ds-n114, ds-n115, ds-n116, ds-n117, ds- n118, ds-n119, ds-n120, ds-n121, ds-n122, ds-n123, ds-n124, ds-n125, d s-n126, ds-n127, ds-n128, ds-n129, ds-n130, ds-n131, ds-n132, ds-n133,ds-n134, ds-n135, ds-n136, ds-n137, ds-n138, ds-n139, ds-n140, ds-n141, ds-n142, ds-n143, ds-n144, ds-n145, ds-n146 , ds-n147, ds-n148, ds-n149, ds-n150, ds-n151, ds-n152, ds-n153, ds-n154, ds-n155, ds-n156, ds-n157, ds-n158, ds-n159 The structure can be any one of the following: ds-n160, ds-n161, ds-n162, ds-n163, ds-n164, ds-n165, ds-n166, ds-n167, ds-n168, ds-n169, ds-n170, ds-n171, ds-n172, ds-n173, ds-n174, ds-n175, ds-n176, ds-n177, ds-n178, ds-n179, ds-n180, ds-n181, or ds-n182, as shown in Table 1-1.

[0019] Table 1-1 Double-stranded region structure (transcript NM_005538.4)

[0020] In some schemes, the reverse complementary double-stranded region includes any one of the structures shown, such as ds-n22, ds-n23, ds-n26, ds-n29, ds-n43, ds-n44, ds-n58, ds-n59, ds-n66, ds-n81, ds-n89, ds-n106, ds-n107, ds-n108, ds-n111, ds-n112, ds-n116, ds-n156, ds-n157, ds-n164, ds-n165, or ds-n170.

[0021] In some embodiments, the sense strand comprises a first strand, the antisense strand comprises a second strand, and 3' and 5' extensions located on the flanks of the second strand, the first and second strands being anticomplementary to form the anticomplementary double-stranded region; the 5' end nucleotide of the second strand and the 3' end nucleotide of the first strand are complementary; the 3' extension is connected to the 3' end of the second strand and has a length of 0-5 nucleotides, for example, 0, 1, 2, 3, 4, or 5 nucleotides; the 5' extension is connected to the 5' end of the second strand and has a length of at least 3 nucleotides, for example, 3, 4, 5, or 6 nucleotides.

[0022] In some schemes, the aforementioned RNA agent for inhibiting INHBC gene expression includes a sense strand comprising a first strand, an antisense strand comprising a second strand, a 3' extension, and a 5' extension, wherein the first and second strands are anticomplementary, the 5' end nucleotide of the second strand is complementary to the 3' end nucleotide of the first strand, the 3' extension is attached to the 3' end of the second strand and has a length of 0-5 nucleotides, the 5' extension is attached to the 5' end of the second strand and has a length of at least 3 nucleotides, and the antisense strand can be cleaved between the 3' end nucleotide of the 5' extension and the 5' end nucleotide of the second strand, the resulting cleavage product being able to silence gene expression via RNA interference.

[0023] In some embodiments, the sense strand comprises a first strand, and the antisense strand comprises a second strand, and 3' and 5' extensions located flanking the second strand, wherein the first and second strands are anticomplementary to form the anticomplementary double-stranded region; the 5' nucleotide of the second strand and the 3' nucleotide of the first strand are complementary; the 3' extension is attached to the 3' end of the second strand and has a length of 0-5 nucleotides, for example, 0, 1, 2, 3, 4, or 5 nucleotides; the 5' extension is attached to the 5' end of the second strand and has a length of at least 3 nucleotides, for example, 3, 4, 5, or 6 nucleotides; the antisense strand can be cleaved between the 3' nucleotide of the 5' extension and the 5' nucleotide of the second strand, and the resulting cleavage product can silence gene expression through RNA interference.

[0024] In some embodiments, the 3' extension is connected to the 3' end of the second strand and is 2 nucleotides in length, and the 5' extension is connected to the 5' end of the second strand and is 3 or 4 nucleotides in length.

[0025] In some schemes, the structural relationship of the first strand, second strand, 3' extension and 5' extension is shown in Figure 1, and the RNA agent is cleaved at the position indicated by ▲.

[0026] In some embodiments, the aforementioned RNA agent that inhibits INHBC gene expression, wherein, counting from the 3' end of the 5' extension, the two nucleotides of the 5' extension are selected from G, A, natural analogs of G, non-natural analogs of G, natural analogs of A, and non-natural analogs of A.

[0027] In some embodiments, the aforementioned RNA agent that inhibits INHBC gene expression, wherein, counting from the 3' end of the 5' extension, the two nucleotides of the 5' extension are selected from G, natural analogs of G, and non-natural analogs of G.

[0028] In some formulations, the aforementioned RNA agent that inhibits INHBC gene expression, wherein the 5' nucleotide of the second strand is selected from A, U, natural analogs of U, non-natural analogs of U, natural analogs of A, and non-natural analogs of A.

[0029] In some embodiments, the RNA agent that inhibits INHBC gene expression described above, wherein the 3' nucleotide of the first strand is or is replaced with U, a natural analog of U, or a non-natural analog of U.

[0030] In some embodiments, the RNA agent that inhibits INHBC gene expression includes an antisense strand or antisense nucleic acid fragment comprising a continuous nucleotide sequence differing by no more than 3, 2, or 1 nucleotides from any of the sequences shown in SEQ ID NO: 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, or 1003.

[0031] In some embodiments, the RNA agent that inhibits INHBC gene expression includes an antisense strand or antisense nucleic acid fragment comprising any of the sequences shown in SEQ ID NO: 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, or 1003.

[0032] In some embodiments, the RNA agent comprises an antisense strand or antisense nucleic acid fragment and a sense strand or sense nucleic acid fragment; the antisense strand or antisense nucleic acid fragment comprises a sequence as shown in any of SEQ ID NO: 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, or 1003; the sense strand or sense nucleic acid fragment correspondingly comprises a sequence as shown in any of SEQ ID NO: 746, 748, 759, 770, 771, 776, 805, 156, 842, 843, 846, 837, 809, 112, 111, 806, 804, 795, 789, 760, 750, or 745.

[0033] In some schemes, the positive strand or positive nucleic acid fragment correspondingly comprises a continuous nucleotide sequence with a sequence difference of no more than 3, 2, or 1 nucleotide as shown in any of the sequences indicated by SEQ ID NO: 746, 748, 759, 770, 771, 776, 805, 156, 842, 843, 846, 837, 809, 112, 111, 806, 804, 795, 789, 760, 750, or 745.

[0034] In some schemes, the RNA agent that inhibits INHBC gene expression includes, wherein the reverse complementary double-stranded region comprises any one of the following structures: ds-n183, ds-n184, ds-n185, ds-n186, ds-n187, ds-n188, ds-n189, ds-n190, ds-n191, ds-n192, ds-n193, ds-n194, ds-n195, ds-n196, ds-n197, ds-n198, ds-n199, ds-n200, ds-n201, ds-n202, ds-n203, or ds-n204, as shown in Tables 1-2.

[0035] Table 1-2 Double-stranded region structure (transcript NM_005538.4)

[0036] In some embodiments, the RNA agent that inhibits INHBC gene expression described above has a blocking group attached to the 5' nucleotide of the 5' extension; further, the blocking group is M06 or InvAB.

[0037] In some schemes, the aforementioned RNA agents that inhibit INHBC gene expression include at least one chemically modified nucleotide.

[0038] In some formulations, the aforementioned RNA agent that inhibits INHBC gene expression includes at least one of the following modifications:

[0039] (1) Modification of the phosphodiester bonds linking nucleotides in the nucleotide sequence of the RNA agent that inhibits INHBC gene expression;

[0040] (2) Modification of the ribose in the nucleotide sequence of the RNA agent that inhibits INHBC gene expression;

[0041] (3) Modification of the bases in the nucleotide sequence of the RNA agent that inhibits INHBC gene expression.

[0042] In some schemes, the aforementioned RNA agent that inhibits INHBC gene expression is wherein, counting from the 3' end of the 5' extension, positions 1 and 2 of the 5' extension are nucleotides containing a 2'-F modification.

[0043] In some embodiments, the aforementioned RNA agent for inhibiting INHBC gene expression is wherein the blocking group is linked to the 5'th nucleotide residue of the 5' extension via a phosphate thioester bond, and the nucleotide residues at positions 1 and 2 of the second strand are linked via phosphate thioester bonds, starting from the 5'th end of the second strand.

[0044] In some embodiments, the aforementioned RNA agent that inhibits INHBC gene expression is wherein, counting from the 3' end of the antisense strand, the 1st and 2nd nucleotide residues of the antisense strand are linked by a phosphate thioester bond, and the 2nd and 3rd nucleotide residues of the antisense strand are linked by a phosphate thioester bond.

[0045] In some embodiments, the aforementioned RNA agent that inhibits INHBC gene expression is wherein, counting from the 3' end of the positive strand, the 1st and 2nd nucleotide residues of the positive strand are linked by a phosphate thioester bond, and the 2nd and 3rd nucleotide residues of the positive strand are linked by a phosphate thioester bond.

[0046] In some formulations, the aforementioned RNA agent that inhibits INHBC gene expression also includes a delivery system.

[0047] In some embodiments, the aforementioned RNA agent that inhibits INHBC gene expression is wherein the 3' end nucleotide of the positive strand is linked to the delivery system; further, the delivery system is L96; and even further, the residues of the 3' end nucleotide of the positive strand are linked to the residues of L96 via phosphate ester bonds.

[0048] In some embodiments, the aforementioned RNA agent for inhibiting INHBC gene expression includes a 5' end nucleotide residue of the positive strand linked to an inverted abasic nucleotide residue via a phosphate thioester bond, the inverted abasic nucleotide being linked to a delivery system, and the 1st and 2nd nucleotide residues of the positive strand being linked via a phosphate thioester bond, starting from the 3' end; furthermore, the delivery system is NAG37; and even further, the inverted abasic nucleotide residue is linked to the NAG37 residue via a phosphate thioester bond.

[0049] In some protocols, the aforementioned RNA agent that inhibits INHBC gene expression is delivered to target cells via a delivery system.

[0050] In some embodiments, the aforementioned RNA agent that inhibits INHBC gene expression has the 3' end nucleotide of the sense strand linked to the delivery system.

[0051] In some embodiments, the aforementioned RNA agent for inhibiting INHBC gene expression includes a 5' end nucleotide residue of the positive strand linked to an inverted abasic nucleotide residue via a phosphate thioester bond, the inverted abasic nucleotide being linked to a delivery system, and the 1st and 2nd nucleotide residues of the positive strand being linked via a phosphate thioester bond, starting from the 3' end.

[0052] In some embodiments, the delivery system is a GalNAc (N-acetylgalactosamine) derivative linked to the RNA agent.

[0053] In some schemes, the RNA agent is linked to a divalent or trivalent GalNAc derivative.

[0054] In some embodiments, the GalNAc derivative is selected from GalNAc-L96, G-GalNAc C3 phosphorous amide, and GalNAc-NAG.

[0055] In some embodiments, the GalNAc-L96 is selected from L96, GalNAc L96-PS, and GalNAc L96-CPG.

[0056] In some schemes, GalNAc-NAG is selected from GalNAc-NAG13, GalNAc-NAG18, GalNAc-NAG24, GalNAc-NAG25, GalNAc-NAG26, GalNAc-NAG27, GalNAc-NAG28, GalNAc-NAG29, GalNAc-NAG30, GalNAc-NAG31, GalNAc-NAG32, GalNAc-NAG33, GalNAc-NAG34, GalNAc-NAG35, GalNAc-NAG36, GalNAc-NAG37, GalNAc-NAG38, and GalNAc-NAG39.

[0057] In some implementations, the delivery system is L96 or NAG37.

[0058] In some embodiments, when the delivery system is L96, the residues of the 3' end nucleotide of the positive strand are linked to the L96 residues via a phosphate ester bond or a thiophosphate ester bond.

[0059] In some embodiments, when the delivery system is NAG37, the inverted non-basic nucleotide residues are linked to NAG37 residues via phosphate thioester bonds.

[0060] In some embodiments, the RNA agent comprises any one of the structures shown in Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, Z16, Z17, Z18, Z19, Z20, Z21 or Z22.

[0061] In some schemes, the aforementioned RNA agents that inhibit INHBC gene expression, wherein the modifications are independently selected from one, two, or three combinations of the following: 2'-OMe modification, 2'-F modification, 2'-deoxy modification, VP modification, 5'-MP modification, PS modification, PS2 modification, MP modification, MOP modification, invAb modification, and invAB modification.

[0062] This invention also provides RNA agents for inhibiting INHBC gene expression, including ds-m1, ds-m2, ds-m3, ds-m4, ds-m5, ds-m6, ds-m7, ds-m8, ds-m9, ds-m10, ds-m11, ds-m12, ds-m13, ds-m14, ds-m15, ds-m16, ds-m17, ds-m18, ds-m19, ds-m20, ds-m21, ds-m22, ds-m23, ds-m24, ds-m25, ds-m26, ds-m27, ds-m28, ds-m29, ds-m30, ds-m31, ds-m32, and ds-m33. ds-m34, ds-m35, ds-m36, ds-m37, ds-m38, ds-m39, ds-m40, ds-m41, ds-m42 , ds-m43, ds-m44, ds-m45, ds-m46, ds-m47, ds-m48, ds-m49, ds-m50, ds-m51 , ds-m52, ds-m53, ds-m54, ds-m55, ds-m56, ds-m57, ds-m58, ds-m59, ds-m6 0. ds-m61, ds-m62, ds-m63, ds-m64, ds-m65, ds-m66, ds-m67, ds-m68, ds-m6 9. ds-m70, ds-m71, ds-m72, ds-m73, ds-m74, ds-m75, ds-m76, ds-m77, ds-m 78. ds-m79, ds-m80, ds-m81, ds-m82, ds-m83, ds-m84, ds-m85, ds-m86, ds-m 87. ds-m88, ds-m89, ds-m90, ds-m91, ds-m92, ds-m93, ds-m94, ds-m95, ds- m96, ds-m97, ds-m98, ds-m99, ds-m100, ds-m101, ds-m102, ds-m103, ds-m10 4. ds-m105, ds-m106, ds-m107, ds-m108, ds-m109, ds-m110, ds-m111, ds-m 112, ds-m113, ds-m114, ds-m115, ds-m116, ds-m117, ds-m118, ds-m119, ds- m120, ds-m121, ds-m122, ds-m123, ds-m124, ds-m125, ds-m126, ds-m127, d s-m128, ds-m129, ds-m130, ds-m131, ds-m132, ds-m133, ds-m134, ds-m135,ds-m136, ds-m137, ds-m138, ds-m139, ds-m140, ds-m141, ds-m142, ds-m143, ds-m1 44. ds-m145, ds-m146, ds-m147, ds-m148, ds-m149, ds-m150, ds-m151, ds-m152, ds- m153, ds-m154, ds-m155, ds-m156, ds-m157, ds-m158, ds-m159, ds-m160, ds-m161, d s-m162, ds-m163, ds-m164, ds-m165, ds-m166, ds-m167, ds-m168, ds-m169, ds-m170 , ds-m171, ds-m172, ds-m173, ds-m174, ds-m175, ds-m176, ds-m177, ds-m178, ds-m 179, ds-m180, ds-m181, ds-m182, ds-m183, ds-m184, ds-m185, ds-m186, ds-m187, ds Any of the following structures shown: -m188, ds-m189, ds-m190, ds-m191, ds-m192, ds-m193, ds-m194, ds-m195, ds-m196, ds-m197, ds-m198, ds-m199, ds-m200, ds-m201, ds-m202, ds-m203, or ds-m204.

[0063] Any one of the structures shown in Table 2.

[0064] Table 2 RNA Agents

[0065] This invention also provides RNA agents for inhibiting INHBC gene expression, with structures such as ds-m1, ds-m2, ds-m3, ds-m4, ds-m5, ds-m6, ds-m7, ds-m8, ds-m9, ds-m10, ds-m11, ds-m12, ds-m13, ds-m14, ds-m15, ds-m16, ds-m17, ds-m18, ds-m19, ds-m20, ds-m21, ds-m22, ds-m23, ds-m24, ds-m25, ds-m26, ds-m27, ds-m28, ds-m29, ds-m30, ds-m31, ds-m32, and ds-m33. ds-m34, ds-m35, ds-m36, ds-m37, ds-m38, ds-m39, ds-m40, ds-m41, ds-m42 , ds-m43, ds-m44, ds-m45, ds-m46, ds-m47, ds-m48, ds-m49, ds-m50, ds-m51 , ds-m52, ds-m53, ds-m54, ds-m55, ds-m56, ds-m57, ds-m58, ds-m59, ds-m6 0. ds-m61, ds-m62, ds-m63, ds-m64, ds-m65, ds-m66, ds-m67, ds-m68, ds-m6 9. ds-m70, ds-m71, ds-m72, ds-m73, ds-m74, ds-m75, ds-m76, ds-m77, ds-m 78. ds-m79, ds-m80, ds-m81, ds-m82, ds-m83, ds-m84, ds-m85, ds-m86, ds-m 87. ds-m88, ds-m89, ds-m90, ds-m91, ds-m92, ds-m93, ds-m94, ds-m95, ds- m96, ds-m97, ds-m98, ds-m99, ds-m100, ds-m101, ds-m102, ds-m103, ds-m10 4. ds-m105, ds-m106, ds-m107, ds-m108, ds-m109, ds-m110, ds-m111, ds-m 112, ds-m113, ds-m114, ds-m115, ds-m116, ds-m117, ds-m118, ds-m119, ds- m120, ds-m121, ds-m122, ds-m123, ds-m124, ds-m125, ds-m126, ds-m127, d s-m128, ds-m129, ds-m130, ds-m131, ds-m132, ds-m133, ds-m134, ds-m135,ds-m136, ds-m137, ds-m138, ds-m139, ds-m140, ds-m141, ds-m142, ds-m143, ds-m1 44. ds-m145, ds-m146, ds-m147, ds-m148, ds-m149, ds-m150, ds-m151, ds-m152, ds- m153, ds-m154, ds-m155, ds-m156, ds-m157, ds-m158, ds-m159, ds-m160, ds-m161, d s-m162, ds-m163, ds-m164, ds-m165, ds-m166, ds-m167, ds-m168, ds-m169, ds-m170 , ds-m171, ds-m172, ds-m173, ds-m174, ds-m175, ds-m176, ds-m177, ds-m178, ds-m1 79. ds-m180, ds-m181, ds-m182, ds-m183, ds-m184, ds-m185, ds-m186, ds-m187, ds- Any one of m188, ds-m189, ds-m190, ds-m191, ds-m192, ds-m193, ds-m194, ds-m195, ds-m196, ds-m197, ds-m198, ds-m199, ds-m200, ds-m201, ds-m202, ds-m203, or ds-m204 is shown.

[0066] In some schemes, the aforementioned RNA agents that inhibit INHBC gene expression include structures such as any one of ds-m22, ds-m23, ds-m26, ds-m29, ds-m43, ds-m44, ds-m58, ds-m59, ds-m66, ds-m81, ds-m89, ds-m106, ds-m107, ds-m108, ds-m111, ds-m112, ds-m116, ds-m156, ds-m157, ds-m164, ds-m165, or ds-m170.

[0067] The present invention also provides RNA agents comprising any one of the structures shown in Table 5, such as Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, Z16, Z17, Z18, Z19, Z20, Z21 or Z22.

[0068] The present invention also provides a pharmaceutical composition comprising the above-described RNA agent that inhibits INHBC gene expression, and / or a physiologically acceptable excipient and / or carrier and / or diluent.

[0069] In some embodiments, the pharmaceutically acceptable carrier in the above-described pharmaceutical composition includes or is selected from aqueous carriers, liposomes, polymers, or peptides.

[0070] The present invention also provides the use of the above-mentioned RNA agent for inhibiting INHBC gene expression or the above-mentioned pharmaceutical composition in the preparation of a medicament for the prevention or treatment of diseases or pathologies related to INHBC expression or for reducing the risk of diseases or symptoms related to INHBC expression.

[0071] The present invention also provides the above-mentioned RNA agent or pharmaceutical composition for inhibiting INHBC gene expression for the prevention or treatment of diseases or pathologies associated with INHBC expression or for reducing the risk of diseases or symptoms associated with INHBC expression.

[0072] The present invention also provides a method for preventing or treating diseases or pathologies associated with INHBC gene expression or for reducing the risk of diseases or symptoms associated with INHBC gene expression, comprising administering an effective amount of the above-mentioned RNA agent that inhibits INHBC gene expression or the above-mentioned pharmaceutical composition to a subject in need.

[0073] In some protocols, the aforementioned diseases or pathologies are obesity (e.g., harmful obesity), coronary heart disease, abnormal blood pressure, and metabolic diseases.

[0074] In some protocols, the aforementioned metabolic diseases include diabetes or hyperlipidemia.

[0075] Some solutions in this invention are derived from arbitrary combinations of the above-mentioned variables. Beneficial effects

[0076] The RNA agent provided in this application can significantly or increase the level of target mRNA that inhibits cells.

[0077] definition

[0078] Unless otherwise specified, the following terms and phrases used in this application are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0079] As used herein, the terms “optional,” “optional,” “optional,” or “optionally” are equivalent in meaning, indicating 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” or “alkyl optionally substituted” includes “alkyl” (where the H on the alkyl group is not substituted / replaced by a non-H substituent) and “substituted alkyl” (where the H on the alkyl group is substituted / replaced by a non-H substituent). As used herein, those skilled in the art will understand that for any group comprising 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. For example, “optionally modified” includes both unmodified and modified, and further, “nucleotide optionally modified” includes both unmodified and modified nucleotides.

[0080] As used herein, when any variable (e.g., a substituent R, such as a nucleic acid being modified) appears more than once in the composition or structure of a compound, its definition is independent in each case. For example, if a group is substituted by 0-2 Rs, the group may optionally be substituted by at most two Rs, and the Rs in each case have independent options. As another example, when multiple nucleotides are modified, each nucleotide is independently and optionally modified, and the type and number of modifications to each nucleotide may be the same or different.

[0081] As used herein, unless otherwise stated, “comprising,” “including,” “at least,” “having,” “having,” “containing,” or equivalents are open-ended expressions that mean that in addition to the elements, components, or steps listed, other unspecified elements, components, or steps may be included.

[0082] As used herein, the term "nucleotide" refers to a pentose sugar (ribose or deoxyribose), a phosphate group, and a base (natural or non-natural), and is intended to include both unmodified (i.e., natural) nucleotides and modified nucleotides. In some embodiments, the nucleotide is an unmodified ribonucleotide. In some embodiments, the ribonucleotide is a 3'-ribonucleotide. In some embodiments, the ribonucleotide is a 5'-ribonucleotide. In some embodiments, the modified or unmodified nucleotide may optionally be further modified.

[0083] Natural nucleotides are composed of natural bases, natural ribose, and phosphate. The natural nucleotides used in this article refer to adenine ribonucleotides, adenine deoxyribonucleotides, guanine ribonucleotides, guanine deoxyribonucleotides, cytosine ribonucleotides, cytosine deoxyribonucleotides, uracil ribonucleotides, thymine ribonucleotides, or thymine deoxyribonucleotides. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of its sugar moiety. "Deoxyribonucleoside" refers to a nucleotide having a hydrogen atom at the 2' position of its sugar moiety.

[0084] The natural bases of RNA include A (adenine), G (guanine), C (cytosine), U (uracil), and T (thymine).

[0085] Nucleotides can be substituted with their analogues, including both natural and non-natural analogues. Examples of guanosine analogues include, but are not limited to, 6-thioguanosine, 8-azaguanosine, 8-oxoguanosine, and 2-aminopurine nucleoside. Examples of adenosine analogues include, but are not limited to, cordycepin (3′-deoxyadenosine), n6-benzyladenosine, and 2-chloroadenosine. Examples of cytidine analogues include, but are not limited to, gemcitabine (2',2'-difluoro-2'-deoxycytidine), cytarabine (1-β-d-arabinoseurerylcytosine), and decitabine (5-aza-2'-deoxycytidine). Examples of uracil analogues include, but are not limited to, 5-fluorouracil, pseudouracil, 5-bromouracil, 4-thiouracil, and 5-azidouracil.

[0086] As used herein, “ribonucleic acid” (RNA) is the carrier of genetic information found in cells and some viruses and viroids. RNA is a long chain molecule formed by ribonucleotides linked by nucleotide bonds, including single-stranded RNA and double-stranded RNA. The natural nucleotide bond is the phosphodiester bond. In some embodiments, the ribose, bases, and nucleotide bonds in RNA may be modified independently and optionally.

[0087] As used herein, "double-stranded ribonucleic acid" is a complex consisting of two nucleic acid strands bonded together from natural or non-natural nucleotides, similar in structure and function to natural ribonucleic acid. These two nucleic acid strands contain antiparallel and substantially complementary sequences. "Substantially complementary" means that, while maintaining function, the antiparallel regions of the two nucleic acid strands may contain a certain number of base mismatches and non-matches. In some embodiments, the certain number refers to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the certain number refers to 1, 2, 3, 4, or 5. In some embodiments, the certain number refers to 1, 2, or 3.

[0088] As used herein, the term “oligonucleotide” refers to a nucleic acid molecule (RNA or DNA) that is, for example, less than 100, 200, 300, or 400 nucleotides in length.

[0089] As used herein, “bonding” refers to the connection between residues of two monomers (such as nucleotides) (such as nucleosides) via a single bond or a group (such as a phosphodiester bond, thiophosphate bond, or dithiophosphate bond). In some embodiments, the bonding refers to the connection between residues of two nucleotides via a phosphodiester bond, thiophosphate bond, or dithiophosphate bond.

[0090] As used herein, a "monomer" is a class of compounds that can be assembled into a ribonucleic acid chain and perform a certain function. As used herein, a "monomer" includes, but is not limited to, natural nucleotides, non-natural nucleotides (e.g., modified nucleotides, nucleotide analogs, inverted non-base deoxynucleotides, GNA, LNA, etc.), and M06.

[0091] As used herein, “inhibition” means that, when a given gene is expressed, gene expression is reduced when that cell, cell population, or tissue is treated with the single / double-stranded RNA, single / double-stranded RNA conjugate, or a pharmaceutical composition comprising one or more of these as described in this application, compared to cells, cell populations, or tissues that have not been treated in this way. The terms “inhibition,” “reduction,” “silencing,” “downregulation,” “suppression,” and other similar terms used herein are used interchangeably and include any level of inhibition. Preferably, inhibition includes statistically significant inhibition or clinically significant inhibition.

[0092] As used herein, "conjugation" refers to the covalent connection between two or more chemical parts, each with a specific function; correspondingly, "conjugated compound" refers to a compound formed by the covalent connection of these chemical parts. For example, "double-stranded ribonucleic acid conjugation" refers to a compound or complex formed by covalently linking one or more chemical parts with specific functions (such as a delivery system, ligand group, or conjugation group) to a double-stranded ribonucleic acid. In some embodiments, the delivery system, ligand group, or conjugation group may be attached to a phosphate group, a sugar ring (including the delivery system, ligand group, or conjugation group covalently linked to an atom at the 3' or 5' position of the nucleotide via a phosphodiester bond), a 2'-hydroxyl group, a 5'-hydroxyl group, or a base of any nucleotide of the double-stranded ribonucleic acid. In some embodiments, the delivery system, ligand group, or conjugation group may also be attached to the 2' position of the nucleotide, in which case the nucleotides are connected by a 2'-5' phosphodiester bond. In some embodiments, the delivery system, ligand group, or conjugate group may also be attached to the 3'-position of the nucleotide, in which case the nucleotides are linked by a 3'-5' phosphodiester bond.

[0093] As used herein, "complementary" or "anti-complementary" may be used interchangeably to refer to a structural relationship between two nucleotides (e.g., on two opposing nucleic acid chains or on opposing regions of a single nucleic acid chain) that allows the two nucleotides to form base pairs with each other (e.g., a purine nucleotide of a nucleic acid complementary to a pyrimidine nucleotide of an opposing nucleic acid may form a base pair together by forming hydrogen bonds with each other). In some embodiments of this application, complementary nucleotides may form base pairs in a Watson-Crick manner or in any other manner that allows for the formation of a stable double helix. In some embodiments of this application, the two nucleic acid chains may have multiple regions forming complementary double helixes. In some embodiments of this application, in DNA, adenine (A) always pairs with thymine (T), and in RNA, adenine (A) pairs with uracil (U); guanine (G) always pairs with cytosine (C). In some embodiments of this application, the complementary nucleotide may also comprise or consist entirely of base pairs formed from non-Watson-Crick base pairs and / or from non-natural and modified nucleotides, such non-Watson-Crick base pairs including, but not limited to, G:U swing base pairs or Hoogstein base pairs. In some embodiments of this application, nucleotides containing hypoxanthine as their base may pair with nucleotide bases containing adenine, cytosine, or uracil. In some embodiments of this application, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequence of this application by nucleotides containing, for example, inosine (in this application, the capital letter "I" can represent a hypoxanthine base, inosine, or an inosine-containing nucleotide, depending on the context) (this replacement is referred to as "I modification"). In some schemes of this application, adenine and cytosine anywhere in the oligonucleotide can be replaced by guanine and uracil, respectively, to form a GU swing base pair with the target mRNA.

[0094] The degree of complementarity between two oligonucleotides is called complementarity, which is measured by the percentage of bases in each strand that can form hydrogen bonds with each other, determined by established base pairing rules. Oligonucleotide sequences do not need to be "perfectly complementary" (i.e., "completely complementary") to their corresponding nucleic acid sequences. In some embodiments, a first nucleotide sequence is considered complementary to a second nucleotide sequence if the first nucleotide sequence exhibits at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence complementarity. In one exemplary embodiment, 18 of the 20 nucleobases of the first nucleotide sequence pair with corresponding regions of the second nucleotide sequence, achieving 90% complementarity. Non-complementary nucleobases, also known as "mismatches," may cluster or spread between complementary bases and do not need to be adjacent to each other or adjacent to complementary nucleobases.

[0095] The term "mismatch" as used in this article includes, but is not limited to:

[0096] 1) Two opposing (independent natural or non-natural) nucleotides (other than AT, AU, or GC) pairing;

[0097] 2) No hydrogen bonds are formed between two opposing (independent natural or non-natural) nucleotides;

[0098] 3) A base is missing between two opposing (independent natural or non-natural) nucleotides.

[0099] In some embodiments, mismatches include wobbly base pairing and Hoogstein base pairing.

[0100] The term "fully complementary" refers to a hybrid formed by the first and second nucleotide sequences in a fully complementary region consisting only of Watson-Crick base pairs. "Fully complementary" oligonucleotides may include internal regions (e.g., at least 7, 8, 9, or 10 nucleotides) that are fully complementary to the target RNA. In some embodiments, the targeting region (e.g., the antisense strand or the second strand) provided herein is fully complementary to a portion of the target mRNA encoding the target gene. In some embodiments, the targeting region has at least 80%, 85%, 90%, or 95% complementarity (fully complementary) to a portion of the target mRNA encoding the target gene. In some embodiments, the targeting region has 100% complementarity (fully complementary) to a portion of the target mRNA encoding the target gene.

[0101] As used herein, a blocking group is a group that can be conjugated to an oligonucleotide provided herein, for example, at the 5' end of the antisense strand, which can reduce or inhibit exonuclease cleavage. In some embodiments, the blocking group can reduce or inhibit the RNA interference effect of the oligonucleotide. In some embodiments, the blocking group is cleaved from the oligonucleotide before providing the RNA interference effect. Examples of blocking groups include, but are not limited to, non-basic nucleotide residues, reverse non-basic nucleotide residues, MO3, and MO6.

[0102] As used herein, the double-stranded nucleotide reagent may optionally be conjugated to one or more blocking groups. The blocking group may be attached to the sense strand, antisense strand, or both strands at the 3' end, 5' end, or both ends. In some embodiments, the blocking group is conjugated to the antisense strand, specifically to the 5' end of the antisense strand. In some embodiments, the blocking group is conjugated to an oligonucleotide (e.g., the 5' end of the antisense strand) via a nucleotide bond, and the internucleotide bond is optionally modified as described herein. In some embodiments, the blocking group is linked to the double-stranded nucleotide reagent via a phosphate thioester. In some embodiments, the blocking group is linked to the double-stranded nucleotide reagent via a phosphodiester bond.

[0103] As used herein, the term "modified nucleotide" refers to a nucleotide having modified internucleotide bonds, and / or modified bases, and / or modified sugars. In some embodiments, the modified nucleotide comprises one, two, three, or more modifications. In some embodiments, the nucleotide comprises one modification. In some embodiments, the nucleotide comprises two modifications. In some embodiments, the nucleotide comprises three modifications.

[0104] As used herein, “modification” of nucleotides includes, but is not limited to: 2'-OMe (2'-O-methyl) modification, 2'-F (2'-deoxy-2'-fluorine) modification, 2'-O-MOE (2'-O-methoxyethyl) modification, 2'-deoxy (2'-d) modification, 5'-morpholine (5'-Mo) modification, unlocked nucleic acid (UNA) modification, glycol nucleic acid (GNA) modification, locked nucleic acid (LNA) modification, tricyclic DNA (tcDNA) modification, (S)-restricted ethyl bicyclic nucleic acid ((S)-cEt-BNA) modification, thiophosphate (PS) modification, dithiophosphate (PS2) modification, methylphosphonate (MP) modification, methoxypropylmethylphosphonate (MOP) modification, phosphoselenate modification, phosphodiselenate modification, phosphorylaminosulfate modification, phosphoryl amide salt modification, phosphoramidate modification, peptide nucleic acid (PNA) modification, 5'-(E)-vinyl phosphate ( Modifications include: VP modification, N6-methyladenosine (m6A) modification, 5-methylcytidine (m5C) modification, 3-methyluridine (m3U) modification, 5-methylureaside (m5U) modification, pseudoureaside modification, 2-thioureaside (s2U) modification, propynouracil (5-pU) modification, linking the 5' or 3' end of the nucleotide to an inverted abase-free nucleotide (invAB) modification, replacing the nucleotide with an inverted abase-free nucleotide (invAb) modification, replacing the nucleotide with 2,4-difluorotolyl ribonucleotide (rF) modification, replacing the nucleotide with (S)-glycerol nucleic acid modification, replacing the nucleotide with hypoxanthine nucleotide (I) modification, replacing the nucleotide base with xanthine, replacing the nucleotide base with 7-methylguanine, replacing the nucleotide base with 5,6-dihydrouracil, and linking the 5' or 3' end of the nucleotide to M06 (M06) modification, etc. In some embodiments, at least one nucleotide comprises one, two, three, or more modifications. In some embodiments, at least one nucleotide is unmodified. In some embodiments, at least one nucleotide comprises one modification. In some embodiments, at least one nucleotide comprises two modifications. In some embodiments, at least one nucleotide comprises three modifications. In some embodiments, all nucleotides are modified, and each nucleotide independently comprises one, two, or three modifications.

[0105] As used herein, in some embodiments, "G", "A", "C", "U", and "T" refer to guanine ribonucleotide, cytosine ribonucleotide, adenine ribonucleotide, thymine ribonucleotide, and uracil ribonucleotide, respectively. Exemplary structures are as follows:

[0106] As used herein, in some embodiments, the RNA agent is synthesized using a phosphoramidite solid-phase synthesis technique, wherein the structure is as follows: When “G”, “A”, “C”, “U”, and “T” are attached to the 5' end 1 position of the strand...

[0107] When “G”, “A”, “C”, “U” and “T” are connected at the 3' end of the chain, the structure is as follows:

[0108] As used herein, the prefix "d" before a monomer (such as nucleotides A, U, C, G, and T) indicates that the monomer is 2'-deoxy modified. An example nucleotide structure with 2'-deoxy modification is shown below:

[0109] As used herein, the "f" label following a monomer (such as nucleotides A, U, C, G, and T) indicates that the monomer is 2'-deoxy-2'-fluorine modified (2'-F modified). An example nucleotide structure with 2'-F modification is shown below:

[0110] As used herein, the prefix "GNA-" before monomers (such as nucleotides A, U, C, G, and T) indicates that the monomer has been modified with ethylene glycol-based nucleic acids (GNA modification). As used herein, Tgn is the abbreviation for GNA-T, with the same meaning. An example of a GNA-modified nucleotide structure is shown below:

[0111] As used herein, lowercase letters (a, u, c, g, t, etc.) indicate that the nucleotide represented by the corresponding uppercase letters (A, U, C, G, and T, etc.) is modified with 2'-O-methyl (2'-OMe). An example nucleotide structure modified with 2'-OMe is shown below:

[0112] As used herein, invAB modification refers to the attachment of an inverted, baseless deoxynucleotide to a monomer (e.g., at the 5' or 3' end of the nucleotide). For example, The structure modified by invAB:

[0113] As used herein, invAb modification refers to the replacement of a monomer (such as a nucleotide) with an inverted, non-basic nucleotide (invAb). For example, The structure modified by invAb:

[0114] As used herein, VP modification refers to the modification of a monomer by (E)-vinyl phosphate (e.g., modification of the 5' position of a nucleotide by 5'-(E)-vinyl phosphate). For example, the structures of U, u, and dU after modification are as follows:

[0115] As used in this article, M06 modification refers to the bonding of a linker at the 5' or 3' end of a monomer (such as a nucleotide). For example, Structure modified by M06: In some embodiments, M06 is through It is bonded to a nucleotide.

[0116] As used in this article, marking "*" between monomers (such as nucleotides A, U, C, G, and T) indicates that the two monomers are linked by a thiophosphate bond (i.e., a thiophosphate diester bond), meaning they are modified by thiophosphate (PS).

[0117] As used in this article, the absence of an asterisk (*) between nucleotides (A, U, C, G, and T, etc.) indicates that the two nucleotides are linked by a phosphate ester bond (i.e., a phosphodiester bond).

[0118] For example, “5'-AdUgCf*dT-3'” indicates that, starting from the 5' end, position 1 is an adenine ribonucleotide, position 2 is a uracil deoxyribonucleotide, position 3 is a 2'-methoxy-modified guanine ribonucleotide, position 4 is a 2'-fluorine-modified cytosine ribonucleotide, and position 5 is a thymine deoxyribonucleotide linked to position 4 by a phosphate thioester bond. For example, the structure “5'-(M06)*AdTgCf*(invAB)-3'” is as follows:

[0119] For example, the structure shown as “5'-(M06)*AdTgCf*[D]-3'” is as follows:

[0120] As used in this article, the structures of NAG37 monomer and L96 monomer are respectively

[0121] [NAG37] and [L96] represent its residues, respectively. For example, the sequence [NAG37]AfGfu*[L96] has the following structure:

[0122] As used herein, “small interfering RNA,” “siRNA,” or “iRNA” agents are interchangeable and can be used to trigger RNA interference (RNAi) mechanisms by specifically binding to the target mRNA sequence, leading to the degradation of the target mRNA and thereby inhibiting gene expression of single-stranded or double-stranded ribonucleic acid molecules (e.g., siRNA agents or their cleavage products can downregulate target genes by, for example, inducing RNAi with respect to the target RNA, wherein the target may include endogenous or pathogenic target RNA). In some embodiments, the siRNA is at least partially complementary to the coding sequence in the target gene expressed in cells. In some embodiments, after delivery of the siRNA to cells expressing the gene, the siRNA can inhibit or block gene expression in vitro or in vivo. Typically, the siRNA contains less than 60 The siRNA comprises a double-stranded region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 complementary base pairs. In some embodiments, the sense and antisense strands of the siRNA are each independently 15-30 nucleotides long, forming a complementary double-stranded region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 base pairs in length. In some embodiments, the sense and antisense strands of the siRNA are completely complementary and have a length of 15-30 base pairs. In some embodiments, the sense and antisense strands of the siRNA are completely complementary and have a length of 17, 18, 19, 20, 21, or 22 base pairs.

[0123] In some embodiments, the length of the ribonucleic acid chain is calculated in nt (nucleotides), where 1 nt (1 nucleotide) includes, but is not limited to, 1 natural nucleotide and 1 modified nucleotide.

[0124] As used in this article, when a sequence region has 0 nucleotides, it includes cases where the region does not exist but is directly connected to the two regions on its left and right.

[0125] As used herein, a “pharmaceuticalally acceptable carrier” may include, but is not limited to, excipients and / or other components. An “excipient” is a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert medium used to deliver one or more nucleic acids to animals. Such agents are well known in the art.

[0126] As used herein, “subject” means any animal, such as a mammal or marsupial. Subjects in this application include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, cattle, rats, or any kind of poultry.

[0127] As used herein, the term "treatment," "curing," or "treatment" refers to the management, elimination, reduction, or improvement of a disease and its associated symptoms, and also refers to methods for 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 physiological symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be suffering from the underlying disorder. While the possibility of complete elimination of the disease or associated symptoms is not excluded, treating a disease does not require the complete elimination of the disease or associated symptoms. As used herein, the term "treatment" also includes "preventive treatment," which is applied before the onset of symptoms or disease manifestations to reduce the likelihood of disease occurrence or recurrence, or to reduce the likelihood of recurrence of a disease that is already under control. This applies to individuals who are not yet ill but are at risk or prone to recurrence, or individuals who are at risk or susceptible to disease recurrence. In the sense of this invention, "treatment" also includes prevention of recurrence or a preventive phase, as well as treatment of acute or chronic signs, symptoms, and / or functional impairments. Treatment may target symptoms, such as symptom suppression. Treatment can be administered in the short term, in the medium term, or as a long-term treatment, such as maintenance therapy.

[0128] As used herein, “effective dose” refers to a drug dose that produces the expected local or systemic therapeutic effect at a reasonable benefit / risk ratio, applicable to any treatment alone or in combination with further doses. In treating a specific disease, the desired local or systemic therapeutic effect typically involves the inhibition of disease progression. This includes slowing disease progression, particularly interrupting or reversing it. When used for disease prevention, the dose is sufficient to prevent or delay the onset of disease. An effective dose does not necessarily have a curative effect or completely prevent disease. The effective dose of the aforementioned drugs will depend on the condition being treated, the severity of the disease, the patient’s individual parameters (including age, physiological condition, body size, and weight), the duration of treatment, the type of concomitant treatment (if any), the specific route of administration, and similar factors. Therefore, the drug dose may vary depending on these parameters. If the initial dose is insufficient to elicit a patient’s response, a higher dose may be used (or a higher effective dose achieved through a different, more local route of administration). In some cases, the effective dose of a drug will also depend on factors such as its therapeutic index and solubility.

[0129] The compositions of this application may further include other auxiliary components conventionally present in pharmaceutical compositions at levels established in the art. Thus, for example, the composition may contain additional, compatible pharmaceutically active substances, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional substances suitable for the physical formulation of various dosage forms of the compositions of this application, such as preservatives, antioxidants, and stabilizers. However, when added, such substances should not unduly interfere with the biological activity of the components of the compositions of this application. The formulation may be sterilized, and if necessary, may be mixed with adjuvants that will not harmfully interact with the nucleic acids of the formulation, such as preservatives, stabilizers, humectants, emulsifiers, salts or buffers that affect osmotic pressure, etc. Attached Figure Description

[0130] Figure 1 is a schematic diagram of the structural relationship between the first chain, the second chain, the 3' extension segment, and the 5' extension segment.

[0131] Figure 2 shows the relative changes in INHBC mRNA in the liver of each group of mice after drug administration.

[0132] Figure 3 shows the relative changes in INHBC mRNA in the liver of each group of mice after drug administration.

[0133] Figure 4 shows the weight change relative to the PBS negative control group.

[0134] Figure 5 shows the changes in fat relative to the PBS negative control group.

[0135] Figure 6 shows muscle changes compared to the PBS negative control group. Detailed Implementation

[0136] The compounds of this application can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical or biological synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of this application.

[0137] The following examples are provided to illustrate this application and are intended to provide a better understanding of it, but are not intended to limit the scope of this application. Any modifications or alterations made to the elements of this application without departing from its spirit and substance are within the scope of this application. Unless otherwise specified, the reagents, kits, and biological materials used in this application are commercially available. Unless otherwise specified, the kits are used in accordance with the kit instructions.

[0138] Example 1: Synthesis of siRNA molecules

[0139] Oligonucleotides were synthesized using a phosphoramidite solid-phase synthesis technique. Synthesis was performed on a general-purpose controlled porous glass CPG. All 2'-modified RNA phosphoramidite and auxiliary reagents were commercially available. All phosphoramidite was dissolved in anhydrous acetonitrile and added to a molecular sieve, with coupling time of 12 min using 5-ethylthio-1H-tetrazole (ETT) as an activator. Phosphophosphate bonds were generated using a 50 mM solution of 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazol-3-thione (DDTT) in anhydrous acetonitrile / pyridine (v / v = 1 / 1) for 1.4 min, or oxidized using a 0.05 M solution of iodine in pyridine / water (v / v = 9 / 1) for 1 min. All sequences were synthesized after the final removal of the DMT group.

[0140] Cleavage and deprotection of oligomers bound to CPG: After termination of solid-phase synthesis, the protecting group was removed by treatment with an acetonitrile solution containing 20% ​​diethylamine for 30 minutes without cleaving the oligonucleotide from the CPG. Subsequently, the dried CPG was treated with concentrated ammonia at 55°C for 16 hours. After centrifugation, the supernatant was transferred to a new tube and the CPG was washed with ammonia. The combined solutions were concentrated to obtain a solid mixture.

[0141] Purification of single-stranded oligonucleotides: Ion-pair reversed-phase purification was performed using a C18 column. Buffer A consisted of 0.1 M TEAA in 5% acetonitrile aqueous solution; Buffer B consisted of acetonitrile. Anion exchange was then performed on the target product.

[0142] Oligomers were purified by HPLC using NanoQ anion exchange. Buffer A was 0.1 M ammonium acetate in 15% acetonitrile / water solution, and buffer B was 1.5 M sodium bromide + 0.1 M ammonium acetate in 15% acetonitrile / water solution. The target product was separated and desalted using a reversed-phase C18 column.

[0143] Annealing of single-stranded oligonucleotides to produce siRNA: The single-stranded oligonucleotides to be annealed are dissolved in sterile RNase-free water (free of RNA hydrolase). Equimolar amounts of the single-stranded oligonucleotide solutions are combined to form complementary strands. The annealing reaction system is set up as follows: the mixture is placed in a 95°C water bath for 2 minutes, cooled to room temperature, and freeze-dried to obtain the final product, siRNA.

[0144] Example 2: Cell viability test

[0145] ●Cell line

[0146] HepG2 cells were provided by Nanjing Kebai Biotechnology Co., Ltd. HepG2 cells were cultured in MEM medium (Gibco catalog number 11095080) containing 10% fetal bovine serum (FBS, Gibco catalog number A5669701), 1% glutamine (GlutaMAX, Gibco catalog number 35050061), and 1% NEAA (Gibco catalog number 11140050).

[0147] ●Main Instruments

[0148] The main instruments used in this experiment included a fluorescence qPCR instrument (Kunpeng BioX6), a centrifuge (ThermoFisher, catalog number 75016073), and a cell counter (Countstar Rigel2).

[0149] ●Main reagents and consumables

[0150] The main reagents used in this experiment included Lipofectamine. TM iRNAiMAX transfection reagent (INVITROGEN, catalog number 13778150), RNA extraction kit (Qiagen, catalog number 74182), FastKing cDNA first-strand synthesis kit (TianGen, catalog number KR116-02), 96-well plates (Costar 3599). SuperReal PreMix Plus (SYBR Green) (TIANGEN, catalog number: FP205), hGAPDH and hINHBC qPCR primers were synthesized by GenScript.

[0151] ●Experimental Methods

[0152] 1. Compound transfection plate

[0153] HepG2 cells were seeded (1.5 × 10⁻⁶ cells). 4 Cells were plated into 96-well cell culture plates, and siRNA was simultaneously transfected into the cells using RNAiMAX. Two concentration points were set for the siRNA assay (50 nM, 0.5 nM). Cells were incubated overnight at 37°C in a 5% CO2 incubator for 24 hours. A control group containing RNAiMAX but without the compound was also included.

[0154] 2. RNA extraction and reverse transcription

[0155] 24 hours after transfection, remove the culture medium and collect the cells for RNA extraction. Use according to the kit instructions. Total RNA was extracted using a 96-kit (QIAGEN-74182). cDNA was synthesized using the FastKing RT Kit (With gDNase) (Tiangen-KR116-02) according to the manufacturer's instructions.

[0156] 3. qPCR detection of target gene mRNA expression levels

[0157] The target cDNA will be detected by qPCR, while GAPDH cDNA will be detected as an internal control in parallel. 18 μL of the prepared PCR reaction solution and 2 μL of sample cDNA will be added to each 96-well container. The qPCR program is as follows: heat at 95°C for 15 min, then cycle at 95°C for 10 sec, 60°C for 20 s, followed by 72°C for 32 s, for a total of 40 cycles.

[0158] ●Data Analysis

[0159] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCt relative quantification method. The relative expression level of the target gene was expressed as 2-ΔΔCT.

[0160] The calculation formula is as follows:

[0161] ΔCT = Average Ct value of target gene - Average Ct value of internal reference gene

[0162] ΔΔCT = ΔCT (drug-treated group) - ΔCT (RNAiMAX control group)

[0163] The relative expression level of the target gene INHBC = 2 -ΔΔCT

[0164] Based on the relative expression level of the hINHBC gene after treatment, the inhibition rate of each candidate molecule on the target gene was obtained using the formula (1 - test group / control group)%.

[0165] ●Experimental Results

[0166] The percentage of inhibition rate of each candidate molecule on the target gene (compared to the control group) is shown in Table 3 below.

[0167] Table 3

[0168] Experimental conclusions

[0169] The RNA agent of this invention can significantly inhibit the level of INHBC mRNA in HepG2 cells.

[0170] Example 3: Cell viability test

[0171] Referring to Example 2, the siRNA assay was performed at seven concentration points (10, 2.5, 0.625, 0.15625, 0.0390625, 0.009765625, 0.002441406 nM) to detect the candidate compounds obtained in Example 2. The IC50 of each candidate molecule and the percentage of maximum inhibition of the target gene (compared to the control group) are shown in Table 4 below.

[0172] Table 4

[0173] Example 4: In vitro siRNA uptake experiment in primary cynomolgus monkey hepatocytes (CPH)

[0174] ●Purpose

[0175] Using CPH cell lines, the in vitro activity of siRNA molecules was evaluated by assessing the degree of silencing of target genes by various candidate molecules.

[0176] ●Cell line

[0177] CPH cells, primary hepatocytes derived from cynomolgus monkeys, were provided by Myoshun (Shanghai) Biotechnology Co., Ltd. Hepatocyte culture medium kit (Myoshun, HCTPM-R-002) was required for CPH resuscitation and culture.

[0178] ●Main Instruments

[0179] The main instruments used in this experiment included a fluorescence qPCR instrument (Roche 480 II), a centrifuge (Thermo Fisher catalog number 75016073), and a cell counter (Countstar Rigel 2).

[0180] ●Main reagents and consumables

[0181] The main reagents used in this experiment included an RNA extraction kit (Tiangen, catalog number DP671-T1), a FastKing cDNA first-strand synthesis kit (TianGen, catalog number KR116-02), a 96-well plate (Costar 3599), a SuperReal PreMix Plus (SYBR Green) (TIANGEN, catalog number: FP215-02), qPCR primers synthesized by Genewiz, and a hepatocyte culture medium kit (Miaoshun, HCTPM-R-002).

[0182] ●Experimental Methods

[0183] 1. Cell plating and compound treatment

[0184] Prepare the collagen coating solution 40 minutes in advance. Add 100 μL of rat tail collagen (AR0001-02) to the hepatocyte coating medium into each well of a 96-well cell plate. After 40 minutes, aspirate the coating solution from the cell plate, remove the cells from the liquid nitrogen container and place them in a 37°C water bath. When they are almost completely thawed, pour them into preheated hepatocyte resuscitation medium (HPM-R-40), centrifuge, and resuspend them in hepatocyte plating medium for cell counting.

[0185] Seed CPH cells (3×10) 4 Cells were transferred from one well to a coated 96-well cell plate. Simultaneously, siRNA was added to the cells. Seven concentration points were set for the siRNA assay (starting at 150 nM, 4-fold dilution). Cells were incubated at 37°C in a 5% CO2 incubator. A compound-free control group was also included.

[0186] The siRNA used was a conjugate prepared based on solid-phase synthesis, and the ligand used was L96. The obtained siRNA conjugates are shown in Table 5.

[0187] Table 5

[0188] 2. RNA extraction and reverse transcription

[0189] After 48 hours of free uptake, the culture medium was removed and cells were collected for RNA extraction. Use according to the kit instructions. Total RNA was extracted using the 96 Kit (Tiangen-DP671-T1). cDNA was synthesized using the FastKing RT Kit (With gDNase) (Tiangen-KR116-02) according to the manufacturer's instructions.

[0190] 3. qPCR detection of target gene mRNA expression levels

[0191] The target cDNA will be detected by qPCR, while GAPDH cDNA will be detected as an internal control in parallel. The primer sequences are as follows:

[0192] The GAPDH gene has a forward primer of 5'-TCAACAGCGACACCCACTC-3' (SEQ ID NO:1072) and a reverse primer of 5'-GTCCGGGGGTCTTACTCCTT-3' (SEQ ID NO:1073).

[0193] The target gene INHBC has a forward primer of 5'-TAGCCCAGAGCTCAGTCATCC-3' (SEQ ID NO:1074) and a reverse primer of 5'-ATGCCTCGTCGATGAATCCG-3' (SEQ ID NO:1075).

[0194] ●Data Analysis

[0195] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCt relative quantification method. The relative expression level of the target gene was expressed as 2-ΔΔCT.

[0196] The calculation formula is as follows:

[0197] ΔCT = Average Ct value of target gene - Average Ct value of internal reference gene

[0198] ΔΔCT = ΔCT (drug-treated group) - ΔCT (RNAiMAX control group)

[0199] Relative expression level of target gene = 2 - ΔΔCT

[0200] Based on the relative expression levels of the target genes after treatment, the inhibition rate of each candidate molecule on the target genes was obtained using the formula (1 - test group / control group)%.

[0201] ●Experimental Results

[0202] The IC50 of the candidate molecules and the maximum inhibition rate of the target genes (compared to the control group) are shown in Table 6 below.

[0203] Table 6

[0204] Experimental conclusions

[0205] The double-stranded ribonucleic acid of this invention can significantly inhibit the level of INHBC mRNA in CPH cells.

[0206] Example 5: In vitro human primary hepatocyte (PHH) free uptake experiment of siRNA

[0207] The test compound was prepared into a 100 μM stock solution using RNase-free water, and PHH cells were provided by Shanghai WuXi AppTec New Drug Development Co., Ltd.

[0208] The main instruments used in this experiment included a fluorescence qPCR instrument (Quanstudio 7flex), a centrifuge (Beckman Allegra-X15RCentrifuge), and a cell counter (Countstar Rigel S2).

[0209] The main reagents used in this experiment included AceQ Universal U+Probe Master Mix V2 (Vazyme, catalog number Q513-03), EZB-Press 96RNA Purification (EZB catalog number EZ4001-L), HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, catalog number R323-01), 96-well plates (Costar catalog number 3599), and qPCR-specific primers for GAPDH and INHBC were provided by WuXi AppTec Co., Ltd.

[0210] 5.1 Experimental Methods

[0211] 5.1.1 Cell Plating

[0212] On day 1, siRNA was diluted with nuclease-free water to a starting point of 100 nM, followed by 4-fold serial dilutions for a total of 7 concentration points. 10 μL of each solution was then transferred to a collagen-coated 96-well cell culture plate. One vial of PHH (1 ml / vial) was transferred to 10 ml of preheated InvitroGRO CP Medium containing 10% FBS and 1% Penicilin-Streptomyci, and seeded at a density of 54,000 cells per well (90 μL / well). The final culture medium in each well was 100 μL. Simultaneously, cell control wells were set up: no compound treatment was performed, PBS was used instead of the compound, and all other conditions remained the same. Cells were incubated in a 5% CO2, 37°C incubator for 48 hours.

[0213] 5.1.2 RNA extraction and reverse transcription

[0214] Forty-eight hours later, the culture medium was removed and cells were collected for RNA extraction. Total RNA was extracted using EZB-Press 96 RNA Purification (EZB catalog number EZ4001-L) according to the kit instructions. cDNA was synthesized using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, catalog number R323-01) according to the kit instructions.

[0215] 5.1.3 qPCR detection of target gene mRNA expression levels

[0216] The target cDNA will be detected by qPCR, and the internal reference gene GAPDH cDNA will also be detected. The qPCR reaction system is shown in Table 7. Add 8 μL of the prepared PCR reaction solution and 2 μL of sample cDNA to each of 384 wells. The qPCR reaction program is as follows: 40 cycles of heating at 95°C for 10 min, then at 95°C for 15 sec, then at 60°C for 1 min, followed by heating at 95°C for 15 sec, 60°C for 1 min, and then at 95°C for 15 sec.

[0217] Table 7 RT-PCR reaction system

[0218] 5.2 Data Analysis

[0219] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCt relative quantification method. The relative expression level of the target gene was calculated using a 2-1T / T ratio. -ΔΔCT express.

[0220] The calculation formula is as follows:

[0221] ΔCT = Average Ct value of target gene - Average Ct value of internal reference gene

[0222] ΔΔCT = ΔCT (drug-treated group) - ΔCT (cell control group)

[0223] The relative expression level of the target gene INHBC = 2 -ΔΔCT

[0224] INHBC inhibition rate % = (1 - value of sample / Ave. value of cell control) * 100

[0225] Data were processed using GraphPad Prism software to calculate the 50% inhibitory concentration (EC50) of the compound against INHBC. 50 )value.

[0226] 5.3 Experimental Results

[0227] The IC50 of the candidate molecules and the maximum inhibition rate of the target genes (compared to the control group) are shown in Table 8 below.

[0228] Table 8

[0229] Experimental conclusion: The double-stranded ribonucleic acid of this invention can significantly inhibit the level of INHBC mRNA in PHH cells.

[0230] Example 6: In vivo efficacy of INHBC siRNA in mice

[0231] The in vivo efficacy of INHBC siRNA was evaluated using a mouse model of high-pressure tail vein injection of full-length human INHBC plasmid DNA. Six- to seven-week-old female BALB / c mice were randomly assigned to groups and administered the drug (single subcutaneous injection; 1 mg / kg). Five mice were in the PBS group, and four mice were in each of the drug-treated groups. Blank PBS was used as a negative control. Day 0 was defined as the day of drug administration, with the previous day designated as Day -1, and the day following as Day 1, and so on. On Day 6, all mice were injected intravenously over 5 seconds with 8% of their body weight in plasmid DNA solution (injection volume (mL) = mouse body weight (g) × 8%), with each mouse receiving 20 μg of plasmid. On Day 7 (24 h after INHBC plasmid injection), all mice were euthanized by CO2 inhalation. After euthanasia, liver samples were collected: two 70 mg portions of liver tissue were immersed in CO2. After incubation overnight at 4°C, the supernatant was discarded and the sample was transferred to a -80°C freezer. All liver samples were stored at -80°C until transferred to the laboratory on dry ice for appropriate testing.

[0232] RNA was extracted from liver tissue using a fully automated nucleic acid extraction and purification system. The concentration of extracted liver RNA was detected using a Nanodrop ONE (Thermo) instrument and adjusted to a uniform concentration. Subsequently, it was... III. RT SuperMix for qPCR (+gDNA wiper) (Vazyme-R323): RNA was reverse transcribed into cDNA according to the instruction manual. cDNA was quantified by qPCR. INHBC gene expression was detected during qPCR. NEO mRNA (sequence information shown in Table 9 below) was also detected as an internal control.

[0233] Table 9

[0234] The dosing regimen is shown in Table 10.

[0235] Table 10

[0236] The experimental results are shown in Figure 2. Compounds Z22, Z21, Z20 and Z12 can knock down INHBC mRNA by more than 50% in mice, with Z20 knocking down more than 75%, indicating strong in vivo efficacy.

[0237] Example 7: In vivo efficacy of INHBC siRNA in mice

[0238] Referring to Example 6, the in vivo efficacy of Z20 and Z1-Z10 in mice was tested.

[0239] The dosing regimen is shown in Table 11 below:

[0240] Table 11

[0241] The results are shown in Figure 3. The results indicate that compounds Z20, Z10, Z8, Z5, Z4, Z3, Z2, and Z1 can knock down INHBE mRNA by more than 50% in mice, with Z20, Z3, and Z1 knocking down by more than 75%, suggesting strong in vivo efficacy.

[0242] Example 8: In vivo testing of the inhibitory effect of INHBC in obese mice

[0243] The in vivo efficacy of INHBC inhibition was tested using a diet-induced obesity (DIO) mouse model. DIO mice, aged 14-15 weeks, were fed with 60% HFD for about 9 weeks and were male. After entering the animal facility, all mice were acclimatized for 3 days or more, and (1) their body weight was measured once; (2) their 24-hour food intake was measured once; and (3) their body fat percentage was measured once by MRI. Grouping: The mice were randomly grouped based on body weight and body fat percentage (Fat%) as the main grouping indicators, as shown in Table 12. The day of grouping was defined as D-1, and the drug was administered from D0. The drug administration volume was 5 μL / g × mouse body weight (g); the drug preparation frequency was freshly prepared for each use. VSE246B-mC3683 is a siRNA drug that specifically targets mouse INHBC mRNA. The sequence results are shown in Table 13.

[0244] Table 12

[0245] Table 13

[0246] During the efficacy period, mouse body weight and body fat were measured weekly, and food intake was measured twice weekly. After 9 weeks of drug administration, mice were euthanized, and three portions (approximately 50-100 mg each) of the left lobe of the liver from the same location were placed in an RNAlater for RNA extraction and qPCR quantitative analysis of the knockdown level of INHBC in the mouse liver.

[0247] Experimental results

[0248] qPCR results showed that the level of INHBC mRNA in the liver of mice in group 2 was knocked down by 85% compared with the PBS negative control group.

[0249] Figure 4 shows that the INHBC siRNA group reduced body weight by 9%; Figure 5 shows that the INHBC siRNA group reduced fat by 22%; Figure 6 shows that the INHBC siRNA group increased muscle mass by 2%.

[0250] Experimental conclusions

[0251] The inhibition of INHBC in this invention can achieve the effects of fat reduction and muscle gain.

[0252] Example 9: In vivo efficacy of INHBC siRNA in healthy cynomolgus monkeys

[0253] To evaluate the in vivo activity of siRNA against INHBC, in vivo activity assays were performed using healthy cynomolgus monkeys. The siRNA conjugate (RNAi), diluted with physiological saline, was subcutaneously injected with Z20 at a dose of 3 mg / kg on day 1 according to the experimental design. The specific design is shown in Table 14 below. Liver biopsies were collected on days 12 before administration and on days 14, 28, 56, and 84 after administration. A kit (MagMAX) was used. TM mirVana TM Total RNA was extracted using the Total RNA Isolation Kit and commercially available reagents (Qiankun). TM platinum TM SYBR TM INHBC mRNA levels were determined by real-time PCR using Green premixed buffer. The instrument used for real-time PCR was a QuantStudio 7Flex (Thermo Fisher Scientific).

[0254] First, the obtained test results were standardized using an internal reference gene to obtain relative mRNA levels. Individual standardization was then performed on the INHBC mRNA levels of each animal. For individual standardization, the average INHBC level for each animal at a given time point was divided by the average expression level before drug administration to determine the relative expression level. Data are expressed as mean ± standard error for each group of cynomolgus monkey samples, n = 3.

[0255] Table 14

[0256] Expected experimental results and conclusions: Z20 can reduce INHBC mRNA levels by more than 70% in the liver of cynomolgus monkeys, showing a strong inhibitory effect.

[0257] Although the present invention has been described in detail with reference to embodiments thereof, these embodiments are provided for illustration and not limitation. Other embodiments that can be obtained according to the principles of the present invention fall within the scope defined by the claims of the present invention, including but not limited to the development of drugs such as siRNA, ASO, antibodies, and in vivo gene editing for inhibiting INHBC, with dosages including but not limited to 1-10 mg / kg.

Claims

1. An RNA agent for inhibiting INHBC gene expression, wherein one strand of the RNA agent has at least 75% homology or complementarity with any continuous fragment of 16-35 nucleotides in length in the mRNA encoding INHBC (SEQ ID NO:729).

2. The RNA agent according to claim 1, comprising an antisense strand or an antisense nucleic acid fragment, said antisense strand or antisense nucleic acid fragment having at least 75% complementarity with any continuous fragment of 16-35 nucleotides in length in the mRNA encoding INHBC (SEQ ID NO:729); And / or, it comprises a sense strand or sense nucleic acid fragment, said sense strand or sense nucleic acid fragment having at least 75% homology to any continuous fragment of 16-35 nucleotides in length in the mRNA encoding INHBC (SEQ ID NO:1); Best location: The antisense strand or antisense nucleic acid fragment consists of 19-27 consecutive nucleotides, preferably 21-26 consecutive nucleotides, for example, 21, 22, 23, 24, 25 or 26 consecutive nucleotides; the sense strand or sense nucleic acid fragment consists of 19-27 consecutive nucleotides, preferably 21-26 consecutive nucleotides, for example, 21, 22, 23, 24, 25 or 26 consecutive nucleotides.

3. The RNA agent according to claim 2, wherein the antisense strand or antisense nucleic acid fragment comprises, as shown in SEQ ID NO: 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229. 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 32 5. A series of consecutive nucleotide sequences with a difference of no more than 5, 4, 3, 2, or 1 nucleotide from any of the sequences shown, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, or 364. Preferably, the antisense strand or antisense nucleic acid fragment comprises, for example, SEQ ID NO: 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 2 28, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 2 74, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 3 20, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, or 364.

4. The RNA agent according to claim 2, wherein the sense strand or sense nucleic acid fragment comprises, as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 1 09, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 1 A series of consecutive nucleotide sequences differing by no more than 5, 4, 3, 2 or 1 nucleotide from any of the sequences shown in 50, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 or 182; Preferably, the positive chain or positive nucleic acid fragment comprises, for example, SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 5 5, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144 The sequence shown is 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, or 182.

5. The RNA agent according to claim 2, wherein, The RNA agent comprises an antisense strand or an antisense nucleic acid fragment, and a sense strand or a sense nucleic acid fragment; The antisense strand or antisense nucleic acid fragment comprises any of the sequences shown in SEQ ID NO: 204, 205, 208, 211, 225, 226, 240, 241, 248, 263, 271, 288, 289, 290, 293, 294, 298, 338, 339, 346, 347 or 352; The positive strand or positive nucleic acid fragment accordingly includes any of the sequences shown in SEQ ID NO: 22, 23, 26, 29, 43, 44, 58, 59, 66, 81, 89, 106, 107, 108, 111, 112, 116, 156, 157, 164, 165 or 170.

6. The RNA agent according to any one of claims 2-5, wherein, The antisense strand and the sense strand form a reverse complementary double-stranded region, and the sense strand and antisense strand are located on two nucleic acid strands; And / or, the sense nucleic acid fragment and the antisense nucleic acid fragment form an inverse complementary double-stranded region, and the sense nucleic acid fragment and the antisense nucleic acid fragment are located on the same nucleic acid strand.

7. The RNA agent according to claim 6, wherein, The reverse complementary double-stranded region contains, such as, ds-n1, ds-n2, ds-n3, ds-n4, ds-n5, ds-n6, ds-n7, ds-n8, ds-n9, ds-n10, ds-n11, ds-n12, ds-n13, ds-n14, ds-n15, ds-n16, ds-n17, ds-n18, ds-n19, ds-n20, ds-n21, ds-n22, ds-n23, ds-n24, ds-n25, ds-n26, ds-n27, ds-n28, ds-n29, ds-n30, ds-n31, ds-n32, ds-n33, ds-n34, ds-n35, ds-n36, ds-n37, ds-n38, ds-n39, ds-n40, ds-n41, ds-n42, ds-n43, ds-n44, ds-n45, ds-n46, ds-n47, ds-n48, ds-n49, ds-n50, ds-n51, ds-n52, ds-n53, ds-n54, ds-n55, ds-n56, ds-n57, ds-n58, ds-n59, ds-n60, ds-n61, ds-n62, ds-n63, ds-n64, ds-n65, ds-n66, ds-n67, ds-n68, ds-n69, ds-n70, ds-n71, ds-n72, ds-n73, ds-n74, ds-n75, ds-n76, ds-n77, ds-n78, ds-n79, ds-n80, ds-n81, ds-n82, ds-n83, ds-n84, ds-n85, ds-n86, ds-n87, ds-n88, ds-n89, ds-n90, ds-n91, ds-n92, ds-n93, ds-n94, ds-n95, ds-n96, ds-n97, ds-n98, ds-n99, ds-n100, ds-n101, ds-n102, ds-n103, ds-n104, ds-n105, ds-n106, ds-n107, ds-n108, ds-n109, ds-n110, ds-n111, ds-n112, ds-n113, ds-n114, ds-n115, ds-n116, ds-n117, ds-n118, ds-n119, ds-n120, ds-n121, ds-n122, ds-n123, ds-n124, ds-n125, ds-n126, ds-n127, ds-n128, ds-n129, ds-n130, ds-n131, ds-n132, ds-n133, ds-n134, ds-n135, ds-n136, ds-n137,ds-n138, ds-n139, ds-n140, ds-n141, ds-n142, ds-n143, ds-n144, ds-n145, ds-n146, ds-n147, ds-n148, ds-n 149, ds-n150, ds-n151, ds-n152, ds-n153, ds-n154, ds-n155, ds-n156, ds-n157, ds-n158, ds-n159, ds-n160, d The structure shown in any one of s-n161, ds-n162, ds-n163, ds-n164, ds-n165, ds-n166, ds-n167, ds-n168, ds-n169, ds-n170, ds-n171, ds-n172, ds-n173, ds-n174, ds-n175, ds-n176, ds-n177, ds-n178, ds-n179, ds-n180, ds-n181, or ds-n182; Preferably, the reverse complementary double-chain region includes any one of the structures shown in ds-n22, ds-n23, ds-n26, ds-n29, ds-n43, ds-n44, ds-n58, ds-n59, ds-n66, ds-n81, ds-n89, ds-n106, ds-n107, ds-n108, ds-n111, ds-n112, ds-n116, ds-n156, ds-n157, ds-n164, ds-n165, or ds-n170.

8. The RNA agent according to any one of claims 2-7, wherein, The sense strand comprises a first strand, and the antisense strand comprises a second strand, as well as 3' and 5' extensions located on the flanks of the second strand. The first and second strands are anticomplementary to form the anticomplementary double-stranded region. The 5' nucleotide of the second strand and the 3' nucleotide of the first strand are complementary. The 3' extension is connected to the 3' end of the second strand and has a length of 0-5 nucleotides, such as 0, 1, 2, 3, 4, or 5 nucleotides. The 5' extension is connected to the 5' end of the second strand and has a length of at least 3 nucleotides, such as 3, 4, 5, or 6 nucleotides. The antisense strand can be cleaved between the 3' nucleotide of the 5' extension and the 5' nucleotide of the second strand. The resulting cleavage product can silence gene expression through RNA interference.

9. The RNA agent according to claim 8, wherein, Starting from the 3' end of the 5' extension, the 2nd nucleotide of the 5' extension is selected from natural analogs of G, A, G, non-natural analogs of G, natural analogs of A, and non-natural analogs of A; and / or, the 5' end nucleotide of the second strand is selected from natural analogs of A, U, U, U, non-natural analogs of U, natural analogs of A, and non-natural analogs of A; and / or, the 3' end nucleotide of the first strand is or is replaced by a natural analog of U, U, or a non-natural analog of U; Preferably, starting from the 3' end of the 5' extension, the 2 nucleotides of the 5' extension are selected from G, natural analogs of G, and non-natural analogs of G; More preferably, the antisense strand or antisense nucleic acid fragment comprises a continuous nucleotide sequence differing by no more than 3, 2, or 1 nucleotides from any of the sequences shown in SEQ ID NO:982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, or 1003, or comprises a sequence differing by no more than 3, 2, or 1 nucleotides from any of the sequences shown in SEQ ID NO:982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, or 1003; optionally, the sense strand or sense nucleic acid fragment correspondingly comprises a sequence as shown in SEQ ID NO:982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, or 1003. SEQ ID NO:746, 748, 759, 770, 771, 776, 805, 156, 842, 843, 846, 837, 809, 112, 111, 806, 804, 795, 789, 760, 750 or 745 are consecutive nucleotide sequences differing by no more than 3, 2 or 1 nucleotides, or the positive strand or positive nucleic acid fragment accordingly contains the sequence shown in SEQ ID NO:746, 748, 759, 770, 771, 776, 805, 156, 842, 843, 846, 837, 809, 112, 111, 806, 804, 795, 789, 760, 750 or 745; For example, the RNA agent comprises any of the structures shown as ds-m183, ds-m184, ds-m185, ds-m186, ds-m187, ds-m188, ds-m189, ds-m190, ds-m191, ds-m192, ds-m193, ds-m194, ds-m195, ds-m196, ds-m197, ds-m198, ds-m199, ds-m200, ds-m201, ds-m202, ds-m203, or ds-m204.

10. The RNA agent according to claim 8 or 9, wherein, The 5' extension has a blocking group attached to the 5' nucleotide; preferably, the blocking group is M06 or an inverted abasic nucleotide.

11. The RNA agent according to any one of claims 1-10, wherein, In the RNA agent, at least one nucleotide is a chemically modified nucleotide; Preferably, the chemical modification is at least one of the following modifications: (1) Modification of the phosphodiester bonds linking nucleotides in the nucleotide sequence of the RNA agent that inhibits INHBC gene expression; (2) Modification of the ribose in the nucleotide sequence of the RNA agent that inhibits INHBC gene expression, such as one or more of 2'-F modification and 2'-OMe modification; (3) Modification of the bases in the nucleotide sequence of the RNA agent that inhibits INHBC gene expression, for example, by linking an inverted abase-free nucleotide.

12. The RNA agent according to claim 11, wherein, The modifications are each independently selected from one, two, or three combinations of the following: 2'-OMe modification, 2'-F modification, 2'-deoxygenation modification, VP modification, 5'-MP modification, PS modification, PS2 modification, MP modification, MOP modification, invAb modification, and invAB modification; Preferably, starting from the 3' end of the 5' extension, positions 1 and 2 of the 5' extension are nucleotides containing 2'-F modification; And / or, the blocking group is connected to the 5' end nucleotide residue of the 5' extension via a thiophosphate bond, and the nucleotide residues at positions 1 and 2 of the second chain are connected via a thiophosphate bond, starting from the 5' end of the second chain. And / or, starting from the 3' end of the antisense strand, the nucleotide residues at positions 1 and 2 of the antisense strand are linked by a phosphate thioester bond, and the nucleotide residues at positions 2 and 3 of the antisense strand are linked by a phosphate thioester bond. And / or, starting from the 3' end of the positive strand, the nucleotide residues at positions 1 and 2 of the positive strand are linked by a phosphate thioester bond, and the nucleotide residues at positions 2 and 3 of the positive strand are linked by a phosphate thioester bond.

13. The RNA agent according to any one of claims 1-12, wherein it is delivered to target cells via a delivery system; Preferably, the 3' end nucleotide of the positive strand is linked to the delivery system; or, the residue of the 5' end nucleotide of the positive strand is linked to an inverted abasic nucleotide residue via a phosphate thioester bond, the inverted abasic nucleotide being linked to the delivery system, and the 1st and 2nd nucleotide residues of the positive strand are linked via a phosphate thioester bond, starting from the 3' end; and / or, the delivery system is a GalNAc (N-acetylgalactosamine) derivative linked to the RNA agent; More preferably, the GalNAc derivative is selected from GalNAc-L96, G-GalNAc C3 phosphorous amide, and GalNAc-NAG; the GalNAc-L96 is preferably selected from L96, GalNAc L96-PS, and GalNAc. L96-CPG, wherein the GalNAc-NAG is preferably selected from GalNAc-NAG13, GalNAc-NAG18, GalNAc-NAG24, GalNAc-NAG25, GalNAc-NAG26, GalNAc-NAG27, GalNAc-NAG28, GalNAc-NAG29, GalNAc-NAG30, GalNAc-NAG31, GalNAc-NAG32, GalNAc-NAG33, GalNAc-NAG34, GalNAc-NAG35, GalNAc-NAG36, GalNAc-NAG37, GalNAc-NAG38 and GalNAc-NAG39; and / or, the RNA agent is linked to a divalent or trivalent GalNAc derivative; More preferably, the delivery system is L96 or NAG37; preferably, when the delivery system is L96, the residues of the 3' end nucleotide of the positive strand are connected to the L96 residues via phosphate ester bonds; when the delivery system is NAG37, the inverted non-basic nucleotide residues are connected to the NAG37 residues via thiophosphate ester bonds. For example, the RNA agent includes any one of the structures shown in Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, Z16, Z17, Z18, Z19, Z20, Z21 or Z22.

14. The RNA agent according to any one of claims 1-13, which comprises, such as ds-m1, ds-m2, ds-m3, ds-m4, ds-m5, ds-m6, ds-m7, ds-m8, ds-m9, ds-m10, ds-m11, ds-m12, ds-m13, ds-m14, ds-m15, ds-m16, ds-m17, ds-m18, ds-m19, ds-m20, ds-m21, ds-m22, ds-m23, ds-m24, ds-m25, ds-m26, ds-m27, ds-m28, ds-m29, ds-m30, ds-m31, ds-m32, ds-m33, ds-m34, ds-m35, ds-m36, ds-m37, ds-m38, ds-m39, ds-m40, ds-m41, ds-m42, ds-m43, ds-m44, ds-m45, ds-m46, ds-m47, ds-m48, ds-m49, ds-m50, ds-m51, ds-m52, ds-m53, ds-m54, ds-m55, ds-m56, ds-m57, ds-m58, ds-m59, ds-m60, ds-m61, ds-m62, ds-m63, ds-m64, ds-m65, ds-m66, ds-m67, ds-m68, ds-m69, ds-m70, ds-m71, ds-m72, ds-m73, ds-m74, ds-m75, ds-m76, ds-m77, ds-m78, ds-m79, ds-m80, ds-m81, ds-m82, ds-m83, ds-m84, ds-m85, ds-m86, ds-m87, ds-m88, ds-m89, ds-m90, ds-m91, ds-m92, ds-m93, ds-m94, ds-m95, ds-m96, ds-m97, ds-m98, ds-m99, ds-m100, ds-m101, ds-m102, ds-m103, ds-m104, ds-m105, ds-m106, ds-m107, ds-m108, ds-m109, ds-m110, ds-m111, ds-m112, ds-m113, ds-m114, ds-m115, ds-m116, ds-m117, ds-m118, ds-m119, ds-m120, ds-m121, ds-m122, ds-m123, ds-m124, ds-m125, ds-m126, ds-m127, ds-m128, ds-m129, ds-m130, ds-m131, ds-m132, ds-m133, ds-m134, ds-m135,ds-m136, ds-m137, ds-m138, ds-m139, ds-m140, ds-m141, ds-m142, ds-m143, ds-m14 4. ds-m145, ds-m146, ds-m147, ds-m148, ds-m149, ds-m150, ds-m151, ds-m152, ds-m 153, ds-m154, ds-m155, ds-m156, ds-m157, ds-m158, ds-m159, ds-m160, ds-m161, ds -m162, ds-m163, ds-m164, ds-m165, ds-m166, ds-m167, ds-m168, ds-m169, ds-m170, d s-m171, ds-m172, ds-m173, ds-m174, ds-m175, ds-m176, ds-m177, ds-m178, ds-m179 , ds-m180, ds-m181, ds-m182, ds-m183, ds-m184, ds-m185, ds-m186, ds-m187, ds-m1 Any one of the following structures: 88, ds-m189, ds-m190, ds-m191, ds-m192, ds-m193, ds-m194, ds-m195, ds-m196, ds-m197, ds-m198, ds-m199, ds-m200, ds-m201, ds-m202, ds-m203, or ds-m204; Preferably, it includes, for example, ds-m22, ds-m23, ds-m26, ds-m29, ds-m43, ds-m44, ds-m58, ds-m59, ds-m66, ds-m81, ds-m89, ds-m106, ds-m107, ds-m108, ds-m111, ds-m112, ds-m116, ds-m156, ds-m157, ds-m164, ds-m165, ds-m170, and ds-m18.

3. Any one of the following structures shown: ds-m184, ds-m185, ds-m186, ds-m187, ds-m188, ds-m189, ds-m190, ds-m191, ds-m192, ds-m193, ds-m194, ds-m195, ds-m196, ds-m197, ds-m198, ds-m199, ds-m200, ds-m201, ds-m202, ds-m203, or ds-m204.

15. A pharmaceutical composition comprising an RNA agent according to any one of claims 1-14, and / or a physiologically acceptable excipient and / or carrier and / or diluent; Preferably, the pharmaceutically acceptable carrier includes or is selected from aqueous carriers, liposomes, polymers, or peptides.

16. Use of the RNA agent according to any one of claims 1-14 or the pharmaceutical composition according to claim 15 in the preparation of a medicament, wherein the medicament is used to prevent or treat diseases or pathologies associated with INHBC gene expression or to reduce the risk of diseases or symptoms associated with INHBC gene expression; Preferably, the disease or pathology is selected from obesity (e.g., harmful obesity), coronary heart disease, abnormal blood pressure, and metabolic diseases; more preferably, the metabolic diseases include diabetes or hyperlipidemia.

17. The RNA agent according to any one of claims 1-14 or the pharmaceutical composition according to claim 15, wherein the RNA or pharmaceutical composition is used to prevent or treat diseases or pathologies associated with INHBC gene expression or to reduce the risk of diseases or symptoms associated with INHBC gene expression; Preferably, the disease or pathology is selected from obesity (e.g., harmful obesity), coronary heart disease, abnormal blood pressure, and metabolic diseases; more preferably, the metabolic diseases include diabetes or hyperlipidemia.

18. A method for preventing or treating diseases or pathologies associated with INHBC gene expression, or for reducing the risk of diseases or symptoms associated with INHBC gene expression, wherein, The method includes administering an effective amount of the RNA agent according to any one of claims 1-14 or the pharmaceutical composition according to claim 15 to a subject in need; Preferably, the disease or pathology is selected from obesity (e.g., harmful obesity), coronary heart disease, abnormal blood pressure, and metabolic diseases; more preferably, the metabolic diseases include diabetes or hyperlipidemia.

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