Double-stranded siRNA analogue for inhibiting expression of INHBE as well as preparation method and application of double-stranded siRNA analogue

Abstract

The present invention relates to a double-stranded siRNA analogue for inhibiting expression of statin subunit betaE (INHBE) and a preparation method and use thereof, the double-stranded siRNA analogue can inhibit expression of INHBE, and is used for preventing and / or treating INHBE-related diseases and disorders, the diseases and disorders comprise metabolic diseases and / or disorders, cardiovascular diseases and / or disorders, and cardiovascular diseases and / or disorders. Such as type 2 diabetes mellitus, obesity, hypertension, coronary heart disease and the like.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and more specifically, to a double-stranded siRNA analog acting on inhibin subunit βE, and a preparation method and use thereof. Background Art

[0002] Inhibin subunit βE (INHBE) is a member of the TGF-β (transforming growth factor β) protein superfamily. It is mainly expressed in the liver and is involved in regulating the growth and differentiation of hepatocytes. According to studies, inhibin subunit βE is a liver-expressed negative regulator of fat storage, and its blockade may be beneficial for metabolic diseases related to fat distribution (Akbari P, et al. Multiancestry exome sequencing reveals INHBE mutations associated with favorable fat distribution and protection from diabetes. Nat Commun. 2022; 13(1): 4844). Analysis of WES data of more than 360,000 individuals of European ancestry showed that INHBE LOF mutations (loss of function mutations) help reduce the prevalence of abdominal obesity, metabolic syndrome, coronary heart disease, and T2D (Deaton AM, et al. Rare loss of function variants in the hepatokine gene INHBE protect from abdominal obesity. Nat Commun. 2022; 13(1): 4319). In humans and mice, increased INHBE expression is associated with insulin resistance and obesity, and selective silencing of the INHBE gene by siRNA has been shown to inhibit weight and fat mass gain in db / db mice (Sugiyama M, et al. InhibinβE (INHBE) is a possible insulin resistance-associated hepatokine identified by comprehensive gene expression analysis in human liver biopsy samples. PLoS One. 2018; 13(3): e0194798). Furthermore, an association was found between INHBE and NAFLD (Cao J, et al. Identification and validation of INHBE and P4HA1 as hub genes in non-alcoholic fatty liver disease. BiochemBiophys Res Commun. 2023; 686: 149180).

[0003] Existing research suggests that inhibiting INHBE may help improve metabolic and cardiovascular diseases. Furthermore, INHBE is expressed in the liver, which further facilitates the development of siRNA therapeutics using the GalNAc delivery platform, potentially complementing existing treatments for related diseases. Currently, there are no clinically available drugs targeting INHBE. Therefore, providing novel siRNA structures to inhibit INHBE expression and / or activity for the treatment of INHBE-related conditions remains an urgent challenge in this field. Summary of the Invention

[0004] The present invention provides a double-stranded siRNA analog for inhibiting INHBE expression, a pharmaceutical composition thereof, and uses thereof. The double-stranded siRNA analog can specifically inhibit the expression of the INHBE gene and has good inhibitory activity on INHBE mRNA.

[0005] In a first aspect, the present invention provides a double-stranded siRNA analog for inhibiting INHBE expression, wherein the double-stranded siRNA analog comprises a sense strand and an antisense strand, wherein the length of the sense strand and the antisense strand are each independently 15-30 nucleotides, the antisense strand comprises a complementary region that complementarily pairs with the sense strand, and the antisense strand comprises at least 15 consecutive nucleotides of a sequence that differs by no more than 3 nucleotides from the sequence shown in any one of SEQ ID NOs: 1068-2134, 2136.

[0006] As a preferred technical solution of the present invention, the length of the sense strand and the antisense strand is each independently 17-27 nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides, and most preferably 19-21 nucleotides. In a specific embodiment, the length of the sense strand and the antisense strand is each independently 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. Wherein, the length of the nucleotides of the sense strand and the antisense strand may be the same or different, for example, the sense strand comprises 19 nucleotides and the antisense strand comprises 21 nucleotides, or the sense strand comprises 19 nucleotides and the antisense strand also comprises 19 nucleotides, or alternatively, the sense strand comprises 21 nucleotides and the antisense strand comprises 19 nucleotides.

[0007] As a preferred embodiment of the present invention, the sense strand and the antisense strand may be partially, substantially, or completely complementary to each other. For example, the sense strand and the antisense strand may be 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary. The region of complementarity is at least 15 nucleotide pairs in length. In a specific embodiment, the region of complementarity is 15-25 nucleotide pairs in length. In a specific embodiment, the region of complementarity is 17-23 nucleotide pairs in length. In a specific embodiment, the region of complementarity is 19 nucleotide pairs in length. Generally, if any nucleotide overhangs as defined herein are present, the sequences of these overhangs are not considered when determining the degree of complementarity between two sequences. For example, a sense strand of 21 nucleotides in length and an antisense strand of 21 nucleotides in length that hybridize to form a 19-nucleotide pair region of complementarity with a 2 nucleotide overhang at the 3' end of each strand would be considered 100% complementary.

[0008] As a preferred technical solution of the present invention, the sense strand and the antisense strand each independently comprise a 3' overhang and / or a 5' overhang having at least one nucleotide, for example, one or both of the sense strand and the antisense strand comprise a 3' overhang and / or a 5' overhang having at least two nucleotides. In a specific embodiment, the nucleotides of the overhang are selected from thymidine deoxyribonucleotides or uracil ribonucleotides, for example, the nucleotides of the overhang are selected from 2 consecutive thymidine deoxyribonucleotides or 2 consecutive uracil ribonucleotides. In certain embodiments, when the overhang is present in the antisense strand, the nucleotides in the overhang can be complementary to the target gene sequence, forming a mismatch with the target gene sequence or comprising some other sequences (for example, polypyrimidine or polypurine sequences, UU, TT, AA, GG, etc.).

[0009] As a preferred technical solution of the present invention, the antisense strand comprises a sequence that differs from any one of SEQ ID NOs: 1068-2134 and 2136 by no more than 3 nucleotides, for example, the antisense strand comprises a sequence that differs from any one of SEQ ID NOs: 1068-2134 and 2136 by no more than 2 nucleotides, or the antisense strand comprises a sequence that differs from any one of SEQ ID NOs: 1068-2134 and 2136 by no more than 1 nucleotide. In a specific embodiment, the antisense strand comprises a sequence shown in any one of SEQ ID NOs: 1068-2134 and 2136. In a specific embodiment, the antisense strand consists of a sequence shown in any one of SEQ ID NOs: 1068-2134 and 2136.

[0010] As a preferred technical solution of the present invention, the antisense strand comprises SEQ ID NO: 1076, 1089-1090, 1092, 1094-1095, 1097-1101, 1103-1105, 1117, 1119-1120, 1126, 1128, 1130, 1132, 1230-1231, 1234-1235, 1238, 1241, 1243-1247, 1249, 1251, 1313-1314, 1316-1317 7. 1319-1321, 1323, 1325, 1331-1332, 1334-1335, 1388, 1390, 1393-1394, 1515, 1522, 1528, 1595, 1629, 1634, 1636, 1662-1663, 1688-1689, 1694-1695, 1703, 1705-1706, 1709, 1712, 1720- 1721, 1779, 1782, 1784, 1786, 1789-1790, 1792, 1810, 1830-1832, 1835, 1840-1841, 1845, 1847, 1849, 1853, 1855, 1860-1861, 1891, 1894, 1898, 1954, 1996-2003, 2008, 2012-2015, 2020, 022-2023, 2025, 2028-2035, 2037, 2039, 2042-2050, 2052, 2054-2055, 2057, 2060-2061, 2064, 2075, 2107, 2111, 2129, or comprises a sequence that differs from any of the above sequences by no more than 3, 2 or 1 nucleotides, or consists of any of the above sequences.

[0011] As a preferred technical solution of the present invention, the antisense strand comprises SEQ ID NO: 1076, 1089-1090, 1092, 1094-1095, 1097-1101, 1103-1105, 1117, 1119-1120, 1126, 1128, 1130, 1132, 1230-1231, 1234-1235, 1238, 1241, 1243-1247, 1249, 1251, 1313-1314 、1316-1317、1319-1321、1323、1325、1331-1332、1334-1335、1388、1390、1393-1394、1515、1522、1528、1595、1629、1634、1636、1662-1663、1688-1689、1694-1695、1703、1705-17 06, 1709, 1712, 1720-1721, 1779, 1782, 1784, 1786, 1789, 1790, 1792, 1810, 1830-1832, 1835, 1840, 1841, 1845, 1847, 1849, 1853, 1855, 1860, 1861, 1891, 1894, 1898, 1954, 1996- The sequence shown in any one of 2003, 2008, 2012-2015, 2129, 2020, 2022, 2023, 2025, 2028-2035, 2037, 2039, 2042-2050, 2052, 2054, 2055, 2057, 2060, 2061, 2064, 2075, 2107, and 2111, or consisting of the sequence shown in any one of the above.

[0012] As a preferred technical solution of the present invention, the antisense strand comprises an antisense strand sequence of any one of the siRNAs listed in Table 5.

[0013] As a preferred technical solution of the present invention, the antisense strand comprises SEQ ID The invention relates to a sequence of any one of NO:1230, 1234, 1241, 1244, 1314, 1317, 1319, 1323, 1332, 1335, 1390, 1393-1394, 1522, 1528, 1595, 1634, 1636, 1695, 1706, 1712, 1789, 1790, 1792, 1830, 1841, 1847, 1855, 1996, 1998, 2012, 2015, 2020, 2028, 2032, 2034, 2037, 2107, 2111, 2001, 2002, 2030, 2046, 2129, or a sequence consisting of any one of the above sequences.

[0014] As a preferred technical solution of the present invention, the antisense strand comprises a sequence shown in any one of SEQ ID NO: 1230, 1241, 1244, 1317, 1319, 1323, 1393, 1695, 1712, 1789, 1790, 1792, 1841, 1847, 1860, 1996, 1998, 2001, 2002, 2012, 2015, 2020, 2028, 2029, 2030, 2032, 2034, 2037, 2107, 2111, and 2129, or consists of any one of the sequences shown above.

[0015] As a preferred technical solution of the present invention, the sense strand comprises a sequence shown in any one of SEQ ID NOs: 1-1067 and 2135. In a specific embodiment, the sense strand consists of a sequence shown in any one of SEQ ID NOs: 1-1067 and 2135.

[0016] As a preferred technical solution of the present invention, the positive chain comprises SEQ ID NO: 9, 22-23, 25, 27-28, 30-34, 36-38, 50, 52-53, 59, 61, 63, 65, 163-164, 167-168, 171, 174, 176, 177-180, 182, 184, 246-247, 249-250, 252-254, 256, 258, 264-265, 267-268, 321, 323, 326, 327, 448, 455, 461, 528, 562, 567, 569, 595-596, 621-622, 627-628, 636, 638-639, 642, 645, 653, 654, 712, 715, 71 7, 719, 722, 723, 725, 743, 763-765, 768, 773-774, 778, 780, 782, 786, 788, 793-794, 824, 827, 831, 887, 929-936, 941, 945-948, 1062, 953, 955, 956, 958, 961-968, 970, 972, 975-983, 985, 987-988, 990, 993-994, 997, 1008, 1040, 1044, or a sequence that differs from any of the above sequences by no more than 3, 2 or 1 nucleotides, or consists of any of the above sequences.

[0017] As a preferred technical solution of the present invention, the sense strand comprises a sense strand sequence of any one of the siRNAs selected from Table 5.

[0018] As a preferred technical solution of the present invention, the positive strand comprises a sequence shown in any one of SEQ ID NO: 163, 167, 174, 177, 247, 250, 252, 256, 265, 268, 323, 326, 327, 455, 461, 528, 567, 569, 628, 639, 645, 722, 723, 725, 763, 774, 780, 788, 929, 931, 945, 948, 953, 961, 965, 967, 970, 1044, 1040, 934, 935, 963, 979, 1062, or consists of any one of the sequences shown above.

[0019] As a preferred technical solution of the present invention, the positive chain comprises a sequence shown in any one of SEQ ID NO: 163, 174, 177, 250, 252, 256, 326, 628, 645, 722, 723, 725, 774, 780, 793, 929, 931, 934, 935, 945, 948, 953, 961, 962, 963, 965, 967, 970, 1040, 1044, 1062, or is composed of any one of the above sequences.

[0020] As a preferred technical solution of the present invention, the double-stranded siRNA analog comprises any pair of paired sense strand sequence and antisense strand sequence as shown in Table 1, or consists of them.

[0021] As a preferred technical solution of the present invention, the double-stranded siRNA analog is selected from any one of the siRNAs in Table 5.

[0022] As a preferred technical solution of the present invention, the double-stranded siRNA analog is selected from any one of the following siRNAs: siHB363, siHB367, siHB404, siHB407, siHB500, siHB503, siHB505, siHB509, siHB518, siHB521, siHB642, siHB645, siHB646, siHB884, siHB890, siHB1043, siHB1105, siHB1107, siHB1211, siHB1222, siHB 1228, siHB1309, siHB1310, siHB1312, siHB1394, siHB1405, siHB1432, siHB1440, siHB1857, siHB1859, siHB2162, siHB2165, si HB2298, siHB2347, siHB2405, siHB2407, siHB2411, siHB1354, siHB1446, siHB1862, siHB1863, siHB2349, siHB2420, siHB2188.

[0023] As a preferred technical solution of the present invention, the double-stranded siRNA analog is selected from any one of the following siRNAs: siHB404, siHB407, siHB503, siHB505, siHB509, siHB645, siHB1211, siHB1228, siHB363, siHB1309, siHB1310, siHB1312, siHB1405, siHB1432, siHB1445, siHB1857, siHB1859, siHB186 2. siHB1863, siHB2162, siHB2165, siHB2298, siHB2347, siHB2348, siHB2349, siHB2405, siHB2407, siHB2411, siHB1 354, siHB1446, siHB2188, siHB30, siHB34, siHB36, siHB38, siHB39, siHB52, siHB55, siHB2417, siHB2424, siHB2435.

[0024] As a preferred technical solution of the present invention, the double-stranded siRNA analog includes at least one modified nucleotide. In a specific embodiment, the sense strand and / or the antisense strand each independently include at least one modified nucleotide. In a specific embodiment, the sense strand includes at least one modified nucleotide and the nucleotides of the antisense strand are not modified, or the nucleotides of the sense strand are not modified and the antisense strand includes at least one modified nucleotide. In some preferred embodiments, the nucleotides of the sense strand and the antisense strand are both modified nucleotides.

[0025] In some embodiments, the modified nucleotides are selected from: alkyl modified nucleotides, methoxy modified nucleotides (e.g., 2'-O-methyl modified nucleotides), ethoxy modified nucleotides (e.g., 2'-O-ethyl modified nucleotides), 2'-O-allyl modified nucleotides, 2'-hydroxy modified nucleotides, methoxyethyl modified nucleotides, amino modified nucleotides, fluoro modified nucleotides (e.g., 2'-fluoro modified nucleotides), deoxy nucleotides, 5'-methyl phosphate nucleotides, 5'-C-methyl phosphate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinylphosphonate deoxyribonucleotides, nucleotides containing thiophosphate groups, nucleotides containing dithiophosphate groups, locked nucleic acids (LNA), morpholino oligonucleotides (PMO). In some embodiments, the alkyl modified nucleotides are selected from methyl modified nucleotides and ethyl modified nucleotides.

[0026] As a preferred technical solution of the present invention, the sense strand and / or the antisense strand independently include at least one 2'-O-methyl modified nucleotide, at least one 2'-fluoro modified nucleotide and at least one nucleotide containing a phosphorothioate group.

[0027] As a preferred technical solution of the present invention, the number of the 2'-O-methyl modified nucleotides is no more than 17, preferably 17 or 15. The number of the 2'-fluoro modified nucleotides is no more than 4, preferably 4. The number of the nucleotides containing phosphorothioate groups is no more than 4, preferably 4 or 2.

[0028] As a preferred technical solution of the present invention, the sense strand and / or the antisense strand each independently comprises at least one 2'-O-methyl modified nucleotide. In a specific embodiment, from the 5' end to the 3' end, the nucleotides at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18 and / or 19 of the sense strand are each independently a 2'-O-methyl modified nucleotide. In a specific embodiment, from the 5' end to the 3' end, the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20 and / or 21 of the antisense strand are each independently a 2'-O-methyl modified nucleotide.

[0029] As a preferred technical solution of the present invention, the sense strand and / or the antisense strand each independently comprises at least one 2'-fluoro-modified nucleotide. In a specific embodiment, from the 5' end to the 3' end, the 5th, 7th, 8th and / or 9th nucleotides of the sense strand are each independently a 2'-fluoro-modified nucleotide. In a specific embodiment, from the 5' end to the 3' end, the 2nd, 6th, 14th and / or 16th nucleotides of the antisense strand are each independently a 2'-fluoro-modified nucleotide.

[0030] As a preferred technical solution of the present invention, the sense strand and / or the antisense strand each independently includes at least one nucleotide containing a phosphorothioate group. In a specific embodiment, the phosphorothioate group is present in at least one position selected from the following: in the direction from the 5' end to the 3' end, between the first and second nucleotides of the sense strand, between the second and third nucleotides of the sense strand, between the first and second nucleotides of the antisense strand, and between the second and third nucleotides of the antisense strand; in the direction from the 3' end to the 5' end, between the first and second nucleotides of the antisense strand, and between the second and third nucleotides of the antisense strand.

[0031] As a preferred technical solution of the present invention, the sense strand and the antisense strand of the double-stranded siRNA analog have a modified form shown in the following formula:

[0032] Justice chain: NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm

[0033] Antisense strand: NmsNfsNmNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm

[0034] Wherein: "N" is a nucleotide; m indicates that the nucleotide adjacent to its left is a 2'-O-methyl modified nucleotide; f indicates that the nucleotide adjacent to its left is a 2'-fluoro modified nucleotide; s indicates that the two nucleotides adjacent to the letter s are connected by thiophosphate groups.

[0035] As a preferred technical solution of the present invention, the antisense strand comprises any modified antisense strand nucleotide sequence shown in Table 2, and / or the sense strand comprises any modified sense strand nucleotide sequence shown in Table 2. In some preferred embodiments, the double-stranded siRNA analog comprises or consists of a paired modified sense strand nucleotide sequence and a modified antisense strand nucleotide sequence shown in Table 2.

[0036] As a preferred technical solution of the present invention, the double-stranded siRNA analog is selected from any one of the siRNAs in Table 3.1, Table 3.2, Table 4.1, and Table 4.2. As a preferred technical solution of the present invention, the double-stranded siRNA analog is linked to a targeting ligand.

[0037] Wherein, the targeting ligand is selected from:

[0038]

[0039] In some specific embodiments, the targeting group can be connected to the 3' end of the sense strand or antisense strand of a double-stranded siRNA analog. In some specific embodiments, the targeting group can be connected to the 5' end of the sense strand or antisense strand of a double-stranded siRNA analog. In some specific embodiments, the targeting group is connected to the 5' end of the sense strand. In some specific embodiments, the targeting group is connected to the 3' end of the sense strand. In some specific embodiments, the targeting group can also be internally connected to nucleotides on the sense strand and / or antisense strand of a double-stranded siRNA analog. In some specific embodiments, the targeting group can also be connected to the double-stranded siRNA analog through a joint, for example, the targeting group can also be connected to the 3' or 5' end of the sense strand through a joint, or, the targeting group can also be connected to the 3' or 5' end of the antisense strand through a joint, or, the targeting group can also be internally connected to nucleotides on the sense strand and / or antisense strand of a double-stranded siRNA analog through a joint.

[0040] In a second aspect, the present invention provides a vector comprising a nucleotide sequence encoding the double-stranded siRNA analog as described above.

[0041] The vector is capable of amplifying or expressing a nucleotide encoding the double-stranded siRNA analog of the present invention linked thereto. The vector can be a viral vector or plasmid capable of transporting nucleic acid molecules, such as: (a) adenoviral vector; (b) retroviral vector; (c) adeno-associated viral vector; (d) herpes simplex virus vector; (e) SV40 vector; (f) polyoma virus vector; (g) papilloma virus vector; (h) picornavirus vector; (i) poxvirus vector; and (j) helper virus-dependent adenovirus or enterovirus.

[0042] In a third aspect, the present invention provides a cell comprising the double-stranded siRNA analog or the vector described above, wherein the double-stranded siRNA analog or the vector of the present invention can be transcribed in the cell.

[0043] In a fourth aspect, the present invention provides a pharmaceutical composition for inhibiting INHBE expression, wherein the pharmaceutical composition comprises the double-stranded siRNA analog as described above, or the vector as described above, or the cell as described above.

[0044] As a preferred technical solution of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0045] In a fifth aspect, the present invention also provides a use of the double-stranded siRNA analog, vector, cell or pharmaceutical composition as described above in the preparation of a medicament for preventing and / or treating diseases, disorders or symptoms mediated by abnormal or overexpression of INHBE.

[0046] As a preferred technical solution of the present invention, the disease, condition or symptom is selected from the group consisting of metabolic and / or cardiovascular diseases, conditions or symptoms. In some specific embodiments, the disease, condition or symptom is selected from the group consisting of metabolic syndrome, type 2 diabetes, hyperlipidemia, hypercholesterolemia, obesity, liver inflammation, fatty liver disease, NASH, hypertension, and coronary heart disease.

[0047] In a sixth aspect, the present invention further provides use of the double-stranded siRNA analog, vector, or pharmaceutical composition described above in the preparation of a medicament for reducing INHBE expression in a cell or subject. Reducing INHBE expression includes reducing the amount of its mRNA, protein, or both. The subject benefits from reduced INHBE expression.

[0048] The double-stranded siRNA analogue for inhibiting INHBE expression provided by the present invention has good inhibitory activity on INHBE and can be used to prevent and / or treat related diseases mediated by INHBE expression. DETAILED DESCRIPTION

[0049] The present invention will be further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0050] The term "comprising" herein is used to mean, and is used interchangeably with, the phrase "including but not limited to," unless the context clearly indicates otherwise.

[0051] The term "or" of the present invention is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise.

[0052] The terms "sequence" and "nucleotide sequence" of the present invention mean the order or sequence of nucleobases or nucleotides, described in alphabetical order using standard nomenclature.

[0053] The term "siRNA analogue" of the present invention refers to a complex of ribonucleic acid molecules having a double-stranded structure that can mediate the silencing of a target RNA (e.g., mRNA) that is complementary thereto. The siRNA analogue comprises two reverse-parallel and substantially complementary nucleic acid chains, the nucleic acid chains comprising an antisense strand that is complementary to the target RNA, and a sense strand that is complementary to the antisense strand. When the two nucleic acid chains are "substantially complementary," they can be completely complementary, or they can form one or more, but generally no more than 5, 4, 3, or 2, mismatched nucleotide pairs for a duplex of up to 30 nucleotide pairs after hybridization, while retaining the ability to hybridize under conditions most relevant to its ultimate application, such as inhibition of gene expression in vitro or in vivo.

[0054] As used herein, "complementary" has the meaning known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand pair with bases on the other strand in a complementary manner. Adenine (A) always pairs with uracil (U); guanine (C) always pairs with cytosine (G). Each base pair consists of a purine and a pyrimidine. When adenine on one strand always pairs with uracil on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary to each other, and the sequence of that strand can be inferred from the sequence of its complementary strand.

[0055] As used herein, the term "region of complementarity" refers to a region of the antisense strand that is substantially complementary to a sequence as defined herein (e.g., a target sequence). When the region of complementarity is not completely complementary to the target sequence, mismatches may occur within or at the terminal regions of the molecule. Typically, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' termini.

[0056] In the present invention, the term "overhang" refers to one or more unpaired nucleotides at the ends of a chain that extend beyond the complementary region. When the 3' end of a chain extends beyond the 5' end of another chain or when the 5' end of a chain extends beyond the 3' end of another chain, a nucleotide overhang is typically formed. Double-stranded siRNA analogs can include an overhang with at least one nucleotide, for example, the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The nucleotide overhang can include or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). One or more overhangs can be on the sense strand, the antisense strand, or any combination thereof. One or more overhangs can be on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand.

[0057] The terms "complementary," "fully complementary," and "substantially complementary" herein can be used with respect to base matching between the sense and antisense strands of a double-stranded siRNA analog, or between two oligonucleotides or polynucleotides (such as the antisense strand of a double-stranded siRNA analog and a target sequence), as understood from the context of their use.

[0058] In the present invention, modified nucleotides include but are not limited to: alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxy nucleotides, 5'-methyl phosphate nucleotides, 5'-C-methyl phosphate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinylphosphonate deoxyribonucleotides (VP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), and inverted abasic deoxyribose residues (invAb).

[0059] Among them, alkyl-modified nucleotides, such as 2'-methyl nucleotides, 2'-ethyl nucleotides, 2'-methoxy modified nucleotides, e.g. 2'-methoxyethyl nucleotides, e.g. 2'-methoxyethoxy nucleotides, e.g. 2'-fluoronucleotides, e.g. 5'-C-methylphosphononucleotides, e.g.

[0060] (E)-vinylphosphonate deoxyribonucleotides (VP), e.g., Phosphorothioate nucleotides (PS), e.g. 2'-deoxyribonucleotides, such as: Inverted abasic deoxyribose residues (invAb), for example:

[0061] Wherein, Base represents a base, R represents an alkyl group or an alkoxy group, Me represents a methyl group, and Et represents an ethyl group.

[0062] The term "locked nucleic acid" is a nucleotide with a modified ribose moiety, wherein the ribose moiety includes an additional bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose in a 3'-endo conformation. Adding locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O. R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0063] Representative U.S. patents for preparing locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125 and 7,399,845, each of which is incorporated herein by reference in its entirety.

[0064] The locked nucleic acid structure is as follows:

[0065]

[0066] In certain embodiments, the sugar surrogate comprises a ring having more than 5 atoms and more than 1 heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

[0067] The term "morpholino" means a sugar surrogate having the formula:

[0068]

[0069] In certain embodiments, the morpholino group can be modified, for example, by adding or changing various substituents according to the above morpholino structure. Such sugar surrogates are referred to herein as "modified morpholinos."

[0070] In the present invention, unless otherwise specified, capital letters C, G, U, A, and T represent the base composition of nucleotides. Lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorine-modified nucleotide; LNA indicates that the nucleotide adjacent to the right of letter f is a locked nucleic acid (LNA) modified; lowercase letter s indicates that the two nucleotides to the left and right of the letter are connected by a thiophosphate group; VP indicates that the nucleotide to the right of the letter VP is a (E)-vinyl phosphate-modified nucleotide. invAb represents an inverted abasic deoxyribonucleotide; dN represents any deoxyribonucleotide; dA represents a deoxyadenine nucleotide; dT represents a deoxythymine nucleotide; dC represents a deoxycytosine nucleotide; and dG represents a deoxyguanine nucleotide.

[0071] It should be emphasized that the "modification" of nucleotides in the present disclosure includes but is not limited to the above examples, and nucleotides can also be replaced with other nucleotides, such as (S)-glycerol nucleic acid.

[0072] The term "targeting ligand" can include naturally occurring substances, such as proteins (e.g., human serum albumin (HAS), low-density lipoprotein (LDL) or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or lipids. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, for example, a synthetic polyamino acid. Examples of polyamino acids include the following polyamino acids: polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene acid-maleic anhydride copolymer, poly-(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly-(2-ethyl acrylic acid), N-isopropylacrylamide polymer or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0073] Targeting ligands can also be cell or tissue targeting agents that bind to a specific cell type, such as renal cells, such as lectins, glycoproteins, lipids or proteins, such as antibodies. The targeting group can be thyroid stimulating hormone, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acid, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic.

[0074] Targeting ligands can also be proteins, for example, glycoproteins, or peptides, for example, molecules with specific affinity for co-ligands, or antibodies, for example, antibodies that bind to a given cell type, for example, hepatocytes. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, for example, lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose or multivalent fucose. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-KKB.

[0075] The targeting ligand can be a substance, e.g., a drug, that can increase the uptake of an iRNA agent into a cell, e.g., by disrupting the cytoskeleton of the cell (e.g., by disrupting cellular microtubules, microfilaments, and / or intermediate filaments). The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, iaplakinolide, erythrosine A, phalloidin, swinholide A, indanocine, or myoservin.

[0076] The term "pharmaceutically acceptable excipient" is a substance that is intentionally included in a drug delivery system other than the active pharmaceutical ingredient (API, therapeutic product, e.g., double-stranded siRNA analog that inhibits INHBE). Excipients do not exert or are not intended to exert a therapeutic effect at the intended dose. Excipients may serve the following functions: a) aid in the handling of the drug delivery system during preparation, b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API; c) aid in product identification; and / or d) enhance any other attribute of the overall safety, efficacy, or delivery of the API during storage or use.

[0077] Among them, excipients include (but are not limited to): absorption enhancers, anti-adherents, defoaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, expanders, fillers, flavorings, glidants, wetting agents, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, waterproofing agents, wetting agents, lubricants such as sodium lauryl sulfate and magnesium stearate, flavoring agents and fragrances.

[0078] Pharmaceutical compositions of the present disclosure include those suitable for oral, nasal, topical, buccal, sublingual, rectal and / or parenteral administration. The preparation can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to prepare a single dose form is generally the amount of the compound that produces the therapeutic effect. Generally speaking, in percent units, this amount is from about 1% to about 99% active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0079] The term "vector" refers to a nucleic acid molecule capable of amplifying or expressing another nucleic acid to which it is linked.

[0080] As used herein, inhibin subunit beta E (INHBE) refers to any INHBE molecule known to those skilled in the art, including variants of the INHBE gene, such as those provided in SNP databases. Many sequence variations within the INHBE gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, for example, www.ncbi.nlm.nih.gov / snp / ?term=INHBE, the entire contents of which are incorporated herein by reference as of the filing date of this application). INHBE is also known as inhibin beta E chain, activin beta E, inhibin beta E subunit, inhibin beta E, and MGC4638.

[0081] The mRNA sequence of human INHBE can be found, for example, in GenBank Accession No. NM_031479.5; the mRNA sequence of mouse INHBE can be found, for example, in GenBank Accession Nos. NM_008382.3 and NM_031815.2. The mRNA sequence of monkey INHBE can be found, for example, in GenBank Accession Nos. XM_005571319.3 and XM_001115958.3. Other examples of INHBE mRNA sequences can be obtained using, for example, GenBank, UniProt, and OMIM. As of the filing date of this application, the entire contents of each of the aforementioned GenBank Accession Nos. are incorporated herein by reference.

[0082] The term "treatment" is used to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventative in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0083] The term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms and includes any level of inhibition.

[0084] The phrase "inhibiting the expression of INHBE" in the present invention includes inhibiting the expression of any INHBE gene (eg, mouse, rat, monkey or human INHBE gene) as well as variants or mutants of the INHBE gene encoding the INHBE protein.

[0085] "Inhibiting the expression of INHBE" includes any level of inhibition of the INHBE gene, such as at least partial inhibition of the expression of the INHBE gene, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0086] The expression of the INHBE gene can be assessed based on the level of any variable associated with INHBE gene expression, such as INHBE mRNA level or INHBE protein level. Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-dose baseline level or a level determined from a similar subject, cell, or sample that has not been treated or that has been treated with a control (e.g., a buffer-only control or an inactive agent control).

[0087] In one embodiment, at least partial inhibition of expression of the INHBE gene can be assessed by a decrease in the amount of INHBE mRNA isolated or detected from a first cell or a group of first cells in which the INHBE gene is transcribed (which has been treated such that expression of the INHBE gene is inhibited), as compared to a second cell or a group of second cells (control cells) that are substantially identical to the first cell or cells but have not been so treated. The degree of inhibition can be expressed as follows:

[0088]

[0089] Example 1 Synthesis of Targeting Ligand L96

[0090] Step A: Hydroxyproline amine (3.00 g, 7.15 mmol) and monomethyl dodecanedioate (1.748 g, 7.15 mmol) were placed in N,N-dimethylformamide (DMF) (50 mL). The peptide coupling reagent (HBTU) (3.25 g, 8.56 mmol) and N,N-diisopropylethylamine (DIEA) (3.7 mL, 21.24 mmol) were added and the reaction was stirred overnight.

[0091] The reaction mixture was poured into an ice-water mixture and extracted with dichloromethane (DCM). The mixture was washed with bicarbonate solution, water, brine, and dried over sodium sulfate. The solvent was removed and the residue was purified by chromatography (eluting with 50% ethyl acetate / hexane, ethyl acetate, and then 5% methanol / dichloromethane) to afford the desired compound 115 as a white solid (4.30 g, 93%). MS, S:C 39 H 51 NO7, calculated as 645.37, found as 646.35 (M+H).

[0092]

[0093] Step B: Compound 101 (4.25 g, 6.58 mmol) was dissolved in a mixture of tetrahydrofuran / methanol / water (50 mL, 2:1:1). Lithium hydroxide (LiOH) (1.90 g, 45.2 mmol) was added and the mixture was stirred overnight.

[0094] After checking with thin layer chromatography silica gel plates (TLC), acetic acid was added to neutralize the reaction mixture. The solvent was removed and the residue was extracted with dichloromethane (DCM). Triethanolamine (TEA, excess) was added to the dichloromethane (DCM) solution and the solution was filtered through a small silica gel pad to obtain the desired product 102 as its triethanolamine (TEA) salt (4.15 g, 86%). MS: C 38 H 49 NO7, calculated value is 631.35; measured value is 630.34 (MH).

[0095]

[0096] Step C: Compound 102 (1.30 g, 2.06 mmol) and a peptide coupling reagent (HBTU) (0.821 g, 1.05 eq.) were added to N,N-dimethylformamide (DMF) (30 mL). To this N,N-diisopropylethylamine (DIEA) (1.07 ml, 3 eq.) was added and the reaction mixture was stirred for 3-4 minutes. A solution of the amine (3.00 g, 1.58 mmol) was added, followed by 1 eq. of DIEA. The reaction mixture was stirred at room temperature overnight.

[0097] The solvent was removed under reduced pressure and the residue was dissolved in dichloromethane (DCM) and washed with bicarbonate and water. The dichloromethane (DCM) was dried over sodium sulfate and the solvent was removed. The residue was purified by chromatography (eluting first with ethyl acetate and then with 5-20% methanol / dichloromethane) to give the product 103 as a white solid (3.35 g, 88%). MS: for C 117 H 175 N 11 O 42 , the calculated value is 2406.19; the measured value is 2429.10 (M+Na).

[0098]

[0099] Among them, ligand 104 (L96) can be linked to siRNA via a phosphate group, a phosphorothioate group, or another linking group.

[0100] For the specific synthesis route, please refer to the document with International Patent Publication No. WO2009073809.

[0101] Example 2 Synthesis of siRNA analogs

[0102] siRNA was prepared using an OligoMaker ApS192 RNA synthesizer (made in Denmark). The specific synthesis route can be found in, for example, patent document WO2023044094. The sequences of siRNA analogs are shown in Table 1.

[0103] Table 1 is a list of siRNA analog sequences

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] Example 3 Synthesis of modified sequences of siRNA analogs

[0118] The modified siRNA analogs were prepared using an OligoMaker ApS192 RNA synthesizer (made in Denmark). Specifically, the double-stranded siRNA analogs shown in Table 1 were prepared into double-stranded siRNA analogs having the modified form shown in the following formula I:

[0119] Justice chain: NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm,

[0120] Antisense strand: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm;

[0121] Where: "N" represents nucleotides, such as A, G, U and C;

[0122] m indicates that the nucleotide adjacent to its left is a 2'-O-methyl modified nucleotide, for example, Am, Um, Gm and Cm represent 2'-O-methyl modified A, U, G and C respectively;

[0123] f indicates that the nucleotide adjacent to its left is a 2'-fluoro-modified nucleotide, for example, Af, Uf, Gf and Cf represent 2'-fluoro-modified A, U, G and C, respectively;

[0124] s indicates that the two nucleotides adjacent to the letter s are connected by a phosphorothioate group.

[0125] The specific synthesis route can be found in patent document WO2023044094. Table 2 lists the sequences of exemplary double-stranded siRNAs including the modified forms shown in Formula I. Furthermore, the ligand L96 was linked to the 3' end of the sense strand of the modified siRNA shown in Table 2 to evaluate its in vivo activity.

[0126] Table 2 is a list of exemplary modified siRNA sequences

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133] Example 4 In vitro testing of siRNA analogs of INHBE in HuH7 cells

[0134] In order to determine the inhibitory efficiency at the mRNA level, INHBE siRNA was transfected into HuH7 cells, and q-PCR experiments were performed to measure the expression level of INHBE mRNA. Specifically, HuH7 cells were cultured in DMEM medium (Gibco 11965-092) containing 10% fetal bovine serum (ExCell BioFSP500), 1% glutamine (Gibco 35050061), 1% NEAA (Gibco 11140050), and 1% penicillin-streptomycin (HyClone SV30010). HuH7 cells in the logarithmic growth phase were taken and cultured at 1×10 4Cells were plated at a density of 1000 cells / well in a 96-well cell plate. siRNA was mixed with Lipofectamine™ RNAiMAX (INVITROGEN 13778150) at the same time, so that the final concentration of the RNAi agent mixture was 10nM, 1nM, and 0.1nM. The RNAi agent mixture was then transfected into the cells. The cells were cultured overnight in a 37°C 5% CO2 incubator, and two replicate wells were measured. TM RNAiMAX compound-free control. 48 hours after transfection, culture medium was removed, and total RNA was extracted (QIAGEN-74182) and reverse transcribed (Vazyme-R323-1). Target cDNA was detected using TaqMan assays, with GAPDH cDNA used in parallel as an internal control.

[0135] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCT relative quantification method. The relative expression of the target gene is expressed as 2-ΔΔCt. The specific method is to subtract the CT value of the internal reference gene (GAPDH) from the CT value of the target gene in each sample to obtain the ΔCT value. Then, the ΔCT value of the control group containing only the transfection reagent (RNAiMAX Control) is subtracted from the ΔCT value of the drug group (sample) to obtain the ΔΔCT value. Finally, the ΔΔCT value is converted to 2-ΔΔCt to obtain the relative expression level of INHBE mRNA (value of sample).

[0136] The inhibition rate of INHBE gene mediated by siRNA analogs was calculated according to the following formula.

[0137] INHBE inhibition rate %=(1-value of sample / Average value of RNAiMAX Control)*100.

[0138] Example 5 In vitro testing of siRNA analogs of INHBE in HuH7 cells

[0139] Human INHBE cDNA (GenBank accession number NM_031479.5) was cloned into the reporter-based screening plasmid psiCHECK2 (Promega-C8021) to produce Renilla luciferase / INHBE fusion mRNA. Huh7 cells were cultured in DMEM (Gibco-10313021) medium supplemented with 10% fetal bovine serum (Gibco-10099141C), 1% glutamine (Gibco-35050061), 1% non-essential amino acids (Gibco-11140050), and 1% penicillin-streptomycin (Gibco-15070063). INHBE-psiCHECK2 plasmid, siRNA analogs and Lipo2000 (Invitrogen-11668019) transfection reagent diluted in Opti-MEM (Gibico-11058021) were added to the Huh7 cell suspension and the cells were transfected at a rate of 1×10 5 Cells were plated at a density of 100 μg / mL in 96-well plates to give final concentrations of 1 nM and 0.05 nM siRNA analogs. After 24 hours of culture, relative levels of Renilla luciferase normalized to the level of constitutively expressed firefly luciferase also present on the psiCHECK2 plasmid were measured using a dual-luciferase reporter assay (Promega-E2920).

[0140] The inhibition rate of INHBE gene mediated by siRNA analogs was calculated according to the following formula:

[0141] INHBE gene inhibition rate (%) = (1-relative level of Renilla luciferase in the sample / relative level of Renilla luciferase in the control group) × 100;

[0142] The calculated INHBE inhibition rates are shown in Tables 3.1, 3.2, 4.1, and 4.2 for the modified siRNAs at various final concentrations. Table 5 summarizes unmodified siRNAs with >60% INHBE inhibition at a final concentration of 1 nM. Some of these unmodified siRNAs tested exhibited at least approximately 50% INHBE inhibition at a low concentration of 0.05 nM.

[0143] Table 3.1 mRNA inhibition rate of modified double-stranded siRNA at 1 nM

[0144]

[0145] Table 3.2 mRNA inhibition rate of modified double-stranded siRNA at 1 nM

[0146]

[0147]

[0148] Table 4.1 mRNA inhibition rate of modified double-stranded siRNA at 0.05 nM

[0149]

[0150] Table 4.2 mRNA inhibition rate of modified double-stranded siRNA at 0.05 nM

[0151] serial number Average Inhibition % serial number Average Inhibition % serial number Average Inhibition % siHBM2335 69.60 siHBM2417 73.42 siHBM2439 66.73 siHBM2339 74.65 siHBM2418 59.98 siHBM2442 70.95 siHBM2347 79.94 siHBM2419 63.69 siHBM371 56.48 siHBM2348 61.55 siHBM2420 78.08 siHBM1354 61.38 siHBM2349 78.35 siHBM2421 64.62 siHBM1446 65.43 siHBM2404 52.85 siHBM2422 70.73 siHBM2405 74.76 siHBM2423 44.72 siHBM2406 42.90 siHBM2424 76.29 siHBM2407 77.38 siHBM2428 57.51 siHBM2408 37.15 siHBM2430 30.69 siHBM2411 77.57 siHBM2432 72.61 siHBM2413 53.80 siHBM2435 83.25 siHBM2416 61.65 siHBM2438 60.56

[0152] Table 5 mRNA inhibition rate of unmodified double-stranded siRNA at 1 nM

[0153]

[0154]

[0155] Example 6 In vitro testing of siRNA analogs of INHBE in primary human hepatocytes (PHH)

[0156] q-PCR was used to examine the effect of siRNA on INHBE gene expression in PHH cells. To determine the inhibitory efficiency at the mRNA level, INHBE siRNA was transfected into PHH cells, and q-PCR was performed to measure the expression level of INHBE mRNA. Specifically, PHH cells were cultured in CP medium (BIOIVT Catalog No. BIOIVTS03316) containing 10% fetal bovine serum (ExCell Bio FSP500). PHH cells (5.4×10 4 cells / well) to a 96-well cell plate coated with collagen in advance, and siRNA was added to the plate with Lipofectamine TM RNAiMAX (INVITROGEN 13778-150) was mixed to make the final concentration of RNAi agent mixture 1nM, 0.1nM or 0.02nM, and then the RNAi agent mixture was transfected into cells. TM RNAiMAX compound-free control. 48 hours after transfection, culture medium was removed, and total RNA was extracted (QIAGEN-74182) and reverse transcribed (Vazyme-R323-1). Target cDNA was detected using TaqMan assays, with GAPDH cDNA used in parallel as an internal control.

[0157] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCT relative quantification method. The relative expression of the target gene is expressed as 2-ΔΔCt. The specific method is to subtract the CT value of the internal reference gene (GAPDH) from the CT value of the target gene in each sample to obtain the ΔCT value. Then, the ΔCT value of the control group containing only the transfection reagent (RNAiMAX Control) is subtracted from the ΔCT value of the drug group (sample) to obtain the ΔΔCT value. Finally, the ΔΔCT value is converted to 2-ΔΔCt to obtain the relative expression level of INHBE mRNA (value of sample).

[0158] The inhibition rate of INHBE gene mediated by siRNA analogs was calculated according to the following formula:

[0159] INHBE inhibition rate %=(1-value of sample / Average value of RNAiMAX Control)*100.

[0160] Table 6 mRNA inhibition rate of double-stranded siRNA in PHH cells at 1 nM

[0161]

[0162] Example 7 In vitro testing of siRNA analogs of INHBE in primary cynomolgus monkey hepatocytes (PCH)

[0163] To determine the inhibitory efficiency at the mRNA level, INHBE siRNA was transfected into PCH cells, and q-PCR experiments were performed to measure the expression level of INHBE mRNA. Specifically, PCH cells were cultured in CP medium (BIOIVT Catalog No. BIOIVT, S03316) containing 10% fetal bovine serum (ExCell Bio F, SP500). PCH cells (5.4×10 4 cells / well) to a 96-well cell plate coated with collagen in advance, and siRNA was added to the plate with Lipofectamine TM RNAiMAX (INVITROGEN 13778150) was mixed to make the final concentration of RNAi agent mixture 1nM, 0.02nM, and then the RNAi agent mixture was transfected into cells. TMRNAiMAX compound-free control. 48 hours after transfection, culture medium was removed, and total RNA was extracted (QIAGEN-74182) and reverse transcribed (Vazyme-R323-1). Target cDNA was detected using TaqMan assays, with GAPDH cDNA used in parallel as an internal control.

[0164] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCT relative quantification method. The relative expression of the target gene is expressed as 2-ΔΔCt. The specific method is to subtract the CT value of the internal reference gene (GAPDH) from the CT value of the target gene in each sample to obtain the ΔCT value. Then, the ΔCT value of the control group containing only the transfection reagent (RNAiMAX Control) is subtracted from the ΔCT value of the drug group (sample) to obtain the ΔΔCT value. Finally, the ΔΔCT value is converted to 2-ΔΔCt to obtain the relative expression level of INHBE mRNA (value of sample).

[0165] The inhibition rate of INHBE gene mediated by siRNA analogs was calculated according to the following formula:

[0166] INHBE inhibition rate % = (1-value of sample / Average value of RNAiMAX Control) * 100;

[0167] The INHBE inhibition rates of some siRNAs of the present invention were tested at final concentrations of 1 nM and 0.02 nM, and are summarized in Table 7.

[0168] Table 7 mRNA inhibition rate of double-stranded siRNA in PCH cells

[0169]

[0170] Example 8 In vivo testing of INHBE siRNA analogs in animal models

[0171] To evaluate the effect of the INHBE-targeting duplex on reducing INHBE mRNA levels in vivo, C57BL / 6 mice were administered with AAV8 adeno-associated virus (2 × 10 11 vg / mouse / 0.2 mL, iv, single dose) model was established. One week after model establishment, mice were randomly divided into groups according to body weight and given a single subcutaneous injection of 3 mg / kg INHBE siRNA. Two weeks after administration, mice were euthanized by CO2, and liver tissue was collected for quantification of INHBE mRNA levels in liver tissue by qPCR. The specific qPCR experimental method is as follows:

[0172] Liver tissue was taken for RNA extraction using MagicPure 96Total RNA Kit (Qianshijin-EC521-96-11), and then Prime, Script TM The extracted tissue RNA was reverse transcribed using RT Master Mix (Takara-RR036B) to convert RNA into cDNA. Premix Ex Taq II TM (Takara-RR820B) The obtained cDNA was subjected to qPCR experiments.

[0173] The target gene mRNA expression level for each sample was calculated using the ΔΔCT relative quantification method. The relative expression of the target gene was expressed as 2-ΔΔCT. Specifically, the ΔCT value was calculated by subtracting the CT value of the internal reference gene (GAPDH) from the CT value of the target gene in each sample. The ΔCT value of the control group was then subtracted from the ΔCT value of the treatment group to obtain ΔΔCT. Finally, the ΔΔCT value was converted to 2-ΔΔCT to obtain the relative expression level of INHBE mRNA (value of sample).

[0174] The inhibition rate of the INHBE gene mediated by RNAi agents was calculated according to the following formula:

[0175] INHBE gene inhibition rate %=(1-value of sample / Average value of Control)*100.

[0176] The sequence listing is compiled in accordance with WIPO Sequence STANDARD ST.26, wherein Table 8 – Conventional Nucleotide Symbols, and Definition:

[0177] Table 8

[0178]

[0179] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A double-stranded siRNA analog for inhibiting INHBE expression, characterized in that: The double-stranded siRNA analog comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length, the antisense strand comprises a complementary region that complementarily pairs with the sense strand, and the antisense strand comprises at least 15 consecutive nucleotides of a sequence that differs by no more than 3 nucleotides from the sequence shown in any one of SEQ ID NOs: 1068-2134 and 2136; Preferably, the length of the sense strand and the antisense strand is independently 17-27 nucleotides, more preferably 19-25 nucleotides, and most preferably 19-23 nucleotides; Preferably, the length of the complementary region is at least 15 nucleotide pairs, more preferably 15-25 nucleotide pairs, most preferably 17-23 nucleotide pairs; Preferably, the sense strand and the antisense strand each independently comprise a 3' overhang and / or a 5' overhang of at least 1 nucleotide, more preferably, one or both of the sense strand and the antisense strand comprise a 3' overhang and / or a 5' overhang of at least 2 nucleotides; most preferably, the antisense strand comprises a 3' overhang of 2 nucleotides; Preferably, the antisense strand comprises SEQ ID NO: 1076, 1089-1090, 1092, 1094-1095, 1097-1101, 1103-1105, 1117, 1119-1120, 1126, 1128, 1130, 1132, 1230-1231, 1234-1235, 1238, 1241, 1243-1247, 1249, 1251, 1313-1314, 1316-1317, 131 9-1321, 1323, 1325, 1331-1332, 1334-1335, 1388, 1390, 1393-1394, 1515, 1522, 1528, 1595, 1629, 1634, 1636, 1662-1663, 1688-1689, 1694-1695, 1703, 1705-1706, 1709, 1712, 1720-1721, 1779 , 1782, 1784, 1786, 1789-1790, 1792, 1810, 1830-1832, 1835, 1840-1841, 1845, 1847, 1849, 1853, 1855, 1860-1861, 1891, 1894, 1898, 1954, 1996-2003, 2008, 2012-2015, 2020, 2022-2023, 2025, 2028-2035, 2037, 2039, 2042-2050, 2052, 2054-2055, 2057, 2060-2061, 2064, 2075, 2107, 2111, 2129, or a sequence that differs from any of the above sequences by no more than 3, 2 or 1 nucleotides; more preferably, the antisense strand comprises the antisense strand sequence of any one of the siRNAs listed in Table 5.

2. The double-stranded siRNA analog according to claim 1, characterized in that The sense strand comprises a sequence shown in any one of SEQ ID NOs: 1-1067 and 2135; Preferably, the sense strand comprises SEQ ID NO:9, 22-23, 25, 27-28, 30-34, 36-38, 50, 52-53, 59, 61, 63, 65, 163-164 ,167-168,171,174,176,177-180,182,184,246-247,249-250,252-254, 256, 258, 264-265, 267-268, 321, 323, 326, 327, 448, 455, 461, 528, 562, 5 67, 569, 595-596, 621-622, 627-628, 636, 638-639, 642, 645, 653, 654, 71 2, 715, 717, 719, 722, 723, 725, 743, 763-765, 768, 773-774, 778, 780, 782, 786, 788, 793-794, 824, 827, 831, 887, 929-936, 941, 945-948, 1062, 953, 955, 956, 958, 961-968, 970, 972, 975-983, 985, 987-988, 990, 993-994, 997, 1008, 1040, 1044, or a sequence comprising a sequence that differs from any of the above sequences by no more than 3, 2, or 1 nucleotides; More preferably, the sense strand comprises a sense strand sequence of any one of the siRNAs selected from Table 5.

3. The double-stranded siRNA analog according to claim 1, characterized in that The double-stranded siRNA analogs comprise any pair of paired sense strand sequences and antisense strand sequences as shown in Table 1; Preferably, the double-stranded siRNA analog is selected from any one of the siRNAs in Table 5, more preferably siHB30, siHB34, siHB36, siHB38, siHB39, siHB52, siHB55, siHB363, siHB367, siHB404, siHB407, siHB500, siHB503, siHB505, siHB509, siHB518, siHB521, siHB642, siHB645, siHB646, siHB884, siHB890, siHB1043, siHB1105, siHB1107, siHB1211, siHB1222, or siHB1117. 228.siHB1309,siHB1310,siHB1312,siHB1394,siHB1405,siHB1432,siHB1440,siHB1857,siHB1859,siHB2162,siHB2165,siHB2298,siHB2347,siHB240 5. siHB2407, siHB2411, siHB1354, siHB1446, siHB1862, siHB1863, siHB2349, siHB2420, siHB2188, siHB1445, siHB2348, siHB2417, siHB2424, siHB2435.

4. The double-stranded siRNA analog according to any one of claims 1 to 3, characterized in that The double-stranded siRNA analog comprises at least one modified nucleotide selected from: Alkyl-modified nucleotides, methoxy-modified nucleotides, ethoxy-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-hydroxy-modified nucleotides, methoxyethyl-modified nucleotides, amino-modified nucleotides, fluoro-modified nucleotides, deoxynucleotides, 5'-methylphosphate nucleotides, 5'-C-methylphosphate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinylphosphonate deoxyribonucleotides, nucleotides containing phosphorothioate groups, nucleotides containing phosphorodithioate groups, locked nucleic acids (LNA), morpholino oligonucleotides (PMO); Preferably, the sense strand and / or the antisense strand each independently comprises at least one 2'-O-methyl modified nucleotide, at least one 2'-fluoro modified nucleotide and at least one nucleotide comprising a phosphorothioate group; More preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18 and / or 19 of the sense strand are each independently a 2'-O-methyl modified nucleotide; and / or From the 5' end to the 3' end, the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20 and / or 21 of the antisense strand are each independently a 2'-O-methyl modified nucleotide; and / or From the 5' end to the 3' end, the 5th, 7th, 8th and / or 9th nucleotides of the sense strand are each independently a 2'-fluoro-modified nucleotide; and / or In the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14 and / or 16 of the antisense strand are each independently a 2'-fluoro-modified nucleotide; And / or, the phosphorothioate group is present in at least one position selected from the following: In the direction from the 5' end to the 3' end, between the first nucleotide and the second nucleotide of the sense strand, between the second nucleotide and the third nucleotide of the sense strand, between the first nucleotide and the second nucleotide of the antisense strand, and between the second nucleotide and the third nucleotide of the antisense strand; In the direction from the 3' end to the 5' end, between the first nucleotide and the second nucleotide of the antisense strand, and between the second nucleotide and the third nucleotide of the antisense strand; Most preferably, the sense strand and antisense strand have the modified form shown in the following formula: Justice chain: NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm; Antisense strand: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm; Among them: "N" represents a nucleotide; m indicates that the nucleotide adjacent to its left is a 2'-O-methyl modified nucleotide; f indicates that the nucleotide adjacent to its left is a 2'-fluoro modified nucleotide; s indicates that the two nucleotides adjacent to the letter s on the left and right are connected by a thiophosphate group.

5. The double-stranded siRNA analog according to claim 4, characterized in that The double-stranded siRNA analog comprises a paired modified sense strand nucleotide sequence and a modified antisense strand nucleotide sequence as shown in any one of Table 2, wherein m indicates that the nucleotide adjacent to its left is a 2'-O-methyl modified nucleotide; f indicates that the nucleotide adjacent to its left is a 2'-fluoro modified nucleotide; s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate group; Preferably, the double-stranded siRNA analog is selected from any one of the siRNAs in Table 3.1, Table 3.2, Table 4.1, and Table 4.

2.

6. The double-stranded siRNA analog according to any one of claims 1 to 9, characterized in that The double-stranded siRNA analog is linked to a targeting ligand; Preferably, the targeting ligand is selected from the following structures: Preferably, the targeting ligand is linked to the 3' or 5' end of the sense strand.

7. A vector comprising a nucleotide sequence encoding the double-stranded siRNA analog according to any one of claims 1 to 3. A cell comprising the double-stranded siRNA analog according to any one of claims 1 to 6 or the vector according to claim 7.

9. A pharmaceutical composition for inhibiting INHBE expression, characterized in that: The pharmaceutical composition comprises the double-stranded siRNA analog according to any one of claims 1 to 6, or the vector according to claim 7, or the cell according to claim 8; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

10. Use of the double-stranded siRNA analog according to any one of claims 1 to 6, the vector according to claim 7, the cell according to claim 8 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for preventing and / or treating a disease, disorder or symptom mediated by abnormal or overexpression of INHBE; preferably, the disease, disorder or symptom is selected from metabolic and / or cardiovascular diseases, disorders or symptoms.

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