Inhibitors that suppress the expression of repressin Beta E gene and their uses

Abstract

A dsRNA and a double-stranded RNAi agent that inhibit INHBE expression, and their uses, wherein the dsRNA and the double-stranded RNAi agent can be used to inhibit INHBE expression, thereby preventing and treating metabolic disorders (e.g., metabolic syndrome).

Description

Inhibitors for inhibiting inhibin Beta E gene expression and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to Chinese patent application number 202311333351.X, filed on October 16, 2023, and U.S. provisional application US63 / 590,562, filed on October 16, 2023. The entire contents of the Chinese patent application are hereby incorporated by reference into this disclosure. Technical Field

[0003] The present disclosure relates to an inhibitor of inhibin Beta E gene (INHBE), such as an RNAi agent or RNA, and pharmaceutical compositions thereof, as well as methods for preventing, treating and / or inhibiting diseases and disorders associated with the INHBE gene. Background Art

[0004] Metabolic syndrome is a pathological condition characterized by abdominal obesity, insulin resistance, hypertension, and hyperlipidemia. Metabolic syndrome can cause metabolic disorders and is not only a risk factor for cardiovascular disease, diabetes, and kidney disease, but also increases the risk of cancer and all-cause mortality. Approximately 20% to 25% of adults worldwide suffer from metabolic syndrome. The incidence of metabolic syndrome caused by obesity is increasing annually among children and young adults, which is associated with a high-calorie, low-fiber diet and sedentary lifestyle. In addition to medication management for each disease group, current treatments for metabolic syndrome primarily involve dietary and lifestyle changes, but clinical compliance is poor in most patients.

[0005] The inhibin beta E gene (INHBE) encodes a secreted protein called activin E. INHBE mRNA is primarily expressed in the liver, where it participates in the regulation of hepatocyte growth and differentiation. A study (Hashimoto O. et al., Life sciences. 2009; 85(13–14): 534–40) reported that insulin stimulates the expression of INHBE in hepatocytes, and that INHBE mRNA is upregulated in the liver of diet-induced obese mice, suggesting that INHBE is involved in glucose metabolism. Further studies (Sugiyama M. et al., PLoS One, 2018; 13(3): e0194798) have shown that activin E is a putative factor inducing insulin resistance, and that INHBE gene expression is positively correlated with insulin resistance and body mass index (BMI) in humans. In addition, some literature indicates (Akbari, P. et al., Nat Commun 13, 4844 (2022).) that INHBE is a negative regulator of fat storage expressed in the liver, and blocking INHBE may be beneficial in treating metabolic diseases related to fat distribution.

[0006] WO2023003922A1 discloses double-stranded RNA (dsRNA) agents that inhibit the expression of target genes associated with metabolic disorders and target INHBE. WO2023044094A1 discloses INHBE modulator compositions and methods of use. However, there is still a need to develop RNAi drugs with better efficacy targeting INHBE.

[0007] Summary of the Invention

[0008] The present disclosure provides an inhibitor for inhibiting the expression of the inhibin Beta E gene (INHBE), such as RNAi or RNA, and a pharmaceutical composition thereof, and use thereof in preparing a medicament for preventing, treating, and / or inhibiting related diseases such as metabolic syndrome.

[0009] RNAi agents and RNA disclosed herein are designed to target the INHBE gene, including portions of the gene that are conserved in orthologs of other mammalian species. RNAi agents typically comprise a sense strand and an antisense strand that form a duplex, double-stranded RNA (referred to herein as "dsRNA"). RNAi agents comprising dsRNA are also referred to herein as "dsRNAi" agents.

[0010] Without intending to be limited by theory, the RNAi agents and RNAs disclosed herein, as well as specific target sites and / or modifications therein, impart improved efficacy, stability, potency, durability, and / or safety. For example, but not limited to, in some embodiments, the RNAi agents and / or RNAs disclosed herein exhibit: (1) improved efficacy and / or potency, for example, by stronger hybridization with target gene mRNA (e.g., as determined by an increase in the Tm of the antisense strand / target mRNA duplex, such as an increase in the theoretical Tm of the antisense strand); and / or, (2) improved safety, for example, by reducing off-target effects, for example, by reducing or attenuating hybridization with off-target RNAs (e.g., as determined by a decrease in the Tm of the duplex formed by the antisense strand and the off-target RNA).

[0011] The use of RNAi agents disclosed herein can enable targeted degradation of the mRNA of the INHBE target gene in mammals. The inventors have demonstrated that the RNAi agents disclosed herein can trigger RNA-induced silencing complex (RISC)-mediated cleavage of INHBE RNA transcripts, thereby resulting in significant inhibition of INHBE target gene expression. In certain embodiments, the RNAi agents disclosed herein are more effective (e.g., more potent) and / or more specific (e.g., safer, with fewer off-target effects) than previous RNAi agents targeting the same gene. In certain embodiments, the RNAi agents are targeted to specific sites in the INHBE mRNA and / or include RNA modifications (e.g., non-canonical base pairing nucleotides, modified nucleotides, chemical modifications) to increase efficacy, effectiveness, specificity, and / or safety. In some such embodiments, the RNAi agent comprises at least one modified nucleotide, such as a non-canonical base pairing nucleotide. Methods and compositions comprising these RNAi agents can be used to treat subjects with INHBE-related diseases or conditions, such as metabolic disorders. Thus, the present disclosure provides methods for treating, preventing, or inhibiting metabolic disorders in a subject who would benefit from inhibiting or reducing INHBE expression using the RNAi agents and compositions of the present disclosure.

[0012] In one aspect, the present disclosure provides a double-stranded RNA interference (dsRNAi) agent for inhibiting the expression of inhibin subunit beta E (INHBE) in a cell, wherein the dsRNAi agent comprises a sense strand and an antisense strand forming a double-stranded RNA (dsRNA) region, the antisense strand comprising a region complementary to INHBE mRNA, wherein the complementary region comprises at least 15 consecutive nucleotides. In certain embodiments, the dsRNAi agent comprises at least one non-canonical base pairing nucleotide, as further described below.

[0013] In some embodiments, the dsRNAi agent comprises one, two, three, four, five or more non-canonical base pairing nucleotides. Non-canonical base pairing nucleotides and other modified nucleotides can be present in any other position in the complementary region, dsRNA region or dsRNAi agent. Non-canonical base pairing nucleotides and other modified nucleotides can be included in the antisense strand, sense strand or both. In some embodiments, in addition to non-canonical base pairing nucleotides, the dsRNAi agent also comprises at least one other modified nucleotide; Such other modified nucleotides may or may not be on the same oligonucleotide chain as the non-canonical base pairing nucleotides.

[0014] In some embodiments, the dsRNAi agent or RNA has increased efficacy, potency, specificity, and / or safety, and / or reduced off-target effects compared to a dsRNAi agent or RNA having the same nucleotide sequence but without non-canonical base pairing and / or modified nucleotides. In some such embodiments, the melting temperature (Tm) of the dsRNAi agent or RNA is altered by at least 2°C compared to a dsRNAi agent or RNA having the same nucleotide sequence but without non-canonical base pairing and / or modified nucleotides.

[0015] In some embodiments, the dsRNAi agents or RNAs of the present disclosure comprise non-canonical base-pairing nucleotides and / or modified nucleotides that alter the Tm by at least 2° C., e.g., 2° C., more than 2° C., 3° ​​C., 4° C., 5° C., or more. In some such embodiments, the Tm is calculated using the formulas or algorithms described herein.

[0016] In some embodiments, the dsRNAi agents or RNAs of the present disclosure comprise at least one non-canonical base-pairing nucleotide and / or modified nucleotide in positions 1-11 of the oligonucleotide strand (eg, antisense strand) in the direction from the 5' end to the 3' end.

[0017] In some embodiments, the dsRNAi agent or RNA of the present invention comprises at least one non-canonical base pairing nucleotide and / or modified nucleotide in the 12th-21st position of the oligonucleotide chain (e.g., antisense chain) from the 5' end to the 3' end. In some embodiments, in the sequence fragment N1N2G0N3N4, N1, N2, N3 and N4 are each independently a nucleotide comprising adenine (A), cytosine (C), guanine (G), thymine (T) or uracil (U) as a base, G0 is a nucleotide comprising guanine as a base, and when at least three bases in N1, N2, N3 and N4 are adenine or uracil, the guanine of G0 in the sequence is replaced by hypoxanthine (I). In some such embodiments, at least one of N1, N2, N3, N4 and G0 also has a modified sugar group and / or a modified internucleotide linkage.

[0018] In some embodiments, the dsRNAi agent or RNA of the present disclosure comprises at least one non-canonical base-pairing nucleotide and / or modified nucleotide at position 6, 7, or 8 in the direction from the 5' end to the 3' end of the oligonucleotide chain, and if at least four nucleotides from positions 2 to 8 are A or U and at least one nucleotide from positions 6 to 8 is G, the G at positions 6, 7, and / or 8 is replaced by a non-canonical base-pairing nucleotide. In some embodiments, the dsRNAi agent or RNA of the present disclosure comprises at least one non-canonical base-pairing nucleotide and / or modified nucleotide at position 6, 7, or 8 in the direction from the 5' end to the 3' end of the oligonucleotide chain (e.g., the antisense strand and / or the sense strand), if the number of adenine and uracil in the nucleotides from positions 2 to 8 is 4 or more and at least one base in the nucleotides from positions 6 to 8 is guanine, the guanine at positions 6, 7, and / or 8 is replaced by a non-canonical base (e.g., hypoxanthine).

[0019] In some such embodiments, the dsRNAi agent or RNA of the present disclosure comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any of the sequences shown in Table 1, Table 2a, or Table 2b. In some such embodiments, the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any of the sequences shown in Table 1, Table 2a, or Table 2b, and the sense strand comprises at least 15 nucleotides that are complementary to the antisense strand. In some such embodiments, the sense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any of the sequences shown in Table 1, Table 2a, or Table 2b, and the antisense strand comprises at least 15 nucleotides that are complementary to the sense strand. In some such embodiments, the antisense strand and / or the sense strand differ by no more than 1, 2, or 3 nucleotides from any of the sequences shown in Table 1, Table 2a, or Table 2b. In some embodiments, the dsRNAi agent comprises a dsRNA comprising any duplex sequence in Table 1, Table 2a, or Table 2b, such as any one of IN-001 to IN-488 and INI-001 to INI-308, or any one of IN-489 to IN-546. In the present application, it should be understood that "difference" can refer to differences in nucleotide sequence length, differences in nucleotides, or a combination of the two.

[0020] Exemplary non-canonical base pairing nucleotides include, but are not limited to, bases selected from inosine (I), xanthosine (X), 7-methylguanosine (m7G), N6-methyladenosine (m6A), dihydrouridine, 5-methylcytosine (m5C), pseudouridine (Ψ), and N1-methylpseudouridine (m1Ψ). In certain embodiments, the non-canonical base pairing nucleotide is inosinic acid (I). In some embodiments, at least one guanine (G) in a dsRNAi agent or RNA (e.g., sense strand and / or antisense strand) of the present disclosure is replaced by hypoxanthine (I). Examples of other modified nucleotides are described elsewhere herein.

[0021] In another aspect, the present disclosure provides a dsRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ from any one of the sequences shown in Table 1, Table 2a, or Table 2b by no more than 3 (0, 1, 2, or 3) nucleotides, and the sense strand has at least 15, 16, 17, 18, 19, 20, or 21 nucleotides that are complementary to the antisense strand.

[0022] In some embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides that differ from any of the sequences shown in Table 1, Table 2a, or Table 2b by 0, 1, 2, or 3 nucleotides.

[0023] In some embodiments, the sense strand sequence is at least substantially complementary to the antisense strand sequence. In some preferred embodiments, the sense strand sequence is fully complementary to the antisense strand sequence (ie, 100% complementary).

[0024] In some embodiments, the length of the sense strand and the antisense strand are each independently 17-25 nucleotides; preferably, the length of the sense strand and the antisense strand are each independently 19-23 nucleotides; more preferably, the length of the sense strand and the antisense strand are each independently 19-21 nucleotides.

[0025] In some embodiments, the sense strand is 19 nucleotides in length and includes any one of the following:

[0026] 15 consecutive nucleotides represented by SEQ ID NO 1621: GGGUCAAGCACAGCU;

[0027] 15 consecutive nucleotides represented by SEQ ID NO 1622: AAGCACAGACUAUCCA;

[0028] 15 consecutive nucleotides represented by SEQ ID NO 1623: CAGCUAUCCAUCAGA;

[0029] 15 consecutive nucleotides represented by SEQ ID NO 1624: AUCCAUCAGAUGAUC;

[0030] 15 consecutive nucleotides represented by SEQ ID NO 1625:UCAGAUGAUCUACUU;

[0031] 15 consecutive nucleotides represented by SEQ ID NO 1626: UGAUCUACUUUCAGC;

[0032] 15 consecutive nucleotides represented by SEQ ID NO 1627:UGAGUCCCAGACAAU;

[0033] 15 consecutive nucleotides represented by SEQ ID NO 1628: AGCCAAGCAGCAAAU;

[0034] 15 consecutive nucleotides represented by SEQ ID NO 1629: GUCGUCCCAGAAUAA;

[0035] 15 consecutive nucleotides represented by SEQ ID NO 1630: AUCAGCUUUGCUACU;

[0036] 15 consecutive nucleotides represented by SEQ ID NO 1631:UGCUACUGUCACAGA;

[0037] 15 consecutive nucleotides represented by SEQ ID NO 1632: UUCCUGGCACUCUUU;

[0038] 15 consecutive nucleotides represented by SEQ ID NO 1633: UCUUUGCUUGAGGAU;

[0039] 15 consecutive nucleotides represented by SEQ ID NO 1634:UGGUGUCCUGAAACU;

[0040] 15 consecutive nucleotides represented by SEQ ID NO 1635: CCUGAAACUGCAACU;

[0041] 15 consecutive nucleotides represented by SEQ ID NO 1636:AGGCAACAGCACAGU;

[0042] 15 consecutive nucleotides represented by SEQ ID NO 1637: CCUAGAGCUUAAGAU;

[0043] 15 consecutive nucleotides represented by SEQ ID NO 1638: CUUAAGAUCCGAGCC;

[0044] 15 consecutive nucleotides represented by SEQ ID NO 1639: ACCAUUACGUAGACU;

[0045] 15 consecutive nucleotides represented by SEQ ID NO 1640: CUCAAAGCCAACAAU;

[0046] 15 consecutive nucleotides represented by SEQ ID NO 1641: CUCUACCUGGAUCAU;

[0047] 15 consecutive nucleotides represented by SEQ ID NO 1642: GAUCAUAAUGGCAAU;

[0048] 15 consecutive nucleotides represented by SEQ ID NO 1643: AAGAGACCAAGAUGA;

[0049] 15 consecutive nucleotides represented by SEQ ID NO 1644: CUGGCAAUAUGACUC;

[0050] 15 consecutive nucleotides represented by SEQ ID NO 1645:UGGGCACUUUCUUGU;

[0051] 15 consecutive nucleotides represented by SEQ ID NO 1646: UUCUUGUCUGAGACU;

[0052] 15 consecutive nucleotides represented by SEQ ID NO 1647:UGAGACUCUGGCUUA;

[0053] 15 consecutive nucleotides represented by SEQ ID NO 1648: UUGGGAGAUGGGUAA;

[0054] 15 consecutive nucleotides represented by SEQ ID NO 1649:GAUGGGUAAAGCGUU;

[0055] 15 consecutive nucleotides represented by SEQ ID NO 1650:AAAGCGUUUCUUCUA;

[0056] 15 consecutive nucleotides represented by SEQ ID NO 1651: CUUCUAAAGGGGUCU;

[0057] 15 consecutive nucleotides represented by SEQ ID NO 1652: UACCCAGAAAGCAUG;

[0058] 15 consecutive nucleotides represented by SEQ ID NO 1653: AAGUCCUGUGAGAAG;

[0059] 15 consecutive nucleotides represented by SEQ ID NO 1654:GAAGGCAGAGAAAAA;

[0060] 15 consecutive nucleotides represented by SEQ ID NO 1655: CAGAGAAAAAUUACU;

[0061] 15 consecutive nucleotides represented by SEQ ID NO 1656:UCCCAAGAUGAGAAA;

[0062] 15 consecutive nucleotides represented by SEQ ID NO 1657: GAGGAAGCAGAUAGA;

[0063] 15 consecutive nucleotides represented by SEQ ID NO 1658: CUGUUGAGGUACCUU;

[0064] 15 consecutive nucleotides represented by SEQ ID NO 1659:AGGUACCUUAAGGGA;

[0065] 15 consecutive nucleotides represented by SEQ ID NO 1660: CAGGAGUCAGGAAAA;

[0066] 15 consecutive nucleotides represented by SEQ ID NO 1661: GGGAGACAAGCAUUU;

[0067] 15 consecutive nucleotides represented by SEQ ID NO 1662:ACAAGCAUUUAUACU;

[0068] 15 consecutive nucleotides represented by SEQ ID NO 1663: CAUUUAUACUUUCUU;

[0069] 15 consecutive nucleotides represented by SEQ ID NO 1664: AAAGAAAAUCAACAA;

[0070] 15 consecutive nucleotides represented by SEQ ID NO 1665: CAAAUGUGAGUCAUA;

[0071] 15 consecutive nucleotides represented by SEQ ID NO 1666: GUCAUAAAGAAGGGU;

[0072] 15 consecutive nucleotides represented by SEQ ID NO 1667:AGAGCAACAGUUCUU;

[0073] 15 consecutive nucleotides represented by SEQ ID NO 1668: GGGUGUCCACAAAGU;

[0074] 15 consecutive nucleotides represented by SEQ ID NO 1669: UCCACAAAGUCAAAG;

[0075] 15 consecutive nucleotides represented by SEQ ID NO 1670: AAAGUCAAAGCUAUU;

[0076] 15 consecutive nucleotides represented by SEQ ID NO 1671:AAAGCUAUUUUCAUA;

[0077] 15 consecutive nucleotides represented by SEQ ID NO 1672: AUUUUCAUAAUAAUA;

[0078] 15 consecutive nucleotides represented by SEQ ID NO 1673: AUAAUACUAACAUGU;

[0079] 15 consecutive nucleotides represented by SEQ ID NO 1674:ACUAACAUGUUAUUU;

[0080] 15 consecutive nucleotides represented by SEQ ID NO 1675: AUGUUAUUUGCCUUU;

[0081] 15 consecutive nucleotides represented by SEQ ID NO 1676: AUUUGCCUUUUGAAU;

[0082] 15 consecutive nucleotides represented by SEQ ID NO 1677: UUUUGAAUUCUCAUU;

[0083] 15 consecutive nucleotides represented by SEQ ID NO 1678: UUCUCAUUAUCUUAA;

[0084] 15 consecutive nucleotides represented by SEQ ID NO 1679: UUAUCUUAAAAUUGU;

[0085] 15 consecutive nucleotides represented by SEQ ID NO 1680: GUGUGACAUGUGAUU;

[0086] 15 consecutive nucleotides represented by SEQ ID NO 1681: AUGUGAUUACAUCAU;

[0087] 15 consecutive nucleotides represented by SEQ ID NO 1682: UUACAUCAUCUUUCU;

[0088] 15 consecutive nucleotides represented by SEQ ID NO 1683: ACUUUCUGACAUCA;

[0089] 15 consecutive nucleotides represented by SEQ ID NO 1684: UCUGACAUCAUUGUU;

[0090] 15 consecutive nucleotides represented by SEQ ID NO 1685: GUUAAUGGAAUGUGU;

[0091] 15 consecutive nucleotides represented by SEQ ID NO 1686: CAGCUUUGCUACUGU;

[0092] 15 consecutive nucleotides represented by SEQ ID NO 1687:AGACUCUGGCUUAUU;

[0093] 15 consecutive nucleotides represented by SEQ ID NO 1688: GCAACAGUUCUUCAA;

[0094] 15 consecutive nucleotides represented by SEQ ID NO 1689: AAAGCUAUUUUCAUA.

[0095] In some embodiments, the antisense strand is 21 nucleotides in length and includes any one of the following:

[0096] 15 consecutive nucleotides represented by SEQ ID NO 1690:AGCUGUGCUUGACCC;

[0097] 15 consecutive nucleotides represented by SEQ ID NO 1691:UGGAUAGCUGUGCUU;

[0098] 15 consecutive nucleotides represented by SEQ ID NO 1692:UCUGAUGGAUAGCUG;

[0099] 15 consecutive nucleotides represented by SEQ ID NO 1693: GAUCAUCUGAUGGAU;

[0100] 15 consecutive nucleotides represented by SEQ ID NO 1694: AAGUAGAUCAUCUGA;

[0101] 15 consecutive nucleotides represented by SEQ ID NO 1695: AAGUAGAUCAUCUGA;

[0102] 15 consecutive nucleotides represented by SEQ ID NO 1696: AUUGUCUGGGACUCA;

[0103] 15 consecutive nucleotides represented by SEQ ID NO 1697: AUUUGCUGCUUGGCU;

[0104] 15 consecutive nucleotides represented by SEQ ID NO 1698: UUAUUCUGGGACGAC;

[0105] 15 consecutive nucleotides represented by SEQ ID NO 1699: AGUAGCAAAGCUGAU;

[0106] 15 consecutive nucleotides represented by SEQ ID NO 1700: UCUGUGACAGUAGCA;

[0107] 15 consecutive nucleotides represented by SEQ ID NO 1701: UCUGUGACAGUAGCA;

[0108] 15 consecutive nucleotides represented by SEQ ID NO 1702: AUCCUCAAGCAAAGA;

[0109] 15 consecutive nucleotides represented by SEQ ID NO 1703: AGUUUCAGGACACCA;

[0110] 15 consecutive nucleotides represented by SEQ ID NO 1704: AGUUGCAGUUUCAGG;

[0111] 15 consecutive nucleotides represented by SEQ ID NO 1705:ACUGUGCUGUUGCCU;

[0112] 15 consecutive nucleotides represented by SEQ ID NO 1706: AUCUUAAGCUCUAGG;

[0113] 15 consecutive nucleotides represented by SEQ ID NO 1707: GGCUCGGAUCUUAAG;

[0114] 15 consecutive nucleotides represented by SEQ ID NO 1708: AGUCUACGUAAUGGU;

[0115] 15 consecutive nucleotides represented by SEQ ID NO 1709: AUUGUUGGCUUUGAG;

[0116] 15 consecutive nucleotides represented by SEQ ID NO 1710: AUGAUCCAGGUAGAG;

[0117] 15 consecutive nucleotides represented by SEQ ID NO 1711: AUUGCCAUUAUGAUC;

[0118] 15 consecutive nucleotides represented by SEQ ID NO 1712:UCAUCUUGGUCUCUU;

[0119] 15 consecutive nucleotides represented by SEQ ID NO 1713:AGUCAUAUUGCCAGG;

[0120] 15 consecutive nucleotides represented by SEQ ID NO 1714: ACAAGAAAGUGCCCA;

[0121] 15 consecutive nucleotides represented by SEQ ID NO 1715: AGUCUCAGACAAGAA;

[0122] 15 consecutive nucleotides represented by SEQ ID NO 1716:UAAGCCAGAGUCUCA;

[0123] 15 consecutive nucleotides represented by SEQ ID NO 1717: UUACCCAUCUCCCAA;

[0124] 15 consecutive nucleotides represented by SEQ ID NO 1718: AACGCUUUACCCAUC;

[0125] 15 consecutive nucleotides represented by SEQ ID NO 1719:UAGAAGAAACGCUUU;

[0126] 15 consecutive nucleotides represented by SEQ ID NO 1720:AGACCCCUUUAGAAG;

[0127] 15 consecutive nucleotides represented by SEQ ID NO 1721: CAUGCUUUCUGGGUA;

[0128] 15 consecutive nucleotides represented by SEQ ID NO 1722: CUUCUCACAGGACUU;

[0129] 15 consecutive nucleotides represented by SEQ ID NO 1723: UUUUUCUCUGCCUUC;

[0130] 15 consecutive nucleotides represented by SEQ ID NO 1724: AGUAAUUUUUUCUCUG;

[0131] 15 consecutive nucleotides represented by SEQ ID NO 1725: UUUCUCAUCUUGGGA;

[0132] 15 consecutive nucleotides represented by SEQ ID NO 1726: UCUAUCUGCUUCCUC;

[0133] 15 consecutive nucleotides represented by SEQ ID NO 1727: AAGGUACCUCAACAG;

[0134] 15 consecutive nucleotides represented by SEQ ID NO 1728: UCCCUUAAGGUACCU;

[0135] 15 consecutive nucleotides represented by SEQ ID NO 1729: UUUUCCUGACUCCUG;

[0136] 15 consecutive nucleotides represented by SEQ ID NO 1730:AAAUGCUUGUCUCCC;

[0137] 15 consecutive nucleotides represented by SEQ ID NO 1731: AGUAUAAAUGCUUGU;

[0138] 15 consecutive nucleotides represented by SEQ ID NO 1732: AAGAAAGUAUAAAUG;

[0139] 15 consecutive nucleotides represented by SEQ ID NO 1733: UUGUUGAUUUUCUUU;

[0140] 15 consecutive nucleotides represented by SEQ ID NO 1734:UAUGACUCACAUUUG;

[0141] 15 consecutive nucleotides represented by SEQ ID NO 1735: ACCCUUCUUUAUGAC;

[0142] 15 consecutive nucleotides represented by SEQ ID NO 1736: AAGAACUGUUGCUCU;

[0143] 15 consecutive nucleotides represented by SEQ ID NO 1737: ACUUUGUGGACACCC;

[0144] 15 consecutive nucleotides represented by SEQ ID NO 1738: CUUUGACUUUGUGGA;

[0145] 15 consecutive nucleotides represented by SEQ ID NO 1739: AAUAGCUUUGACUUU;

[0146] 15 consecutive nucleotides represented by SEQ ID NO 1740:UAUGAAAAUAGCUUU;

[0147] 15 consecutive nucleotides represented by SEQ ID NO 1741: UAUUAUUAUGAAAAU;

[0148] 15 consecutive nucleotides represented by SEQ ID NO 1742: ACAUGUUAGUAUUAU;

[0149] 15 consecutive nucleotides represented by SEQ ID NO 1743:AAAUAACAUGUUAGU;

[0150] 15 consecutive nucleotides represented by SEQ ID NO 1744: AAAGGCAAAUAACAU;

[0151] 15 consecutive nucleotides represented by SEQ ID NO 1745: AUUCAAAAGGCAAAU;

[0152] 15 consecutive nucleotides represented by SEQ ID NO 1746: AAUGAGAAUUCAAAA;

[0153] 15 consecutive nucleotides represented by SEQ ID NO 1747: UUAAGAUAAUGAGAA;

[0154] 15 consecutive nucleotides represented by SEQ ID NO 1748: ACAAUUUUAAGAUAA;

[0155] 15 consecutive nucleotides represented by SEQ ID NO 1749: AAUCAAUGUCACAC;

[0156] 15 consecutive nucleotides represented by SEQ ID NO 1750: AUGAUGUAAUCACAU;

[0157] 15 consecutive nucleotides represented by SEQ ID NO 1751:AGAAAGAUGAUGUAA;

[0158] 15 consecutive nucleotides represented by SEQ ID NO 1752: UGAUGUCAGAAAGAU;

[0159] 15 consecutive nucleotides represented by SEQ ID NO 1753:AACAAUGAUGUCAGA;

[0160] 15 consecutive nucleotides represented by SEQ ID NO 1754:ACACAUUCCAUUAAC;

[0161] 15 consecutive nucleotides represented by SEQ ID NO 1755:ACAGUAGCAAAGCUG;

[0162] 15 consecutive nucleotides represented by SEQ ID NO 1756:AAUAAGCCAGAGUCU;

[0163] 15 consecutive nucleotides represented by SEQ ID NO 1757: UUGAAGAACUGUUGC;

[0164] 15 consecutive nucleotides represented by SEQ ID NO 1758:UAUGAAAAUAGCUUU.

[0165] In some embodiments, the sense strand of the present disclosure is derived from the mRNA sequence of human INHBE (e.g., Gene ID: 83729, NM_031479.5 (SEQ ID NO: 1619)). Alternatively, the sense strand of the present disclosure is a fragment of the mRNA sequence of human INHBE. In some embodiments, the sense strand of the present disclosure is derived from the mRNA sequence of cynomolgus monkey INHBE (e.g., Gene ID: 102127493, XM_005571319.3 (SEQ ID NO: 1620)). Alternatively, the sense strand of the present disclosure is a fragment of the mRNA sequence of cynomolgus monkey INHBE.

[0166] In some embodiments, the dsRNA comprises any one of the antisense or sense strand sequences in Table 1, Table 2a, or Table 2b. In some embodiments, the dsRNA comprises any one of the sense or antisense strand sequences set forth in SEQ ID NOs: 1-1590. In some embodiments, the dsRNA comprises any one of the sense or antisense strand sequences set forth in SEQ ID NOs: 1759-1874.

[0167] In some embodiments, the dsRNA comprises an antisense strand sequence and a sense strand sequence as shown in the duplex sequences in Table 1, Table 2a, or Table 2b. In some embodiments, the dsRNA comprises any duplex selected from IN-001 to IN-488 and INI-001 to INI-308. In some embodiments, the dsRNA comprises any duplex selected from IN-489 to IN-546.

[0168] In some embodiments, at least one nucleotide in the sense strand and / or the antisense strand is a modified nucleotide. In some such embodiments, the modified nucleotide is a non-canonical base pairing nucleotide.

[0169] In some embodiments, substantially all nucleotides in the sense strand and / or the antisense strand are modified nucleotides. In some such embodiments, at least one of the modified nucleotides is a non-canonical base pairing nucleotide.

[0170] In some embodiments, all nucleotides in the sense strand and / or the antisense strand are modified nucleotides. In some such embodiments, at least one of the modified nucleotides is a non-canonical base pairing nucleotide.

[0171] In some embodiments, the modified nucleotides of the present disclosure are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-F-arabinonucleotides, phosphorothioate modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleic acids, inverted nucleotides, and inosine base substituted nucleotides.

[0172] In some embodiments, the modified nucleotides of the present disclosure are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, phosphorothioate modified nucleotides, and inverted nucleotides. In some preferred embodiments, the inverted nucleotides are selected from the group consisting of inverted A nucleotides, inverted dA nucleotides, inverted dT nucleotides, inverted C nucleotides, and inverted U nucleotides.

[0173] In some embodiments, the modified nucleotides disclosed herein include any one or a combination of the following:

[0174] (1) From the 5' end to the 3' end, the nucleotides at positions 2, 4, 12, and 14 of the antisense strand are 2'-fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides;

[0175] (2) in the direction from 5' to 3', the nucleotides at positions 7, 8, and 9 of the sense strand are 2'-fluorinated nucleotides;

[0176] (3) From the 5' end to the 3' end, the guanine (the base in G) in positions 2-8 of the antisense strand is replaced with hypoxanthine (the base of inosine or inosinic acid I).

[0177] In some such embodiments, the modified nucleotides substituted with hypoxanthine in positions 2-8 of the antisense strand further satisfy one or a combination of the following characteristics:

[0178] (i) guanine at positions 6-8 of the antisense strand is replaced with hypoxanthine in the direction from 5' to 3' end (I);

[0179] (ii) guanine in the sequence segment N1N2GN3N4 of the antisense strand is replaced by hypoxanthine (I) in the direction from the 5' end to the 3' end, provided that at least three bases among N1, N2, N3 and N4 are adenine or uracil;

[0180] (iii) the number of adenines and uracils in positions 2 to 8 of the antisense strand is 4 or more in the 5' to 3' direction; and / or

[0181] (iv) the base guanine in at least one nucleotide in the antisense strand is substituted with hypoxanthine, wherein the hypoxanthine substitution results in a difference in dissociation temperature of at least 2°C, 2°C, or greater than 2°C between the dsRNA comprising the antisense strand and the corresponding dsRNA without the hypoxanthine substitution.

[0182] In some embodiments, the antisense strand comprises a sequence selected from SEQ ID NO: 86, 182, 212, 350, 376, 402, 440, 460, 462, 464, 466, 468, 470, 472, 47 4, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504 ,506,508,510,512,514,516,518,520,522,524,526,528,530,532,534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 5 66, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 88, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930,932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778, 1780, 1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800, 1801 802, 1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826, 1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848, 1850, 1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872, or 1874 of completely consecutive nucleotides.

[0183] In some embodiments, the sense strand comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 181, 211, 349, 375, 401, 439, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 520, 521, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 551, 552, 553, 554, 555 21, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 5 99, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753,755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 846 47, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 93 9, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 1759, 1761, 1763, 1765, 1767, 1769, 1771, 1773, 1775, 1777, 1779, 1781, 1783, 1785, 1787, 1789, 1791, 1793, 1795, 1797, 1799, 1801, 1803, completely consecutive nucleotides of 1805, 1807, 1809, 1811, 1813, 1815, 1817, 1819, 1821, 1823, 1825, 1827, 1829, 1831, 1833, 1835, 1837, 1839, 1841, 1843, 1845, 1847, 1849, 1851, 1853, 1855, 1857, 1859, 1861, 1863, 1865, 1867, 1869, 1871 or 1873.

[0184] In some embodiments, the dsRNA comprises a sense strand sequence and an antisense strand sequence selected from the group consisting of: SEQ ID NOs: 85 and 86; SEQ ID NOs: 181 and 182; SEQ ID NOs: 211 and 212; SEQ ID NOs: 349 and 350; SEQ ID NOs: 375 and 376; SEQ ID NOs: 401 and 402; SEQ ID NOs: 439 and 440; SEQ ID NOs: 459 and 460; SEQ ID NOs: 461 and 462; SEQ ID NOs: 463 and 464; SEQ ID NOs: 465 and 466; SEQ ID NOs: 467 and 468; SEQ ID NOs: 469 and 470; SEQ ID NOs: 471 and 472; SEQ ID NOs: 473 and 474; SEQ ID NOs: 475 and 476; SEQ ID NOs: 477 and 478; SEQ ID NOs: 479 and 480; SEQ ID NOs: 481 and 482; SEQ ID NOs: 481 and 482; SEQ ID NOs: 483 and 484; SEQ ID NOs: 485 and 486; SEQ ID NOs: 487 and 488; SEQ ID NOs: 489 and 490; SEQ ID NOs: 491 and 492; SEQ ID NOs: 493 and 494; SEQ ID NOs: 495 and 496; SEQ ID NOs: 497 and 498; SEQ ID NOs: 499 and 500; SEQ ID NOs: 501 and 502; SEQ ID NOs: 503 and 504; SEQ ID NOs: 505 and 506; SEQ ID NOs: 507 and 508; SEQ ID NOs: 509 and 510; SEQ ID NOs: 511 and 512; SEQ ID NOs: 513 and 514; SEQ ID NOs: 515 and 516; SEQ ID NOs: 517 and 518; SEQ ID NOs: 519 and 520; SEQ ID NOs: 521 and 522; SEQ ID NOs: 521 and 522; SEQ ID NOs: 523 and 524; SEQ ID NOs: 525 and 526; SEQ ID NOs: 527 and 528; SEQ ID NOs: 529 and 530; SEQ ID NOs: 531 and 532; SEQ ID NOs: 533 and 534; SEQ ID NOs: 535 and 536; SEQ ID NOs: 537 and 538; SEQ ID NOs: 539 and 540; SEQ ID NOs: 541 and 542; SEQ ID NOs: 543 and 544; SEQ ID NOs: 545 and 546; SEQ ID NOs: 547 and 548; SEQ ID NOs: 549 and 550;SEQ ID NOs: 551 and 552; SEQ ID NOs: 553 and 554; SEQ ID NOs: 555 and 556; SEQ ID NOs: 557 and 558; SEQ ID NOs: 559 and 560; SEQ ID NOs: 561 and 562; SEQ ID NOs: 563 and 564; SEQ ID NOs: 565 and 566; SEQ ID NOs: 567 and 568; SEQ ID NOs: 569 and 570; SEQ ID NOs: 571 and 572; SEQ ID NOs: 573 and 574; SEQ ID NOs: 575 and 576; SEQ ID NOs: 577 and 578; SEQ ID NOs: 579 and 580; SEQ ID NOs: 581 and 582; SEQ ID NOs: 583 and 584; SEQ ID NOs: 585 and 586; SEQ ID NOs: 587 and 588; SEQ ID NOs: 589 and 590; SEQ ID NOs: SEQ ID NOs: 591 and 592; SEQ ID NOs: 593 and 594; SEQ ID NOs: 595 and 596; SEQ ID NOs: 597 and 598; SEQ ID NOs: 599 and 600; SEQ ID NOs: 601 and 602; SEQ ID NOs: 603 and 604; SEQ ID NOs: 605 and 606; SEQ ID NOs: 607 and 608; SEQ ID NOs: 609 and 610; SEQ ID NOs: 611 and 612; SEQ ID NOs: 613 and 614; SEQ ID NOs: 615 and 616; SEQ ID NOs: 617 and 618; SEQ ID NOs: 619 and 620; SEQ ID NOs: 621 and 622; SEQ ID NOs: 623 and 624; SEQ ID NOs: 625 and 626; SEQ ID NOs: 627 and 628; SEQ ID NOs: 629 and 630; SEQ ID NOs: 631 and 632; SEQ ID NOs: 631 and 632; SEQ ID NOs: 633 and 634; SEQ ID NOs: 635 and 636; SEQ ID NOs: 637 and 638; SEQ ID NOs: 639 and 640; SEQ ID NOs: 641 and 642; SEQ ID NOs: 643 and 644; SEQ ID NOs: 645 and 646; SEQ ID NOs: 647 and 648; SEQ ID NOs: 649 and 650; SEQ ID NOs: 651 and 652; SEQ ID NOs: 653 and 654; SEQ ID NOs: 655 and 656; SEQ ID NOs: 657 and 658; SEQ ID NOs: 659 and 660;SEQ ID NOs: 661 and 662; SEQ ID NOs: 663 and 664; SEQ ID NOs: 665 and 666; SEQ ID NOs: 667 and 668; SEQ ID NOs: 669 and 670; SEQ ID NOs: 671 and 672; SEQ ID NOs: 673 and 674; SEQ ID NOs: 675 and 676; SEQ ID NOs: 677 and 678; SEQ ID NOs: 679 and 680; SEQ ID NOs: 681 and 682; SEQ ID NOs: 683 and 684; SEQ ID NOs: 685 and 686; SEQ ID NOs: 687 and 688; SEQ ID NOs: 689 and 690; SEQ ID NOs: 691 and 692; SEQ ID NOs: 693 and 694; SEQ ID NOs: 695 and 696; SEQ ID NOs: 697 and 698; SEQ ID NOs: 699 and 700; SEQ ID NOs: SEQ ID NOs: 701 and 702; SEQ ID NOs: 703 and 704; SEQ ID NOs: 705 and 706; SEQ ID NOs: 707 and 708; SEQ ID NOs: 709 and 710; SEQ ID NOs: 711 and 712; SEQ ID NOs: 713 and 714; SEQ ID NOs: 715 and 716; SEQ ID NOs: 717 and 718; SEQ ID NOs: 719 and 720; SEQ ID NOs: 721 and 722; SEQ ID NOs: 723 and 724; SEQ ID NOs: 725 and 726; SEQ ID NOs: 727 and 728; SEQ ID NOs: 729 and 730; SEQ ID NOs: 731 and 732; SEQ ID NOs: 733 and 734; SEQ ID NOs: 735 and 736; SEQ ID NOs: 737 and 738; SEQ ID NOs: 739 and 740; SEQ ID NOs: 741 and 742; SEQ ID NOs: 741 and 742; SEQ ID NOs: 743 and 744; SEQ ID NOs: 745 and 746; SEQ ID NOs: 747 and 748; SEQ ID NOs: 749 and 750; SEQ ID NOs: 751 and 752; SEQ ID NOs: 753 and 754; SEQ ID NOs: 755 and 756; SEQ ID NOs: 757 and 758; SEQ ID NOs: 759 and 760; SEQ ID NOs: 761 and 762; SEQ ID NOs: 763 and 764; SEQ ID NOs: 765 and 766; SEQ ID NOs: 767 and 768; SEQ ID NOs: 769 and 770;SEQ ID NOs: 771 and 772; SEQ ID NOs: 773 and 774; SEQ ID NOs: 775 and 776; SEQ ID NOs: 777 and 778; SEQ ID NOs: 779 and 780; SEQ ID NOs: 781 and 782; SEQ ID NOs: 783 and 784; SEQ ID NOs: 785 and 786; SEQ ID NOs: 787 and 788; SEQ ID NOs: 789 and 790; SEQ ID NOs: 791 and 792; SEQ ID NOs: 793 and 794; SEQ ID NOs: 795 and 796; SEQ ID NOs: 797 and 798; SEQ ID NOs: 799 and 800; SEQ ID NOs: 801 and 802; SEQ ID NOs: 803 and 804; SEQ ID NOs: 805 and 806; SEQ ID NOs: 807 and 808; SEQ ID NOs: 809 and 810; SEQ ID NOs: SEQ ID NOs: 811 and 812; SEQ ID NOs: 813 and 814; SEQ ID NOs: 815 and 816; SEQ ID NOs: 817 and 818; SEQ ID NOs: 819 and 820; SEQ ID NOs: 821 and 822; SEQ ID NOs: 823 and 824; SEQ ID NOs: 825 and 826; SEQ ID NOs: 827 and 828; SEQ ID NOs: 829 and 830; SEQ ID NOs: 831 and 832; SEQ ID NOs: 833 and 834; SEQ ID NOs: 835 and 836; SEQ ID NOs: 837 and 838; SEQ ID NOs: 839 and 840; SEQ ID NOs: 841 and 842; SEQ ID NOs: 843 and 844; SEQ ID NOs: 845 and 846; SEQ ID NOs: 847 and 848; SEQ ID NOs: 849 and 850; SEQ ID NOs: 851 and 852; SEQ ID NOs: 851 and 852; SEQ ID NOs: 853 and 854; SEQ ID NOs: 855 and 856; SEQ ID NOs: 857 and 858; SEQ ID NOs: 859 and 860; SEQ ID NOs: 861 and 862; SEQ ID NOs: 863 and 864; SEQ ID NOs: 865 and 866; SEQ ID NOs: 867 and 868; SEQ ID NOs: 869 and 870; SEQ ID NOs: 871 and 872; SEQ ID NOs: 873 and 874; SEQ ID NOs: 875 and 876; SEQ ID NOs: 877 and 878; SEQ ID NOs: 879 and 880;SEQ ID NOs: 881 and 882; SEQ ID NOs: 883 and 884; SEQ ID NOs: 885 and 886; SEQ ID NOs: 887 and 888; SEQ ID NOs: 889 and 890; SEQ ID NOs: 891 and 892; SEQ ID NOs: 893 and 894; SEQ ID NOs: 895 and 896; SEQ ID NOs: 897 and 898; SEQ ID NOs: 899 and 900; SEQ ID NOs: 901 and 902; SEQ ID NOs: 903 and 904; SEQ ID NOs: 905 and 906; SEQ ID NOs: 907 and 908; SEQ ID NOs: 909 and 910; SEQ ID NOs: 911 and 912; SEQ ID NOs: 913 and 914; SEQ ID NOs: 915 and 916; SEQ ID NOs: 917 and 918; SEQ ID NOs: 919 and 920; SEQ ID NOs: SEQ ID NOs: 921 and 922; SEQ ID NOs: 923 and 924; SEQ ID NOs: 925 and 926; SEQ ID NOs: 927 and 928; SEQ ID NOs: 929 and 930; SEQ ID NOs: 931 and 932; SEQ ID NOs: 933 and 934; SEQ ID NOs: 935 and 936; SEQ ID NOs: 937 and 938; SEQ ID NOs: 939 and 940; SEQ ID NOs: 941 and 942; SEQ ID NOs: 943 and 944; SEQ ID NOs: 945 and 946; SEQ ID NOs: 947 and 948; SEQ ID NOs: 949 and 950; SEQ ID NOs: 951 and 952; SEQ ID NOs: 953 and 954; SEQ ID NOs: 955 and 956; SEQ ID NOs: 957 and 958; SEQ ID NOs: 959 and 960; SEQ ID NOs: 961 and 962; NO:961 and 962; SEQ ID NO:963 and 964; SEQ ID NO:965 and 966; SEQ ID NO:967 and 968; SEQ ID NO:969 and 970; SEQ ID NO:971 and 972; SEQ ID NO:973 and 974; SEQ ID NO:1759 and 1760; SEQ ID NO:1761 and 1762; SEQ ID NO:1763 and 1764; SEQ ID NO:1765 and 1766; SEQ ID NO:1767 and 1768; SEQ ID NO:1769 and 1770; SEQ ID NO:1771 and 1772;SEQ ID NOs: 1773 and 1774; SEQ ID NOs: 1775 and 1776; SEQ ID NOs: 1777 and 1778; SEQ ID NOs: 1779 and 1780; SEQ ID NOs: 1781 and 1782; SEQ ID NOs: 1783 and 1784; SEQ ID NOs: 1785 and 1786; SEQ ID NOs: 1787 and 1788; SEQ ID NOs: 1789 and 1790; SEQ ID NOs: 1791 and 1792; SEQ ID NOs: 1793 and 1794; SEQ ID NOs: 1795 and 1796; SEQ ID NOs: 1797 and 1798; SEQ ID NOs: 1799 and 1800; SEQ ID NOs: 1801 and 1802; SEQ ID NOs: 1803 and 1804; SEQ ID NOs: 1805 and 1806; SEQ ID SEQ ID NOs: 1807 and 1808; SEQ ID NOs: 1809 and 1810; SEQ ID NOs: 1811 and 1812; SEQ ID NOs: 1813 and 1814; SEQ ID NOs: 1815 and 1816; SEQ ID NOs: 1817 and 1818; SEQ ID NOs: 1819 and 1820; SEQ ID NOs: 1821 and 1822; SEQ ID NOs: 1823 and 1824; SEQ ID NOs: 1825 and 1826; SEQ ID NOs: 1827 and 1828; SEQ ID NOs: 1829 and 1830; SEQ ID NOs: 1831 and 1832; SEQ ID NOs: 1833 and 1834; SEQ ID NOs: 1835 and 1836; SEQ ID NOs: 1837 and 1838; SEQ ID NOs: 1839 and 1840; SEQ ID NOs: 1840 and 1841; NO: 1841 and 1842; SEQ ID NO: 1843 and 1844; SEQ ID NO: 1845 and 1846; SEQ ID NO: 1847 and 1848; SEQ ID NO: 1849 and 1850; SEQ ID NO: 1851 and 1852; SEQ ID NO: 1853 and 1854; SEQ ID NO: 1855 and 1856; SEQ ID NO: 1857 and 1858; SEQ ID NO: 1859 and 1860; SEQ ID NO: 1861 and 1862; SEQ ID NO: 1863 and 1864; SEQ ID NO: 1865 and 1866; SEQ ID NO: 1867 and 1868; SEQ ID NO: 1869 and 1870; SEQ ID NO: 1871 and 1872;Alternatively, SEQ ID NOs: 1873 and 1874.;

[0185] In some embodiments, the double-stranded RNA comprises the sense and antisense strands of any duplex selected from IN-001 to IN-488 and INI-001 to INI-308. In some embodiments, the double-stranded RNA comprises the sense and antisense strands of any duplex selected from IN-489 to IN-546.

[0186] In some embodiments, the antisense oligonucleotides of the present disclosure are substantially complementary to a target mRNA (e.g., INHBE mRNA) and comprise a contiguous nucleotide sequence that is at least about 85% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, to any one of the sense strand oligonucleotides or portions of the sense strand oligonucleotides provided herein, based on the entire length.

[0187] In some embodiments, the antisense oligonucleotides of the present disclosure are substantially complementary to any of the sense strand oligonucleotides provided herein and comprise a contiguous nucleotide sequence that is at least about 85% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, to any of the sense strand oligonucleotides or portions of the sense strand oligonucleotides provided herein, based on the entire length.

[0188] In some embodiments, the dsRNA of the present disclosure comprises a sense strand that is substantially complementary to an antisense oligonucleotide that is complementary to a target mRNA, e.g., INHBE mRNA, wherein the sense strand is at least about 85% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, to any one of the antisense oligonucleotides or portions of the antisense oligonucleotides based on their entire length.

[0189] In some embodiments, the double-stranded region of the dsRNA is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotide pairs in length.

[0190] In some embodiments, the antisense strand of the dsRNA is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length.

[0191] In some embodiments, the sense strand of the dsRNA is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length.

[0192] In some embodiments, the sense and antisense strands of the dsRNA are independently 15 to 30 nucleotides in length.

[0193] In some embodiments, the sense and antisense strands of the dsRNA are independently 19 to 25 nucleotides in length.

[0194] In some embodiments, the sense and antisense strands of the dsRNA are independently 21 to 23 nucleotides in length.

[0195] In some embodiments, the sense strand of the dsRNA is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, wherein the strands form a double-stranded region of 21 consecutive base pairs with a 2 nucleotide-long single-stranded overhang at the 3'-end.

[0196] On the other hand, the present disclosure provides an RNAi agent (e.g., a dsRNAi agent) comprising any of the aforementioned dsRNAs and optionally comprising a targeting ligand. The targeting ligand is typically conjugated to the dsRNA and serves to target the RNAi agent to cells.

[0197] In some embodiments, the double-stranded RNAi agent comprises any of the aforementioned dsRNAs and further comprises at least one targeting ligand, the sense strand of the dsRNA being conjugated to the targeting ligand. In some preferred embodiments, the 3' end of the sense strand is conjugated to the targeting ligand.

[0198] In some embodiments, the targeting ligand of the present disclosure is specifically targeted to the asialoglycoprotein receptors (ASGPR) on the surface of hepatocytes. Preferably, the targeting ligand comprises N-acetyl-galactosamine (GalNAc), or the targeting ligand is a GalNAc derivative. More preferably, the targeting ligand is any targeting ligand disclosed in WO2022266753A1 (targeting moiety). Unless otherwise clearly contradictory, WO2022266753A1 is incorporated herein by reference in its entirety.

[0199] In some embodiments, the structure of the double-stranded RNAi agent is selected from Formula 1 to Formula 33, wherein R 2is the dsRNA. According to common knowledge in the art, R 2 The dsRNA agent is conjugated to a targeting ligand via the 3' end or the 5' end of the sense strand; preferably, the dsRNA agent is conjugated to the targeting ligand via the 3' end of the sense strand.

[0200] Table A1. Structure of the double-stranded RNAi agent of the present application

[0201] In another aspect, the present disclosure also provides a cell, a vector, a host cell, and a pharmaceutical composition comprising the double-stranded RNAi agent of the present disclosure.

[0202] In some embodiments, the pharmaceutical composition comprises any of the aforementioned double-stranded RNAi agents or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present disclosure can be used to prevent and / or treat various corresponding diseases or conditions, as further described below.

[0203] In some embodiments, the pharmaceutical composition is formulated for administration by injection or infusion, e.g., for intravenous, subcutaneous, intraperitoneal, or intramuscular administration. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0204] In some embodiments, the carrier of the pharmaceutical composition is a non-buffered solution or a buffered solution. Typical non-buffered solutions are saline or water, and buffered solutions include one or more of acetate, citrate, prolamin, carbonate, and phosphate. A preferred buffered solution is phosphate-buffered saline (PBS).

[0205] In another aspect of the present disclosure, methods for inhibiting INHBE expression in cells are also provided. These methods comprise: contacting the cells with a double-stranded RNAi agent or RNA of the present disclosure to degrade the mRNA transcript of the INHBE gene, thereby inhibiting the expression of the INHBE gene in the cells.

[0206] In some embodiments, the cell is in a subject. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is an adipocyte. In some embodiments, the subject is a human.

[0207] In some embodiments, INHBE expression is inhibited by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0208] In another aspect, the present disclosure also provides a method for treating a subject having a condition mediated by INHBE expression (e.g., an INHBE-related disease or condition), comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent of the present disclosure, thereby inhibiting the expression of the INHBE gene in the cell.

[0209] In some embodiments, the subject is a human.

[0210] In some embodiments, the subject has a metabolic disorder.

[0211] In some embodiments, the metabolic disorder is metabolic syndrome, type 2 diabetes, obesity, prediabetes, elevated triglyceride levels, lipodystrophy, liver inflammation, fatty liver, hypercholesterolemia, elevated liver enzymes, non-alcoholic steatohepatitis, cardiovascular disease, kidney disease. The metabolic syndrome includes, but is not limited to, one or more of abdominal obesity, insulin resistance, hypertension, dyslipidemia, and hyperlipidemia.

[0212] In some embodiments, the expression of the INHBE gene in the cell is inhibited so that the protein level of the INHBE gene in the serum of the subject is reduced by at least 50%, 60%, 70%, 80%, 90% or 95% compared to before administration of the dsRNAi agent.

[0213] In some embodiments, the double-stranded RNAi agent is administered to a subject at a dose of about 0.10 mg / kg to about 50 mg / kg, for example, at a dose of about 0.01 mg / kg to about 10 mg / kg (e.g., 3 mg / kg, 5 mg / kg, or 9 mg / kg), about 0.5 mg / kg to about 50 mg / kg, about 10 mg / kg to about 30 mg / kg, about 10 mg / kg to about 20 mg / kg, about 15 mg / kg to about 20 mg / kg, about 15 mg / kg to about 25 mg / kg, about 15 mg / kg to about 30 mg / kg, or about 20 mg / kg to about 30 mg / kg.

[0214] In some embodiments, the method further comprises determining the level of INHBE in a sample from the subject. In some embodiments, the level of INHBE in a sample from the subject is determined before, during, and / or after administration of the dsRNAi agent to the subject. Any suitable sample can be used, such as, but not limited to, a blood sample, a serum sample, or a liver tissue sample.

[0215] In some embodiments, the method further comprises administering to the subject an additional therapeutic agent to treat the metabolic disorder, the therapeutic agent including but not limited to insulin, glucagon-like peptide 1 (GLP-1) agonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists, glucagon receptor agonists, sulfonylureas, seglininides, biguanides, thiazolidinediones, α-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, statins, and combinations of any of the foregoing.

[0216] In some embodiments, the double-stranded RNAi agent of the present disclosure can be administered simultaneously or sequentially with another therapeutic agent. In some embodiments, the double-stranded RNAi agent is administered before or after administration of another therapeutic agent, such as a standard therapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0217] Figures 1-5b show the results of detecting the knockdown activity of dsRNA against human INHBE in HEP3B using rt-PCR (full curve).

[0218] FIG6 shows the results of detecting the knockdown activity of dsRNA against human and mouse INHBE (single spot) using a reporter gene assay.

[0219] Figures 7-8 show the results of detecting the knockdown activity of dsRNA against human INHBE (3 points) using a reporter gene assay.

[0220] FIG9 shows the results of detecting the knockdown activity of dsRNA against mouse INHBE using a reporter gene assay (3 points).

[0221] Figures 10 to 15 show the results of detecting the knockdown activity of dsRNA against human INHBE (full curve) using a reporter gene assay.

[0222] FIG16 shows the results of detecting the knockdown activity of dsRNA against mouse INHBE using a reporter gene assay (full curve).

[0223] Figures 17-19 and 21 show the results of detecting the knockdown activity of dsRNA against human INHBE (3 points) using a reporter gene assay.

[0224] FIG20 and FIG22 show the results of detecting the knockdown activity of dsRNA against human INHBE (2 points) using a reporter gene method.

[0225] FIG23 and FIG24 show the results of detecting the knockdown activity of dsRNA against human INHBE (full curve) using a reporter gene assay.

[0226] FIG25 shows the results of siRNA stability test in serum.

[0227] FIG26 shows the results of siRNA stability test in human liver S9.

[0228] FIG27 shows the changes in siRNA content in the liver of mice in each group (3 mice) after administration at different time points.

[0229] FIG28 shows the percentage of different siRNAs in mouse liver after normalization based on the content of each RNAi in the liver tissue of 3 mice 7 days after administration. DETAILED DESCRIPTION

[0230] The present disclosure provides RNAi agents and compositions that trigger RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the target gene inhibin subunit beta E (INHBE). The gene can be in cells, such as adipocytes and / or hepatocytes or other liver cells (or liver cells), such as cells in a subject (e.g., a human). The use of these RNAi agents and compositions enables targeted degradation of INHBE mRNA in mammals. As inhibitors of INHBE expression, the RNAi agents and compositions of the present disclosure can be used to prevent, treat, and / or inhibit INHBE-related diseases or conditions, such as metabolic syndrome and related conditions.

[0231] Thus, the present disclosure provides methods for treating, preventing, or inhibiting INHBE-related diseases or conditions, such as, but not limited to, metabolic disorders, e.g., metabolic syndrome; carbohydrate disorders, e.g., type 2 diabetes, prediabetes; lipid metabolism disorders, e.g., hyperlipidemia, hypertension, lipodystrophy; kidney disease; cardiovascular disease; or, weight disorders, e.g., obesity, overweight, using RNAi compositions that trigger RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of inhibin subunit beta E (INHBE) target genes.

[0232] The RNAi agents of the present disclosure comprise an antisense RNA strand having a region of up to about 30 nucleotides in length, e.g., 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23 , 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 21-22, 15, 15-16, 15-17, 15-20, 15-21, 15-22, 15-23, 15-24, or at least 15 nucleotides, said region being substantially complementary to at least a portion of a transcript mRNA of an INHBE target gene.

[0233] In certain embodiments, one or both strands of a double-stranded RNAi agent of the present disclosure are up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides, and have a region of at least 15 consecutive nucleotides that is substantially complementary to at least a portion of the transcript mRNA of the INHBE target gene. In some embodiments, these RNAi agents with longer antisense strands can, for example, include a second RNA strand (sense strand) of 20-60 nucleotides in length, wherein the sense and antisense strands form a double-stranded region (duplex) of 15-30 consecutive nucleotides.

[0234] The use of RNAi agents disclosed herein enables targeted degradation of INHBE mRNA in mammals. The inventors have demonstrated that RNAi agents disclosed herein can trigger RNA-induced silencing complex (RISC)-mediated cleavage of INHBE RNA transcripts, thereby resulting in significant inhibition of INHBE target gene expression. In certain embodiments, the RNAi agents disclosed herein are more effective (e.g., more potent) and / or more specific (e.g., safer, with fewer off-target effects) than previous RNAi agents targeting the same gene. In certain embodiments, RNAi agents are targeted to specific sites in INHBE mRNA by selecting a specific site, and / or include RNA modifications (e.g., non-canonical base-pairing nucleotides, modified nucleotides, chemical modifications) to increase efficacy, effectiveness, specificity, and / or safety. In some such embodiments, the RNAi agent comprises at least one modified nucleotide, such as a non-canonical base-pairing nucleotide. Methods and compositions comprising these RNAi agents can be used to treat subjects suffering from INHBE-related diseases or conditions, such as metabolic disorders. Thus, the present disclosure provides methods for treating, preventing, or inhibiting metabolic disorders in a subject who would benefit from inhibiting or reducing INHBE expression using the RNAi agents and compositions of the present disclosure.

[0235] The present disclosure also provides methods for preventing at least one symptom in a subject suffering from a condition that would benefit from inhibition or reduction of INHBE expression. The following detailed description will disclose how to prepare and use RNAi agents and compositions thereof to inhibit expression of INHBE target genes, as well as compositions, uses, and methods for treating subjects who would benefit from inhibition and / or reduction of INHBE target gene expression, such as subjects susceptible to or diagnosed with a metabolic disorder.

[0236] definition

[0237] In order to provide a clear and consistent understanding of the terms used in the specification of the present invention, some definitions are provided below. In addition, unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0238] When used in conjunction with the term "comprising" in the claims and / or the specification, the use of the word "a" can mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." Similarly, the word "another" can mean at least a second or more.

[0239] The term "or" is used herein to mean the term "and / or" and can be used interchangeably with the term "and / or" unless the context clearly indicates otherwise. For example, "sense strand or antisense strand" is understood to mean "sense strand or antisense strand, or sense strand and antisense strand."

[0240] As used in this specification and claims, the words "comprising" (and any forms of including, such as "comprises" and "including"), "having" (and any forms of having, such as "have" and "having"), "including" (and any forms of including, such as "including" and "including"), and "comprising" (and any forms of including, such as "containing" and "including"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0241] The term "about" or "approximately" is used to indicate that the value includes the error introduced by the instruments and methods used in determining the value. The term "about" when used in conjunction with a numerical value is intended to encompass numerical values ​​within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as such variations are appropriate for performing the disclosed methods. When "about" precedes a series of numbers or a range, it should be understood that "about" can modify each number in the series or range.

[0242] The terms "at least," "not less than," or "or more" preceding a number or a range of numbers should be understood to include the number adjacent to the term "at least," as well as all subsequent numbers or integers that can logically be included as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 15 nucleotides in a 17-nucleotide nucleic acid molecule" means 15, 16, or 17 nucleotides having the specified properties. When "at least" appears before a series of numbers or a range, it should be understood that "at least" can modify each number in the series or range.

[0243] As used herein, "not more than" or "or less" is understood to refer to the value adjacent to the phrase and logically lower values ​​or integers, as is logical from the context, to zero. For example, a duplex having an overhang of "not more than 2 nucleotides" has an overhang of 2, 1, or 0 nucleotides. When "not more than" appears before a series of numbers or a range, it is understood that "not more than" can modify each number in the series or range. As used herein, a range includes an upper limit and a lower limit.

[0244] The terms "inhibin Beta E" and "INHBE" are used interchangeably and are also referred to as "inhibin subunit Beta E chain," "inhibin Beta E," "inhibin βE," "activin βE," "activin Beta E," and "MGC4638." The sequence of human INHBE mRNA transcripts can be found, for example, in GenBank Accession No. NM_031479.5. The sequence of mouse INHBE mRNA can be found, for example, in GenBank Accession No. NM_008382.3 (SEQ ID NO: 1875). The predicted sequence of cynomolgus monkey INHBE mRNA can be found, for example, in GenBank Accession No. XM_005571319.3. Other examples of INHBE mRNA sequences are readily available through public databases, such as GenBank, UniProt, OMIM, and the Macaca Genome Project website. For more information on INHBE, for example, visit www.ncbi.nlm.nih.gov / gene / ?term=INHBE. As of the date of filing this application, each of the above-mentioned GenBank accession numbers and gene database numbers is incorporated herein by reference in its entirety.

[0245] The term "target sequence" or "target nucleic acid" or "target mRNA" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene, including mRNA that is a product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence is at least long enough to serve as a substrate for RNAi-directed cleavage at or near the nucleotide sequence portion of the mRNA molecule formed during the transcription of the target gene. In one embodiment, the target sequence is located within the protein coding region of the target gene. In another embodiment, the target sequence is located within the 3'UTR of the target gene. The target nucleic acid can be a cellular gene (or an mRNA transcribed from the gene) whose expression is associated with a specific condition or disease state. The target sequence can be about 19-36 nucleotides in length, e.g., about 19-30 nucleotides in length, e.g., 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In certain embodiments, the target sequence is 15-30 nucleotides in length, e.g., 15-23 nucleotides in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 17-30, 17-29, 18-28, 17-27, 16-26, 15-25, 15-24, 16-23, 16-22, 16-21, 16-30, 16-29, 16-28, 16-27, 16-26, 16-25, 16-24, 17-25, 17-23, or 15-17 nucleotides in length. In certain embodiments, the target sequence is 17-25 nucleotides, 19-21 nucleotides, 19-23 nucleotides, or 21-23 nucleotides in length. Ranges and lengths intermediate to the above ranges and lengths are also considered part of this disclosure.

[0246] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by the sequence referred to using standard nucleotide nomenclature.

[0247] The terms "siRNA," "RNAi agent," "siRNA agent," and "RNA interference agent" are used interchangeably herein and refer to biologically active agents that contain RNA and mediate targeted cleavage of RNA transcripts through an RNA-induced silencing complex (RISC) pathway. RNAi agents direct the specific degradation of mRNA sequences through a process known as RNA interference. RNAi agents modulate (e.g., inhibit) the expression of genes in cells, such as cells in a subject (e.g., a mammalian subject, such as a human). In some embodiments, the RNAi agent used in the compositions, uses, and methods of the present disclosure comprises a double-stranded RNA (dsRNA) or duplex of the present disclosure and may be referred to herein as a "double-stranded RNAi agent," "dsRNAi agent," or "dsRNA agent."

[0248] In certain embodiments, the dsRNAi agent of the present invention includes a double-stranded RNA agent, which, when introduced into a cell, is processed into short interfering RNA by a nuclease called Dicer. Short interfering RNA is integrated into RISC, and one or more helicases unwind the RNA duplex, allowing the complementary antisense strand to guide target recognition. After binding to the target mRNA, one or more nucleases in RISC will cut the target mRNA to induce silencing. Therefore, in other embodiments, the siRNA agent relates to single-stranded RNA produced in the cell and promotes the formation of the RISC complex to achieve silencing of the target gene. In some such embodiments, the RNAi agent is a single-stranded siRNA (ssRNAi), which can be introduced into a cell or organism to inhibit the target mRNA. The single-stranded RNAi agent binds to the RISC nuclease, Argonaute 2, and then cuts the target mRNA. The ssRNAi agent is generally 15-30 nucleotides in length and can be chemically modified. Any antisense oligonucleotide described herein can be used as the ssRNAi agent described herein. In some embodiments, the ssRNAi agent comprises at least one non-canonical base-pairing nucleotide. In some embodiments, the ssRNAi agent comprises at least one modified nucleotide.

[0249] The term "double-stranded RNA" or "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid chains, with "sense" (or "justice") and "antisense" orientations relative to the target RNA. In some embodiments of the present disclosure, dsRNA triggers the degradation of a target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi. Generally speaking, the majority of the nucleotides in each chain of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both chains may also include one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Each chain of a dsRNA molecule may have a length ranging from 12 to 40 nucleotides. For example, each strand can be between 14-40 nucleotides in length, 17-37 nucleotides in length, 25-37 nucleotides in length, 17-25 nucleotides in length, 17-22 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, or 21-23 nucleotides in length, and the sense and antisense strands can be equal or unequal lengths without limitation.

[0250] The term "antisense strand" refers to a strand of an iRNA (e.g., dsRNA) that includes a region that is substantially complementary to a target sequence (e.g., INHBE mRNA). As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence. In cases where the complementary region is not completely complementary to the target sequence, there may be mismatches within the interior or terminal regions of the molecule. Typically, the most tolerated mismatches are within the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5'- and / or 3'-ends of the dsRNA. The antisense and sense strands of a dsRNA may have the same or different lengths, as are known in the art.

[0251] When a first sequence is referred to as being "substantially complementary" to a second sequence, the two sequences can be fully complementary (i.e., complementary over the entire length of one or both nucleotide sequences), or they can form one or more, but generally no more than 5, 4, 3, or 2, mismatched base pairs upon hybridization over a distance of up to 30 base pairs, while retaining the ability to hybridize under appropriate conditions (conditions relevant to their application, such as inhibition of gene expression, such as physiological conditions). It should be noted that when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches for purposes of determining complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, wherein the longer oligonucleotide comprises a 21-nucleotide sequence that is fully complementary to the shorter oligonucleotide, can be referred to as "fully complementary" for the purposes described herein.

[0252] The term "sense strand" refers to the strand of a dsRNA that comprises a region that is substantially complementary to a region of the antisense strand.

[0253] As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence relative to a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions. Such conditions can, for example, be stringent conditions, wherein stringent conditions can include: 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours. Other conditions, such as physiologically relevant conditions that may be encountered in an organism, may also be applicable. For example, complementary sequences are sufficient to enable the relevant function of the nucleic acid, such as RNAi. A skilled person can determine the set of conditions that is most suitable for testing the complementarity of two sequences based on the ultimate use of the hybridizing nucleotides.

[0254] The terms "complementary," "fully complementary," and "substantially complementary" as used herein may be used to describe base matching between the sense and antisense strands of a dsRNA, or between two oligonucleotides or polynucleotides, such as the antisense strand of a dsRNA agent and a target sequence, and their meanings will be understood from the context in which the terms are used.

[0255] The term "melting temperature" or "Tm" is used herein to refer to the temperature at which 50% of double-stranded RNA (dsRNA) molecules open or denature (i.e., 50% of the double-stranded RNA molecules are separated into single strands and 50% of the complementary oligonucleotide chains do not hybridize to each other). For a given oligonucleotide, its corresponding Tm value can be calculated using any acceptable formula or software known in the art. For example, but not limited to, the OligoAnalyzer from Integrated DNA Technologies (IDT) (Coralville, Iowa, USA) can be used. TMTools are used to calculate Tm; Tm calculation tools on the website http: / / insilico.ehu.es / tm.php?formula=basic are used, etc. The term "ΔTm" refers to the Tm (e.g., calculated Tm) difference between two different oligonucleotides (e.g., an unmodified oligonucleotide and an oligonucleotide comprising one or more modified nucleotides having the same sequence). In certain embodiments, ΔTm is used to refer to the difference in melting temperature between two dsRNA regions or duplexes of the present disclosure, wherein one of the dsRNA regions or duplexes comprises a nucleotide substitution by at least one modified nucleotide (e.g., a non-canonical base-paired nucleotide). In some embodiments, ΔTm is used to refer to the difference in melting temperature between two oligonucleotides (e.g., two antisense strands, two sense strands), wherein one of the oligonucleotides comprises at least one nucleotide substituted by a non-canonical base-paired nucleotide.

[0256] "G", "C", "A", "T" and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymine (also known as 5-methyluracil) and uracil as bases. However, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in further detail below. G, C, A, T and U are referred to as "standard" nucleotides in this article, and the definition of standard nucleotides is as shown in Table A2. Accordingly, guanine, cytosine, adenine, thymine and uracil are referred to as "standard" bases in this article, which are the common bases for RNA or DNA construction. In this article, when only stating or describing for base groups or base parts and without ambiguity, "G" can also be used to represent guanine, "C" to represent cytosine, "A" to represent adenine, "T" to represent thymine and "U" to represent uracil. For canonical bases or canonical nucleotides, in most cases, A pairs with U (T in DNA) and G pairs with C, following the Watson-Crick base pairing rules (referred to herein as "canonical base pairing").

[0257] Table A2. Definition of canonical nucleotides

[0258] "Non-canonical" base pairing that does not follow the Watson-Crick base pairing rules is also possible. For example, GU wobble base pairing occurs frequently and has a pairing strength (e.g., thermodynamic stability) comparable to Watson-Crick base pairs. Therefore, adenine and cytosine at any position in the nucleotide sequence of the dsRNAi agent of the present disclosure can be replaced by guanine and uracil, respectively, to form GU wobble base pairing with the target sequence.

[0259] Certain modified nucleotides also exhibit non-canonical base pairing. Such modified nucleotides have base pairing characteristics different from canonical nucleotides. For example, a modified nucleotide (e.g., inosinic acid) with hypoxanthine as a base can be base paired with a nucleotide containing adenine, cytosine, or uracil. Therefore, in the nucleotide sequence of the dsRNAi agent of the present disclosure, a nucleotide containing uracil, guanine, or adenine can be replaced by a modified nucleotide (e.g., inosinic acid) containing the base hypoxanthine. In certain embodiments, one or more canonical nucleotides in the dsRNAi agent of the present disclosure are replaced by modified nucleotides with different base pairing characteristics; such replacement parts are referred to herein as "non-canonical base pairing nucleotides". The non-canonical base pairing nucleotides of the present disclosure include nucleotides capable of non-Watson-Crick or wobble base pairing and / or modified nucleotides with different base pairing characteristics compared to the canonical nucleotides they replace. Sequences containing such non-canonical base pairing nucleotides are suitable for RNAi agents, compositions, and methods of the present disclosure.

[0260] It should be understood that non-canonical base pairing nucleotides (e.g., modified nucleotides having base pairing characteristics different from the canonical nucleotides they replace) can differ not only in the base pairs they form, but also in the strength or stability of the base pairing. Non-canonical base pairing may be stronger or weaker than canonical base pairing. For example, m1Ψ (modified from U) pairs stronger with A than U pairs with A, and m1Ψ-G pairing is even stronger than m1Ψ-A pairing. Therefore, by replacing canonical nucleotides with non-canonical base pairing nucleotides, the base pairing strength can be changed, thereby changing the Tm of the antisense strand duplex and / or the Tm of hybridization between the antisense strand and the target RNA (e.g., INHBE mRNA). Therefore, in some embodiments, canonical nucleotides are replaced by non-canonical base pairing nucleotides, thereby changing the Tm of the oligonucleotide (e.g., changing the calculated Tm of the oligonucleotide, changing the Tm of the resulting double-stranded RNA molecule, such as the Tm of the duplex formed by the antisense strand hybridized with the target mRNA and / or the sense strand). Without wishing to be bound by theory, by changing the Tm of the oligonucleotide by replacing at least one canonical nucleotide with non-canonical base pairing nucleotides, the efficacy, effectiveness, specificity, safety and / or off-target effects of dsRNAi can be regulated and controlled. For example, but not limited to, the efficacy or effectiveness of the dsRNAi agent can be increased by increasing the pairing strength with the desired target mRNA and / or reducing the pairing strength with off-target mRNA. Similarly, the undesirable off-target effects can be reduced by increasing the pairing strength with the desired target mRNA and / or reducing the pairing strength with off-target mRNA. Therefore, in some embodiments, compared with similar RNAi agents that do not comprise at least one non-canonical base pairing nucleotides, RNAi agents of the present disclosure have improved efficacy, effectiveness, specificity and / or safety.

[0261] In certain embodiments of the dsRNAi agents, antisense strands and sense strands of the present disclosure, at least one canonical nucleotide is replaced by a non-canonical base pairing nucleotide. In some such embodiments, one nucleotide is replaced by a non-canonical base pairing nucleotide, i.e., the dsRNAi agent, antisense strand or sense strand comprises a non-canonical base pairing nucleotide. In some embodiments, two, three, four, five or more nucleotides are replaced by non-canonical base pairing nucleotides, i.e., the dsRNAi agent, antisense strand or sense strand comprises two, three, four, five or more non-canonical base pairing nucleotides. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more nucleotides in the oligonucleotide or dsRNAi agent are modified nucleotides, such as non-canonical base pairing nucleotides. In some embodiments, all nucleotides in a dsRNAi agent, e.g., in the sense strand and / or antisense strand, are modified nucleotides, e.g., non-canonical base-pairing nucleotides. In some embodiments, at least one nucleotide in a dsRNAi agent, e.g., in the sense strand and / or antisense strand, is a modified nucleotide, e.g., non-canonical base-pairing nucleotide.

[0262] Therefore, in certain embodiments, RNAi agent of the present disclosure comprises at least one non-canonical base pairing nucleotide, that is, at least one nucleotide in antisense strand and / or sense strand is replaced by non-canonical base pairing nucleotide.In some such embodiments, at least one non-canonical base pairing nucleotide is present on the antisense strand.In some such embodiments, at least one non-canonical base pairing nucleotide is present on the sense strand.In some such embodiments, at least one non-canonical base pairing nucleotide is present on the antisense strand and sense strand simultaneously.In some embodiments, at least one non-canonical base pairing nucleotide is present in the complementarity region, that is, the region substantially complementary to the target sequence in the oligonucleotide, for example, the region complementary to the target sequence (for example, INHBE mRNA) in the antisense strand of the present disclosure.

[0263] In certain embodiments, replacing at least one canonical nucleotide with a non-canonical base pairing nucleotide changes the melting temperature (Tm) of the oligonucleotide or dsRNA duplex. In some embodiments, the Tm changes by at least 2°C (i.e., the ΔTm is at least about 2°C). In some embodiments, the ΔTm is about 2°C. In some embodiments, the ΔTm exceeds 2°C. In some embodiments, the ΔTm is about 2.5°C, 3°C, 3.5°C, 4°C, 4.5°C, or 5°C.

[0264] In certain embodiments, at least one non-canonical base pairing nucleotide is present at positions 1-11, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or 11, from the 5' to the 3' direction of the antisense strand.

[0265] In certain embodiments, at least one non-canonical base pairing nucleotide is present at positions 12-21, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, and / or 21, of the antisense strand in the 5' to 3' direction.

[0266] In certain embodiments of the oligonucleotides and dsRNAi agents of the present disclosure, in the direction from the 5' end to the 3' end, if at least four nucleotides in positions 2-8 of the antisense strand are A or U and at least one of the nucleotides at positions 6-8 is G, then the G at positions 6, 7 and / or 8 is replaced by a non-canonical base pairing nucleotide.

[0267] In certain embodiments of the oligonucleotides and dsRNAi agents of the present disclosure, in the sequence 5′-N1N2G0N3N4-3′, when at least three bases among N1, N2, N3 and N4 are adenine or uracil, the guanine in G0 is replaced by a non-canonical base, such as hypoxanthine, wherein N1, N2, N3 and N4 are each independently a nucleotide comprising adenine (A), cytosine (C), guanine (G) or uracil (U), and G0 is a nucleotide comprising guanine.

[0268] It should be understood that all non-canonical base pairing nucleotides or non-canonical bases disclosed herein or known in the art are suitable for use in the RNAi agents, compositions and methods of the present disclosure. Non-limiting examples of non-canonical base pairing nucleotides (or nucleosides) or non-canonical bases are defined as shown in Table B. In some embodiments, the non-canonical base pairing nucleosides (or nucleotides) or non-canonical bases are the modifying groups shown in Table B. In some embodiments, the non-canonical base is hypoxanthine. In some embodiments, the non-canonical base pairing nucleoside is inosine. In some embodiments, the non-canonical base pairing nucleotide is a canonical nucleotide capable of wobble pairing. The aforementioned combinations are also encompassed.

[0269] Table B. Examples of non-canonical base-paired nucleosides (or nucleotides) and non-canonical bases according to certain embodiments

[0270] The term "modified nucleotide" refers to any nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage and / or a modified nucleobase. Thus, the term "modified nucleotide" encompasses substitution, addition or removal of, for example, a functional group or atom of an internucleoside linkage, a sugar moiety or a nucleobase. Modifications suitable for use in the present disclosure include all types of modifications disclosed herein or known in the art. In some embodiments, the modified nucleotide is a non-canonical base pairing nucleotide as defined herein, i.e., a nucleotide having a pairing characteristic that is different from the canonical nucleotide that it replaces. In other embodiments, the modified nucleotide may not have different pairing characteristics, but may still have desired or advantageous features of the RNAi agents, compositions and methods disclosed herein, such as stability, resistance to degradation (e.g., nuclease resistance), manufacturability, etc. In certain embodiments, the RNAi agent of the present disclosure comprises at least one modified nucleotide in addition to at least one non-canonical base pairing nucleotide, i.e., in addition to comprising at least one non-canonical base pairing nucleotide, at least one additional nucleotide is replaced by an additional modified nucleotide (which may or may not be a non-canonical base pairing nucleotide) in the antisense strand and / or the sense strand. In some such embodiments, at least one additional modified nucleotide is present on the antisense strand. In some such embodiments, at least one additional modified nucleotide is present on the sense strand. In some such embodiments, at least one additional modified nucleotide is present on both the antisense strand and the sense strand. In some such embodiments, at least one additional modified nucleotide is present on the same strand as the at least one non-canonical base pairing nucleotide. In other embodiments, at least one additional modified nucleotide is not present on the same strand as the at least one non-canonical base pairing nucleotide, i.e., is present on another strand. In some embodiments, at least one additional modified nucleotide is present in a complementary region, i.e., a region in an oligonucleotide that is substantially complementary to a target sequence, e.g., a region in an antisense strand of the present disclosure that is complementary to a target sequence mRNA (e.g., INHBE mRNA).

[0271] In the present disclosure, non-limiting examples of common modified nucleotides and related moieties are defined as shown in Table C. In some embodiments, the modified nucleotide is a modified nucleotide shown in Table C. In some embodiments, at least one additional modified nucleotide of the RNAi agent of the present disclosure is a modified nucleotide shown in Table C.

[0272] Table C. Definitions of modified nucleotides in some embodiments.

[0273] Among them, the modification pattern of inverted nucleotides is also called inverted bases in some literature, which refers to those bases with linkages reversed from the normal 5' to 3' linkages (ie, 5' to 5' linkages or 3' to 3' linkages).

[0274] In some embodiments, the inclusion of deoxynucleotides may be considered to constitute modified nucleotides.

[0275] It should be understood that all types of modifications disclosed herein or known in the art are applicable to the RNAi agents, compositions and methods of the present disclosure.

[0276] The term "derivative" as used in the present disclosure should be understood as another compound that is similar in structure but different in some minor structures.

[0277] The term "inhibit" and similar expressions refer to reducing or effectively stopping, and can be used interchangeably with "reduce", "silence", "downregulate", "suppress" and other similar terms, and include any level of inhibition. As a non-limiting example, "inhibit" herein refers to reducing or effectively reducing the onset or progression of a metabolic disorder or related disease in a subject, including a reduction in one or more aspects of the disease (e.g., symptoms, tissue characteristics, cell activity, inflammatory activity or immune activity, etc.), or no detectable worsening.

[0278] As used herein, "inhibited expression" of a gene (e.g., INHBE) refers to a decrease in the amount or level of RNA transcript (e.g., INHBE mRNA) or protein encoded by the gene and / or a decrease in the amount or level of activity of the gene in a cell, cell population, sample, or subject, as compared to an appropriate reference (e.g., a reference cell, cell population, sample, or subject). As used herein, "inhibiting INHBE expression" refers to a decrease in the amount or level of INHBE mRNA and / or activin E in a cell, cell population, sample, or subject, such as an 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%, compared to an appropriate reference (e.g., a reference cell, cell population, sample, or subject).

[0279] As used herein, the phrase "contacting cells with an RNAi agent" (e.g., dsRNAi agent) includes contacting cells by any possible means. Contacting cells with an RNAi agent includes contacting cells with an RNAi agent in vitro or contacting cells with an RNAi agent in vivo. The contact can be carried out directly or indirectly. Therefore, for example, the RNAi agent can be brought into physical contact with the cell, or alternatively, the RNAi agent can be placed in a situation where it will allow or cause it to subsequently contact the cell. Contacting cells in vitro can be carried out by, for example, incubating the cell with the RNAi agent. Contacting cells in vivo can be carried out, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another region (e.g., bloodstream or subcutaneous space) so that the RNAi agent subsequently reaches the tissue where the cell is located. For example, the RNAi agent can contain or be coupled to a targeting ligand, such as GalNAc, which guides the RNAi agent to a site of interest, such as the liver. In other embodiments, the RNAi agent can contain or be coupled to one or more C22 hydrocarbon chains and one or more GalNAc derivatives. In other embodiments, the RNAi agent contains or is coupled to one or more C22 hydrocarbon chains and does not contain or is not coupled to one or more GalNAc derivatives. A combination of in vitro and in vivo contact methods is also possible. For example, cells can also be contacted with the RNAi agent of the present disclosure in vitro and subsequently transplanted into a subject. In certain embodiments, contacting cells with the RNAi agent includes promoting or influencing the uptake or absorption of cells. The absorption or uptake of the RNAi agent can occur by unassisted diffusion or active cell processes, or by adjuvants or devices. The introduction of RNAi agents into cells can be carried out in vitro or in vivo. For example, for in vivo introduction, the RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction of cells includes methods known in the art, such as electroporation and lipofection.

[0280] "Subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. In certain embodiments, the subject is a human.

[0281] In some embodiments, the "treatment" of any disease or condition refers to improving at least one disease or condition. In certain embodiments, "treatment" refers to improving at least one physical parameter, which may or may not be perceived by the patient. In certain embodiments, "treatment" refers to physically (e.g., stabilization of obvious symptoms), physiologically (e.g., stabilization of physical parameters), or both to suppressing a disease or condition. In some embodiments, "treatment" refers to improving the quality of life of a subject in need or reducing the symptoms or side effects of a disease. A "therapeutically effective amount" refers to the amount of a dsRNA or dsRNAi agent sufficient to achieve the treatment or prevention of a disease when administered to a cell, tissue, or subject alone or in combination with other therapeutic drugs. A "therapeutically effective amount" will vary according to the compound or RNAi agent, the disease and its severity, and the age, weight, etc. of the subject suffering from the disease to be treated or prevented. The term "therapeutically effective amount" as used herein refers to the amount of a compound or composition sufficient to prevent, treat, suppress, reduce, improve, or eliminate one or more causes, symptoms, or complications of a disease or condition, such as metabolic syndrome. The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients, whether administered in combination, sequentially, or simultaneously, that results in a therapeutic effect. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter of at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

[0282] In some embodiments, "prevention" or "preventing" of any disease or condition refers to at least reducing the likelihood of acquiring the risk (or susceptibility) of the disease or condition (i.e., so that at least one clinical symptom of the disease does not appear in a patient who may be exposed to or susceptible to the disease but has not yet experienced or displayed symptoms of the disease). When used with respect to a disease, disorder, or condition that would benefit from a decrease in INHBE expression, it means reducing the likelihood that a subject will develop symptoms associated with the disease, disorder, or condition, such as metabolic syndrome. Failure to develop a disease, disorder, or condition, or a reduction in the development of symptoms associated with the disease, disorder, or condition (e.g., a reduction of at least about 10% on a clinically accepted scale for the disease or condition), or a delay in the onset of symptoms (e.g., a delay of days, weeks, months, or years) is considered effective prevention.

[0283] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0284] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., a lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material (involved in carrying or transporting a compound or dsRNAi agent from one organ or part of the body to another organ or part of the body).

[0285] 2. RNA Interference

[0286] The present disclosure provides RNA (siRNA or RNAi) agents that inhibit the expression of metabolic disorder-related target gene INHBE through RNA interference (RNAi) process. In some embodiments, the RNAi agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of INHBE gene in cells (e.g., adipocytes and / or liver cells, e.g., hepatocytes). The siRNA or RNA agent comprising a dsRNA molecule is also referred to herein as a "dsRNAi" agent. In certain embodiments, the cell is present in a subject. In some embodiments, the subject is a mammal, e.g., a human. In some embodiments, the subject has or is susceptible to metabolic disorders (e.g., metabolic syndrome), carbohydrate disorders (e.g., type 2 diabetes, prediabetes), lipid metabolism disorders (e.g., hyperlipidemia, hypertension, lipodystrophy), kidney disease, cardiovascular disease, and / or weight disorders (e.g., obesity, overweight). The dsRNAi agent comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed in the expression of the INHBE gene. In some embodiments, the length of the complementary region is about 19-30 nucleotides (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length). In some embodiments, the length of the complementary region is about 15-30 nucleotides (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 nucleotides in length). In certain embodiments, the dsRNAi agent comprises at least one modified nucleotide, as described herein. In certain embodiments, the dsRNAi agent comprises at least one non-canonical base pairing nucleotide, as described herein.

[0287] According to the method disclosed herein, cells expressing target genes (e.g., humans, primates, non-primates or mice INHBE genes) are contacted with siRNA agents that inhibit target gene expression. In some embodiments, the expression of the target gene is suppressed by at least about 50%. The suppression of target gene expression can be determined using any suitable method, such as, but not limited to, by methods based on PCR or branched DNA (bDNA), or by protein-based methods, such as by immunofluorescence analysis, using, for example, Western blotting or flow cytometry techniques. In certain embodiments, the suppression of expression is determined by the rt-PCR method provided in the examples herein (e.g., as described in the examples below, using, for example, 10 nM concentration of siRNA in a suitable organism cell line). In certain embodiments, animal models are used to determine the suppression of in vivo expression, such as by knocking down human genes in rodents expressing human genes (e.g., mice expressing INHBE genes). In some such embodiments, siRNA is administered to a subject (e.g., an animal model) in a single dose (e.g., 3 mg / kg, 5 mg / kg, 6 mg / kg or 9 mg / kg).

[0288] dsRNA comprises two RNA chains, which are complementary and hybridize to form a duplex structure under the conditions under which the dsRNA will be used (e.g., under physiological conditions). One chain (the antisense strand) of the dsRNA comprises a complementary region that is substantially complementary to the target sequence and is generally fully complementary. The target sequence can be derived from the mRNA sequence formed during the expression of the INHBE gene. The other chain (the positive strand) comprises a region complementary to the antisense strand so that when the two chains are combined under appropriate conditions, they will hybridize and form a duplex structure. As described elsewhere herein and known in the art, the complementary sequence of the dsRNA can be contained in the self-complementary region of a single nucleic acid molecule, rather than on a separate oligonucleotide. Generally, the duplex structure is 15 to 30 base pairs in length, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30 , 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22 base pairs. In certain embodiments, the length of the duplex structure is 17 to 25 base pairs, for example, 17-23, 17-25, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24, or 24-25 base pairs in length, for example, 19-21 base pairs in length. Ranges and lengths intermediate to the above ranges and lengths are also considered part of this disclosure.

[0289] Similarly, the region complementary to the target sequence is 15 to 30 nucleotides in length, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 21-23 or 21-22 nucleotides in length. Ranges and lengths intermediate to the above ranges and lengths are also considered part of this disclosure.

[0290] In some embodiments, the duplex structure is 19 to 30 base pairs in length. Similarly, the region complementary to the target sequence is 19 to 30 nucleotides in length.

[0291] In some embodiments, the duplex structure is 15 to 23 base pairs in length. Similarly, the region complementary to the target sequence is 15 to 23 nucleotides in length.

[0292] In some embodiments, the dsRNA is about 19 to about 23 nucleotide pairs in length, or about 25 to about 30 nucleotides in length. In some embodiments, the dsRNA is about 15 to about 23 nucleotides in length, or about 17 to about 23 nucleotides in length, or about 17 to about 25 nucleotides in length, or about 19 to about 21 nucleotides in length.

[0293] The duplex region is the major functional portion of the dsRNA, e.g., a duplex region of about 15 to about 30 base pairs, or about 17 to about 30 base pairs, or about 19 to about 30 base pairs, e.g., about 15-23, 15-25, 17-25, 17-23, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-28 In one embodiment, the dsRNA can be an RNA molecule or RNA molecule complex having a duplex region of greater than 30 base pairs, to the extent that the dsRNA can be processed into a functional duplex (e.g., 15-30 base pairs or at least 15 base pairs) that can target the desired RNA for cleavage. Thus, in one embodiment, the dsRNA is a miRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, the siRNA agent useful for targeting INHBE gene expression is not produced in the target cell by cleavage of a larger dsRNA.

[0294] DsRNA as described herein can also include one or more single-stranded nucleotide overhangs, for example 1-4, 2-4, 1-3, 2-3, 1, 2, 3 or 4 nucleotides. The dsRNA with at least one nucleotide overhang has excellent inhibitory properties relative to its blunt-ended counterpart. The nucleotide overhangs can comprise or consist of nucleotides / nucleoside analogs comprising deoxynucleotides / nucleosides. The overhangs can be located on sense strand, antisense strand or its combination in any. In addition, the nucleotides of the overhangs can be present in the 5'-end, 3'-end or both ends of the antisense strand or sense strand of the dsRNA.

[0295] "Flat" or "blunt-ended" means that there are no unpaired nucleotides at the ends of the dsRNA, i.e., there are no nucleotide overhangs. A "blunt-ended" dsRNA is double-stranded over its entire length, i.e., there are no nucleotide overhangs at either end of the molecule. The dsRNAi agents of the present disclosure include dsRNAs that have no nucleotide overhangs at one end (i.e., agents having one overhang and one blunt end) or dsRNAs that have no nucleotide overhangs at either end. In some embodiments, such oligonucleotides are double-stranded over their entire length.

[0296] dsRNA can be synthesized by standard methods known in the art. Double-stranded RNAi compounds of the present disclosure can be prepared using a two-step procedure. First, each chain of the double-stranded RNA molecule is prepared separately and then annealed. Each chain of the siRNA compound can be prepared using solution phase or solid phase organic synthesis or both. The advantage of organic synthesis is that oligonucleotide chains containing non-natural or modified nucleotides can be easily prepared. Similarly, single-stranded oligonucleotides of the present disclosure can be prepared using solution phase or solid phase organic synthesis or both.

[0297] In one aspect, dsRNA of the present disclosure comprises at least two nucleotide sequences, sense sequence and antisense sequence.In some embodiments, sense strand is selected from the sequence group that any one provides in table 1, table 2a and table 2b, and the corresponding antisense strand of sense strand is selected from the sequence that any one provides in table 1, table 2a and table 2b.In this respect, one of two sequences is complementary to another in two sequences, and one of them is substantially complementary to the mRNA sequence produced in the expression of relevant target gene.Therefore, in this respect, dsRNA will comprise two oligonucleotides, one of which oligonucleotide is described as any one sense strand in table 1, table 2a and table 2b, and second oligonucleotide is described as the corresponding antisense strand of any one sense strand in table 1, table 2a and table 2b.

[0298] In some embodiments, the sense strand or antisense strand is selected from the sense strand or antisense strand of any one of duplexes IN-043, IN-091, IN-106, IN-176, IN-189, IN-202, and IN-221.

[0299] It should be understood that, although, for example, the sequences in Table 1 are unmodified or conjugated sequences, the siRNA RNAs of the present disclosure, such as the dsRNAs of the present disclosure, can comprise any of the unmodified, unconjugated, modified, or conjugated sequences listed in Table 1, Table 2a, and Table 2b. In other words, the present disclosure encompasses the dsRNAs of Table 1, Table 2a, and Table 2b, which are unmodified, unconjugated, modified, or conjugated, as described herein.

[0300] It is well known to those skilled in the art that dsRNAs having a duplex structure of approximately 20 to 23 base pairs (e.g., 21 base pairs) are considered particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20: 6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14: 1714-1719; Kim et al. (2005) Nat Biotech 23: 222-226). In some embodiments, the dsRNA can include at least one strand that is at least 21 nucleotides in length. It is reasonable to expect that shorter duplexes of the dsRNAs in Tables 1, 2a, and 2b, with only a few nucleotides removed from one or both ends, can also have similar effects compared to the above-mentioned dsRNAs. Thus, dsRNAs having a sequence of at least 12, 13, 14, 15, 19, 20 or more consecutive nucleotides derived from any one of Tables 1, 2a, and 2b, and whose ability to inhibit INHBE gene expression does not differ by more than about 5%, 10%, 15%, 20%, 25% or 30% from a dsRNA comprising the entire sequence, are considered to be within the scope of the present disclosure.

[0301] 3. Modification of RNAi Agents

[0302] In certain embodiments, the RNA of the dsRNAi agent of the present disclosure is unmodified and does not comprise chemical modifications or conjugations such as those known in the art and described herein. In other embodiments, the RNA (e.g., dsRNA) of the dsRNAi agent of the present disclosure is chemically modified to enhance stability or provide other beneficial properties. In certain embodiments, substantially all nucleotides of the RNA of the present disclosure are modified. In other embodiments, all nucleotides of the RNA or substantially all nucleotides of the RNA are modified. In some embodiments, there are no more than 5, 4, 3, 2, or 1 unmodified nucleotides in the chain of the RNA (e.g., oligonucleotide, antisense strand, sense strand, or dsRNA).

[0303] In some embodiments, the dsRNAi agent of the present disclosure comprises at least one nucleic acid modification described herein. For example, at least one modification is selected from the group consisting of modified internucleoside linkages, modified core bases, modified sugars, and any combination thereof. Without limitation, such modifications may be present anywhere in the dsRNAi agent of the present disclosure. In certain embodiments, the dsRNAi agent comprises at least one non-canonical base pairing nucleotide. Without limitation, such nucleotides may be present anywhere in the dsRNAi agent of the present disclosure. In some embodiments, at least one modified nucleotide and / or non-canonical base pairing nucleotide changes the melting temperature of the oligonucleotide, for example, ΔTm is at least 2°C, for example, about 2°C, more than 2°C, about 2-5°C, about 3°C, about 4°C, or about 5°C.

[0304] In one embodiment, the dsRNA agent of the present disclosure comprises one or more targeting ligands, such as one or more GalNAc derivatives, and comprises at least one additional nucleic acid modification described herein. For example, the dsRNAi agent may include at least one modification selected from modified internucleoside linkages, modified core bases, modified sugars, and any combination thereof. Without limitation, such modifications may be present anywhere in the dsRNAi agent of the present disclosure. For example, the modification may be present in one of the RNA molecules. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, reverse connection) or 3'-end modifications (conjugation, DNA nucleotides, reverse connection, etc.); base modifications, for example, replacement with stable bases, destabilizing bases, or bases paired with extended bases, removal of bases (absaccharide nucleotides) or conjugation of bases; sugar modifications (e.g., at 2'- or 4'-positions) or sugar replacements; or main chain modifications, including modification or replacement of phosphodiester bonds. Specific examples of RNAi agents that can be used in the embodiments described herein include, but are not limited to, RNAs containing modified main chains or free of natural internucleoside linkages. RNAs with modified backbones include RNAs that do not have a phosphorus atom in the backbone, etc. In some embodiments, the modified RNAi agent has a phosphorus atom in the internucleoside backbone.

[0305] In some embodiments, backbone modifications refer to internucleoside linkages or backbones including but not limited to phosphorothioate groups, chiral phosphorothioates, phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, chiral phosphonates, phosphinates, phosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters, morpholino linkages, wherein adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0306] In some embodiments, the sense strand of the dsRNA can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages (phosphorothioate modified nucleotides), and the antisense strand of the dsRNA can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages (phosphorothioate modified nucleotides). In some embodiments, the sense strand of the dsRNA can contain 1 or 2 phosphorothioate linkages, and the antisense strand of the siRNA can contain 1, 2, 3, or 4 phosphorothioate linkages.

[0307] In some embodiments, the sense strand of the dsRNA contains two phosphorothioate internucleoside linkages. In some embodiments, the phosphorothioate internucleoside linkage is located between the nucleotides at positions 1-3 from the 5' end of the sense strand. In some embodiments, the phosphorothioate internucleoside linkage is located between the nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, one phosphorothioate internucleoside linkage is located at the 5' end of the sense strand, and another phosphorothioate linkage is located at the 3' end of the sense strand. In some embodiments, the sense strand of the dsRNA contains one phosphorothioate internucleoside linkage. In some embodiments, the phosphorothioate internucleoside linkage is located between the nucleotides at positions 1-2 from the 5' end of the sense strand. In some embodiments, the phosphorothioate internucleoside linkage is located between the nucleotides at positions 2-3 from the 5' end of the sense strand. In some embodiments, the targeting ligand is linked to the sense strand via a phosphorothioate linkage.

[0308] In some embodiments, the antisense strand of the dsRNA contains 4 phosphorothioate nucleoside linkages. In some embodiments, the 4 phosphorothioate nucleoside linkages are located between the nucleotides at positions 1-3 from the 5' end of the antisense strand and between the nucleotides at positions 1-3 from the 3' end. In some embodiments, the antisense strand of the dsRNA contains 3 phosphorothioate nucleoside linkages. In some embodiments, the 3 phosphorothioate nucleoside linkages are located between the nucleotides at positions 1-2 from the 5' end of the antisense strand and between the nucleotides at positions 1-3 from the 3' end. In some embodiments, the 3 phosphorothioate nucleoside linkages are located between the nucleotides at positions 1-3 from the 5' end of the antisense strand and between the nucleotides at positions 1-2 from the 3' end. In some embodiments, the antisense strand of the dsRNA contains 2 phosphorothioate nucleoside linkages. In some embodiments, the two phosphorothioate internucleoside linkages are located between the nucleotides 1-2 from the 5' end and between the nucleotides 1-2 from the 3' end of the antisense strand.

[0309] In some embodiments, the antisense strand of the dsRNA comprises a nucleotide sequence (from 5' end → 3' end) of any antisense strand sequence in Table 1, 2a, or 2b. In some embodiments, the sense strand of the dsRNA comprises a nucleotide sequence (from 5' end → 3' end) of any sense strand in Table 1, 2a, or 2b. In some embodiments, the antisense strand of the dsRNA comprises a nucleotide sequence (from 5' end → 3' end) of any antisense strand in Table 1, 2a, or 2b, and the sense strand comprises a nucleotide sequence (from 5' end → 3' end) of any sense strand in Table 1, 2a, or 2b.

[0310] In some embodiments, the modified nucleotides disclosed herein are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-F-arabino nucleotides, phosphorothioate modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides, inverted nucleotides, and inosine base substituted nucleotides.

[0311] In some embodiments, the modified nucleotides disclosed herein are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, phosphorothioate modified nucleotides, and inverted nucleotides. In some preferred embodiments, the inverted nucleotides are selected from the group consisting of inverted A nucleotides, inverted dA nucleotides, inverted dT nucleotides, inverted C nucleotides, and inverted U nucleotides.

[0312] Exemplary modified nucleotides or nucleobases include, but are not limited to, synthetic and natural nucleosides or nucleobases such as inosine, xanthine, hypoxanthine, nebularine, isoguanosine, tuberculin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N6-(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, )adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxy)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N6-(isopentyl)adenine, N6-(methyl)adenine, N6,N6-(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine , 8-(hydroxy)guanine, 8-(sulfanyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propenyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(Methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidinyl)uracil, 5-(1,3-oxadiazole-1-alkyl) uracil, 5-(cyanoalkyl) uracil, 5-(dialkylaminoalkyl) uracil, 5-(dimethylaminoalkyl) uracil, 5-(halogenated) uracil, 5-(methoxy) uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio) uracil, 5-(methoxycarbonylmethyl) uracil, 5-(propenyl) uracil, 5-(propynyl) uracil, 5-(trifluoromethyl) uracil Pyrimidine, 6-(azo)uracil, dihydrouracil, N3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5 -(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylvinyl)-pseudouracil, 1-(aminocarbonylvinyl)-2(thio)pseudouracil Pyrimidine, 1-(aminocarbonylvinyl)-4-(thio)pseudouracil, 1-(aminocarbonylvinyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl )-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-substituted 1(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1, 3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)phenothiazin-1-yl, 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 1,3,5-(triaza)-2,6 -(dioxa)-naphthalene, inosinyl, 2-azainosinyl, 7-deazainosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isoquinolonyl, 5-(methyl)isoquinolonyl, 3-(methyl)-7-(propynyl)isoquinolonyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidazopyridinyl, 9-(methyl)-imidazopyridinyl, pyrrolopyrazinyl, isoquinolonyl, 7-(propynyl)isoquinolyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4, 6-(dimethyl)indolyl, phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl, stilbene, tetracene, pentacene, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, N2-substituted purine, N6-substituted purine, O6-substituted purine, substituted 1,2,4-triazole, pyrrolopyrimidin-2-one-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted-6-phenyl- Pyrrolo-pyrimidin-2-one-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, di-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, p-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, di-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, or any O-alkylated or N-alkylated derivative thereof. Alternatively, substituted or modified analogs of any of the above nucleotides or nucleobases can be used in the RNAi agents, compositions and methods of the present disclosure.

[0313] In some embodiments, the modified nucleotides disclosed herein include any one or a combination of the following:

[0314] (1) From the 5' end to the 3' end, the nucleotides at positions 2, 4, 12, and 14 of the antisense strand are 2'-fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides;

[0315] (2) in the direction from 5' to 3', the nucleotides at positions 7, 8, and 9 of the sense strand are 2'-fluorinated nucleotides;

[0316] (3) From the 5' end to the 3' end, the G in positions 2-8 of the antisense strand is replaced by I; or, from the 5' end to the 3' end, the guanine (the base in G) in positions 2-8 of the antisense strand is replaced by hypoxanthine (the base of inosine or inosinic acid I).

[0317] In some such embodiments, the modified nucleotides substituted with hypoxanthine in positions 2-8 of the antisense strand further satisfy one or a combination of the following characteristics:

[0318] (i) guanine at positions 6-8 of the antisense strand is replaced with hypoxanthine in the direction from 5' to 3' end (I);

[0319] (ii) in the 5' to 3' direction, the guanine in the sequence N1N2GN3N4 of the antisense strand is replaced by hypoxanthine (I), if at least three bases among N1, N2, N3 and N4 are adenine or uracil;

[0320] (iii) the number of adenines and uracils in positions 2 to 8 of the antisense strand is 4 or more in the 5' to 3' direction; and / or

[0321] (iv) guanine in at least one nucleotide in the antisense strand is substituted with hypoxanthine, when the hypoxanthine substitution results in a difference in the dissociation temperature of the dsRNA comprising the antisense strand of at least 2°C, 2°C, or greater than 2°C compared to the dsRNA without the hypoxanthine substitution.

[0322] In some embodiments, the dsRNAi agent further comprises a phosphate or a phosphate mimic located at the 5'-end of the antisense strand. In some embodiments, the phosphate mimic is 5'-vinyl phosphate (VP). In some embodiments, the 5'-end of the antisense strand of the dsRNAi agent does not comprise 5'-vinyl phosphate (VP).

[0323] The end of RNAi reagent of the present disclosure can be modified, and this modification can be at one end or both ends.For example, dsRNA 3 ' and / or 5 ' end can be conjugated to other functional molecular entities, such as labeling moieties, such as fluorophores (such as pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based on, for example, sulfur, silicon, boron or ester). Functional molecular entities can be connected to sugar by phosphate group and / or linker. The terminal atom of joint can connect or replace the phosphate group of sugar or the connecting atom of C-3 ', C-5 ', O, N, S or C group. Alternatively, joint can connect or replace the terminal atom of nucleotide substitute (for example, PNA). When linker / phosphate functional molecular entity-linker / phosphate array is inserted between two chains of double-stranded oligomeric compound, the array can replace the hairpin loop in hairpin type oligomeric compound. In some embodiments, end modification can also be used for monitoring distribution, and in this case, the preferred group to be added includes fluorophores, such as fluorescein or Alexa dye, such as Alexa 488. In some embodiments, terminal modifications may also be used to enhance uptake, and non-limiting modifications useful for this include targeting ligands.

[0324] The present disclosure also encompasses various salts, mixed salts, and free acid forms of the dsRNA agents. In some embodiments, the dsRNA agent is in free acid form. In some embodiments, the dsRNAi agent is in salt form. In one embodiment, the dsRNAi agent is in sodium salt form. According to common knowledge in the art, when the dsRNAi agent of the present disclosure is in sodium salt form, sodium ions are present in the agent as counterions to the phosphodiester and / or phosphorothioate groups.

[0325] In certain embodiments, the dsRNAi agent of the present disclosure is further modified by covalently linking one or more conjugate groups. Generally speaking, the conjugate group changes one or more properties of the dsRNA agent of the present disclosure connected, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cell distribution, cellular uptake, charge and removal. Conjugate groups are commonly used in the field of chemistry, and are directly or by optional linking moieties or linking groups connected to the parent compound. Conjugate groups preferably include but not limited to polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, bile acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin and dye.

[0326] In some embodiments, the targeting ligand of the present disclosure comprises N-acetyl-galactosamine (GalNAc), or a GalNAc derivative, such as L96 (see siRNA drug Inclisiran). In some embodiments, the targeting ligand is any targeting ligand disclosed in WO2022266753A1. Unless otherwise clearly contradictory, WO2022266753A1 is incorporated herein by reference in its entirety.

[0327] In some embodiments, the structure of the dsRNAi agent is selected from Formula 1 to Formula 33, wherein R 2 According to common knowledge in this field, R 2 The dsRNAi agent is formed by conjugating the 3' end or 5' end of the sense strand of the dsRNA to a targeting ligand.

[0328] IV. Delivery and Use of RNAi Agents

[0329] The RNAi agents of the present invention can be delivered to cells, such as cells in a subject (e.g., a subject with a metabolic disorder) in a variety of ways. For example, delivery can be carried out by contacting cells with the RNAi agents of the present invention in vitro or in vivo. In vivo delivery can also be carried out directly by administering a composition (e.g., a pharmaceutical composition) comprising an RNAi agent (e.g., dsRNA) to the subject. Alternatively, in vivo delivery can be carried out indirectly by administering one or more vectors that encode and guide the expression of the RNAi agent.

[0330] In one embodiment, the cells are liver cells, such as hepatocytes. In one embodiment, the cells are adipocytes. In certain embodiments, the RNAi agent is taken up by one or more tissue or cell types present in an organ (e.g., liver, adipose tissue).

[0331] Another aspect of the present disclosure relates to a method for reducing the expression and / or activity of the INHBE gene in a subject, comprising administering a dsRNAi agent of the present disclosure to the subject. In some embodiments, the method comprises administering a therapeutically effective amount of a dsRNAi agent of the present disclosure to the subject, thereby inhibiting or reducing the expression of the INHBE gene in the subject (e.g., a cell in the subject). In some embodiments, the method comprises contacting the cell with a double-stranded RNAi agent of the present disclosure, such that the expression of the INHBE gene is inhibited or reduced in the cell. In some such embodiments, the mRNA transcript of the target gene, such as the INHBE gene, is degraded in the subject or cell, thereby inhibiting or reducing the expression of the INHBE gene in the subject or cell.

[0332] In another aspect, the disclosure relates to a method of treating a subject having or at risk of having or developing a metabolic disorder, comprising administering to the subject a therapeutically effective amount of a dsRNAi agent of the disclosure, thereby treating the subject.

[0333] In another aspect, the present disclosure relates to a method of treating or preventing a metabolic disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a dsRNAi agent of the present disclosure, such that the metabolic disorder is treated or prevented.

[0334] In another aspect, the present disclosure relates to a method for treating or preventing an INHBE-related disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a dsRNAi agent such that the INHBE-related disease or condition is treated or prevented. The term "INHBE-related disease or condition" includes any disease or condition caused, mediated, or associated with INHBE gene expression or protein production, and includes any disease or condition that would benefit from or be ameliorated by a reduction in INHBE gene expression or protein activity. Examples of INHBE-related diseases or conditions include, but are not limited to, metabolic disorders, metabolic syndrome, type 2 diabetes, obesity, prediabetes, elevated triglyceride levels (hypertriglyceridemia), lipodystrophy, liver inflammation, fatty liver, hypercholesterolemia, diseases associated with elevated liver enzymes, nonalcoholic steatohepatitis, cardiovascular disease, kidney disease, abdominal obesity, insulin resistance, hypertension, hyperlipidemia, cardiometabolic disorders, and cancers associated with INHBE expression.

[0335] In some embodiments, the subject is a human.

[0336] In some embodiments, the subject has a metabolic disorder.

[0337] In some embodiments, the metabolic disorder is one or more of metabolic syndrome, type 2 diabetes, obesity, prediabetes, hypertriglyceridemia, lipodystrophy, liver inflammation, fatty liver, hypercholesterolemia, diseases associated with elevated liver enzymes, nonalcoholic steatohepatitis, cardiovascular disease, and kidney disease. In some embodiments, the metabolic syndrome includes but is not limited to one or more of abdominal obesity, insulin resistance, hypertension, and dyslipidemia.

[0338] In some embodiments, the INHBE-associated disease or condition is one or more of a metabolic disorder, metabolic syndrome, type 2 diabetes, obesity, prediabetes, hypertriglyceridemia, lipodystrophy, liver inflammation, fatty liver, hypercholesterolemia, a disease associated with elevated liver enzymes, nonalcoholic steatohepatitis, cardiovascular disease, kidney disease, abdominal obesity, insulin resistance, hypertension, dyslipidemia, a cardiometabolic disorder, and a cancer associated with INHBE expression.

[0339] Non-limiting examples of metabolic disorders include carbohydrate disorders, such as diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, galactosemia, hereditary fructose intolerance, fructose 1,6-bisphosphatase deficiency, glycogen storage diseases, inborn errors of glycosylation, insulin resistance, insulin insufficiency, hyperinsulinemia, impaired glucose tolerance (IGT), dysglycogen metabolism, amino acid metabolism disorders, such as maple syrup urine disease (MSUD), homocystinuria; organic acid metabolism disorders, such as methylmalonic aciduria, 3-methylglutaric aciduria-Bartter syndrome, glutaric aciduria, 2-hydroxyglutaric aciduria-D and L types; fatty acid β-oxidation disorders, such as medium chain acyl-CoA dehydrogenase deficiency (MCAD), long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LC3D), and hydroxyacyl-CoA dehydrogenase deficiency (LC3D). and peroxisomal disorders, such as Zellweger syndrome (CHS), X-linked adrenoleukodystrophy, and Refsum disease.

[0340] In some embodiments, the metabolic disorder is associated with body fat distribution and includes, but is not limited to, metabolic syndrome, type 2 diabetes, hyperlipidemia or dyslipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C)), hyperlipidemia or dyslipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B, or other lipid components), obesity (particularly abdominal obesity), lipodystrophy (e.g., the inability to store fat locally in body fat (regional lipodystrophy) or throughout the body (lipoatrophy)), insulin resistance or elevated or altered insulin levels during fasting or metabolic challenge, hepatic fat deposition or fatty liver body fat distribution and its complications (e.g., cirrhosis, fibrosis, or inflammation of the liver), non-alcoholic steatohepatitis, other types of liver inflammation, elevated or elevated or altered liver enzyme levels or other markers of liver damage, inflammation or fat deposition in the liver, elevated blood pressure and / or hypertension, elevated blood sugar or glucose or hyperglycemia, metabolic syndrome, coronary artery disease and other atherosclerotic conditions and their complications. In some embodiments, the metabolic disorder is associated with body fat distribution characterized by greater fat accumulation around the waist (e.g., greater abdominal fat or larger waist circumference) and / or less fat accumulation around the hips (e.g., lower gluteofemoral fat or smaller hip circumference), resulting in a larger waist-to-hip ratio (WHR) and higher cardiometabolic risk, independent of body mass index (BMI).

[0341] In one embodiment, metabolic disorder is metabolic syndrome. As used herein, term "metabolic syndrome" refers to a disease comprising a collection of symptoms including overnutrition, sedentary lifestyle, genetic factors, aging and resulting obesity. Metabolic syndrome includes abdominal obesity, insulin resistance, dyslipidemia and elevated blood pressure, and metabolic syndrome is associated with other comorbidities including prothrombotic state, proinflammatory state, non-alcoholic fatty liver disease and reproductive disorders. Metabolic syndrome approximately doubles the risk of cardiovascular disease and 5 times the risk of type 2 diabetes. Abdominal obesity (e.g., excessive waist circumference (waist-to-hip ratio)), hypertension, insulin resistance and dyslipidemia are core components of metabolic syndrome and its various components (e.g., central obesity, fasting blood glucose (FBG) / prediabetes / diabetes, hypercholesterolemia, hypertriglyceridemia and hypertension).

[0342] In one embodiment, the metabolic disorder is a carbohydrate disorder. In one embodiment, the carbohydrate disorder is diabetes. As used herein, the term "diabetes" refers to a collection of metabolic disorders characterized by high blood sugar (glucose) levels, which are caused by defects in insulin secretion or action or both. The two most common types of diabetes, type 1 diabetes (also referred to as "type I diabetes") and type 2 diabetes (also referred to as "type II diabetes"), are both caused by the body's inability to regulate insulin. Insulin is a hormone released by the pancreas in response to elevated blood sugar (glucose) levels.

[0343] As used herein, the term "type 1 diabetes" refers to a chronic disease that occurs when the pancreas produces too little insulin to properly regulate blood sugar levels. Type 1 diabetes is also known as insulin-dependent diabetes mellitus, IDDM, and juvenile-onset diabetes. People with type 1 diabetes (insulin-dependent diabetes) typically produce little or no insulin. Type 1 diabetes may be due to progressive autoimmune destruction of pancreatic beta cells and subsequent insulin deficiency. People with type 1 diabetes must take regular insulin injections.

[0344] In type 2 diabetes (also known as non-insulin-dependent diabetes mellitus, NDDM), the pancreas continues to produce insulin, sometimes even at higher than normal levels, yet the body becomes resistant to its effects, leading to relative insulin deficiency. Obesity is a risk factor for type 2 diabetes, and most people with the condition are obese.

[0345] In some embodiments, diabetes includes prediabetes. "Prediabetes" refers to one or more early diabetic conditions, including impaired glucose utilization, abnormal or impaired fasting blood glucose levels, impaired glucose tolerance, impaired insulin sensitivity, and insulin resistance. Prediabetes is a major risk factor for type 2 diabetes, cardiovascular disease, and mortality. Developing therapeutic interventions to effectively treat prediabetes and prevent the development of type 2 diabetes has received widespread attention.

[0346] Diabetes can be diagnosed by performing a glucose tolerance test. Clinically, diabetes is often divided into several basic categories. The main examples of these categories include autoimmune diabetes, non-insulin dependent diabetes mellitus (type 2 NDDM or NIDDM), insulin dependent diabetes mellitus (type 1 IDDM), non-autoimmune diabetes and maturity-onset diabetes of the juvenile onset (MODY). Another classification is commonly referred to as secondary diabetes, refers to the diabetes caused by some identifiable conditions, and these conditions cause or promote the development of diabetic syndrome. The example of the second category includes but is not limited to, the diabetes caused by pancreatic disease, hormone abnormalities, the diabetes induced by drugs or chemicals, the diabetes caused by insulin receptor abnormalities, the diabetes relevant to genetic syndromes and the diabetes of other reasons.

[0347] In one embodiment, metabolic disorder is lipid metabolism disorder. " lipid metabolism disorder " or " lipid metabolism disorder " as used herein refer to any disease relevant to lipid metabolism disorder or caused by lipid metabolism disorder. The term also includes any disease, disease or symptom that can cause hyperlipidemia or be characterized as any or all lipids and / or lipoprotein levels in blood that are abnormally elevated. The term can refer to hereditary diseases, such as familial hypertriglyceridemia, type 1 familial partial lipodystrophy (FPLDI), or secondary or acquired diseases, such as due to disease, disease or symptom (such as, renal failure), diet or intake of some medicine (such as, due to treatment, such as, AIDS or HIV, and using highly effective antiretroviral therapy (HAART) to induce or obtain the disease) and induce or obtain the disease. The term also refers to the disorder of fat distribution and / or storage, such as lipodystrophy.

[0348] Additional examples of lipid metabolism disorders include, but are not limited to, atherosclerosis, dyslipidemia, hypertriglyceridemia (including drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcohol-induced hypertriglyceridemia, beta-adrenergic blocking agent-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia, vitamin A-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, and familial hypertriglyceridemia. hyperlipidemia, hyperlipidemia (including familial combined hyperlipidemia), hyperlipidemia (including familial combined hyperlipidemia), hyperlipidemia (including familial combined hyperlipidemia), lipodystrophy, gout associated with hypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposits), heterogeneous LPL deficiency hyperlipidemia, high LDL and heterogeneous LPL deficiency hyperlipidemia, fatty liver disease, or nonalcoholic steatohepatitis (NASH).

[0349] In one embodiment, the metabolic disorder is cardiovascular disease. The cardiovascular disease may include, but is not limited to, coronary artery disease (also known as ischemic heart disease), hypertension, inflammation associated with coronary artery disease, restenosis, peripheral vascular disease, or stroke.

[0350] In one embodiment, the metabolic disorder is a kidney disease. The kidney disease may include, but is not limited to, chronic kidney disease, diabetic nephropathy, or gout.

[0351] In one embodiment, the metabolic disorder is a disorder related to weight. Weight disorders may include, but are not limited to, obesity, hypometabolic states, hypothyroidism, uremia, and risk of weight gain (including rapid weight gain), weight loss, sustained weight loss, or weight regain after weight loss.

[0352] In one embodiment, the metabolic disorder is a glycemic disorder. Glycemic disorders may include, but are not limited to, diabetes, hypertension, and polycystic ovary syndrome (PCOS) associated with insulin resistance.

[0353] Other exemplary metabolic disorders include, but are not limited to, renal transplantation, nephrotic syndrome, Cushing's syndrome, acromegaly, systemic lupus erythematosus, dysglobulinemia, lipodystrophy, glycogenotype 1, and Addison's disease.

[0354] In one embodiment, metabolic disorder is essential hypertension." Essential hypertension " may be the result of environmental or genetic causes (e.g., the result without obvious underlying medical causes). In another embodiment, metabolic disorder is secondary hypertension." Secondary hypertension " has an identifiable underlying disease, which may be of multiple etiologies, including kidney, blood vessel and endocrine causes, such as renal parenchymal disease (e.g., polycystic kidney disease, glomerular or interstitial disease), renal vascular disease (e.g., renal artery stenosis, fibromuscular dysplasia), endocrine disease (e.g., adrenocortical steroids or mineralocorticoids are excessive, pheochromocytoma, hyperthyroidism or hypothyroidism, growth hormone is excessive, hyperparathyroidism), coarctation of the aorta or use of oral contraceptives.

[0355] In one embodiment, the metabolic disorder is refractory hypertension. "Refractory hypertension" refers to blood pressure that remains above target (e.g., systolic blood pressure above 130 mmHg or diastolic blood pressure above 90 mmHg) despite simultaneous use of three different classes of antihypertensive drugs (one of which is a thiazide diuretic). Subjects who use four or more medications to control their blood pressure are also considered to have refractory hypertension.

[0356] In some embodiments of the present disclosure, expression of the INHBE gene in a subject or cell reduces the level of INHBE protein in the serum of the subject by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0357] In some embodiments, inhibiting the expression of the INHBE gene in a cell reduces the protein level of the INHBE gene expression in the serum of the subject by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0358] In some embodiments of the present disclosure, the dsRNAi agent is administered to a subject at a dose of about 0.01 mg / kg to about 50 mg / kg, or at a dose of about 0.10 mg / kg to about 50 mg / kg, for example, but not limited to, a dose of about 0.01 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 50 mg / kg, about 10 mg / kg to about 30 mg / kg, about 10 mg / kg to about 20 mg / kg, about 15 mg / kg to about 20 mg / kg, about 15 mg / kg to about 25 mg / kg, about 15 mg / kg to about 30 mg / kg, or about 20 mg / kg to about 30 mg / kg.

[0359] In some embodiments of the present disclosure, the method further comprises determining the level of INHBE in a sample from the subject, e.g., in a blood, serum, liver tissue, or adipose tissue sample. The level of INHBE in a sample from the subject can be determined before, during, and / or after administration of a dsRNAi agent to the subject (e.g., to monitor efficacy or treatment efficiency, to monitor INHBE mRNA and / or protein levels before, during, or after treatment, etc.).

[0360] In some embodiments of the present disclosure, the method further comprises administering to the subject an additional therapeutic agent to treat the metabolic disorder. The therapeutic agent includes but is not limited to insulin, glucagon-like peptide 1 agonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists, glucagon receptor agonists, sulfonylureas, seglininides, biguanides, thiazolidinediones, α-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, statins, and a combination of any of the foregoing drugs.

[0361] In some embodiments, the dsRNAi agent of the present disclosure is administered by injection or by infusion. In one embodiment, the double-stranded RNAi agent is administered subcutaneously. In one embodiment, the double-stranded RNAi agent is administered intramuscularly. In one embodiment, the double-stranded RNAi agent is administered intravenously. In one embodiment, the double-stranded RNAi agent is administered by systemic administration to the lungs, such as intranasal administration or oral inhalation administration.

[0362] In some embodiments, the pharmaceutical composition comprises a dsRNAi agent of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention can be used in practice for the prevention and / or treatment of various corresponding diseases or conditions. An acceptable carrier (or excipient) is a substance that is intentionally included in a drug delivery system in addition to an active pharmaceutical ingredient (API, therapeutic product, such as a dsRNA agent of the present invention). The carrier or excipient does not or is not intended to exert a therapeutic effect at the intended dose. The carrier or excipient may play the following roles: a) aids in the handling of the drug delivery system during preparation; b) protects, supports or enhances the stability, bioavailability or patient acceptability of the API; c) aids in product identification; and / or d) enhances any other properties of the overall safety, efficacy or delivery of the API during storage or use.

[0363] Carriers or excipients include, but are not limited to, the following components: absorption enhancers, anti-adherents, anti-foaming agents, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, expanders, fillers, flavorings, glidants, wetting agents, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained-release matrices, sweeteners, thickeners, tonicity agents, vehicles, waterproofing agents, and wetting agents.

[0364] In some embodiments, the carrier of the pharmaceutical composition is a non-buffered solution or a buffered solution. Typical non-buffered solutions are saline or water, and buffered solutions include one or more of acetate, citrate, prolamin, carbonate, and phosphate. In some embodiments, the buffered solution is phosphate buffered saline (PBS).

[0365] The present disclosure includes all combinations of the specific embodiments described. Further embodiments of the present disclosure and the full scope of applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present disclosure, these descriptions and examples are provided by way of illustration only, as various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including citations, are incorporated herein by reference in their entirety for all purposes.

[0366] The compounds disclosed herein can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining the specific embodiments with other methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples disclosed herein.

[0367] Example

[0368] The present invention will be more readily understood by reference to the following examples, which are provided to illustrate the invention and are not to be construed as limiting the scope of the invention in any way.

[0369] Unless otherwise defined or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. Unless otherwise specified, the materials and instruments used in the present invention were all commercially available.

[0370] Example 1. siRNA synthesis

[0371] 1.1 Target sequence screening

[0372] siRNAs were designed based on the full INHBE mRNA sequence, all of which were obtained from the NCBI gene database (https: / / www.ncbi.nlm.nih.gov / gene / ). All siRNAs were designed to ensure full sequence identity to human (Gene ID: 83729) and cynomolgus macaque (Gene ID: 102127493).

[0373] After scanning the entire sequence, all potential 19-nucleotide siRNA sequences were obtained and simultaneously compared to the cynomolgus macaque sequence to ensure a match. All human / cynomolgus macaque sequences were then compared to the human full transcriptome mRNA sequence using BLAST, and any siRNAs with potential off-target effects were removed. The activity of all siRNAs was evaluated using rational siRNA design principles, and molecules with low theoretical activity were removed.

[0374] 1.2 siRNA synthesis

[0375] siRNAs were designed targeting different regions of INHBE (siRNA sequences are shown in Tables 1, 2a, and 2b) and synthesized and annealed by Suzhou Beixin Biotechnology Co., Ltd. For in vitro activity assays, blunt-ended siRNA sequences (e.g., IN-151 to IN-230) were synthesized by adding two additional complementary nucleotides or two dTs (i.e., dTdT) to the 3' end of the antisense strand.

[0376] Table 1: Sense and antisense strand sequences of unmodified INHBE dsRNA agents

[0377] Table 2a: Sense and antisense strand sequences of modified INHBE dsRNA reagents

[0378] Table 2b: Sense and antisense strand sequences of modified INHBE dsRNA reagents

[0379] 1.3 RNAi synthesis

[0380] The GalNAc-coupled immobilized carrier (CPG or PS) in Formulas 1 to 33 was used, and the fully modified siRNA was designed according to the original sequence and then delivered to Suzhou Beixin Biotechnology Co., Ltd. for synthesis and annealing.

[0381] The synthesis of the targeting ligands in Formulas 1 to 33 is described in WO2022266753A1. The RNAi compound of Formula 5 formed by linking the dsRNAs in Tables 1, 2a, and 2b with the targeting ligands is named "Duplex No. L5."

[0382] Example 2: siRNA in vitro activity detection method

[0383] 2.1 Fluorescence quantitative PCR

[0384] 2.1.1 Cell culture and transfection

[0385] Hep3B cells were cultured in MEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and double-antibody (Gibco) in an atmosphere of 5% CO2 and 37°C. After the cells grew to nearly completely cover the culture flask, they were trypsinized and resuspended. The resuspended cells were adjusted to a density of 1.5 × 10 4Cells were seeded into 96-well plates and suspended in siRNA transfection complexes. The siRNA transfection complexes were prepared by mixing Opti-MEM (Gibco) containing 0.3 μL / well Lipofectamine RNAi Max (Thermo) with siRNA in a 1:1 ratio. After the cells were cultured for a certain period of time, Dynabeads were used according to the instructions. TM Total mRNA was extracted using an mRNA isolation kit (Thermo). Finally, mRNA was eluted with 20 μL of RNase-free H₂O at 80°C for 5 minutes, and 15 μL of the supernatant was quickly transferred to a magnetic rack. Heating was performed using a BIO-RAD T100 Thermal Cycler PCR instrument.

[0386] 2.1.2 Reverse transcription and fluorescent quantitative PCR

[0387] cDNA synthesis and quantitative PCR were performed using a one-step method. Relative mRNA levels of INHBE and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were measured using the ΔΔCt method. To the supernatant transferred from Example 2.1.1 above, the One-Step mix buffer and One-Step Enzyme mix, along with the forward and reverse primers for the target gene, were added according to the manufacturer's instructions (HiScript II One Step RT-PCR Kit, Vazyme).

[0388] The instrument used was a LightCycler 480 II (Roche). The reaction conditions were: (1) reverse transcription at 50°C for 3 minutes; (2) pre-denaturation at 95°C for 30 seconds; (3) denaturation at 95°C for 10 seconds, and annealing and extension at 60°C for 30 seconds. Step (3) was repeated for 40 cycles. The results were normalized using a blank control to obtain relative mRNA levels and knockdown efficiency. If IC50 values ​​were required, they were obtained by performing a four-parameter fit using Graphpad Prism.

[0389] The sequences of NC, NCM, and IN-Ref1 to IN-Ref9, IN-Ref1m, IN-Ref2m, and IN-Ref8m based on WO2023003922A1 are shown in Table 3. IN-Ref1m-L96 is an RNAi formed by ligating the dsRNA of IN-Ref1m to L96. The structure and preparation of L96 are described in WO2014089313A1.

[0390] Table 3: NC, NCm and other reference sequences

[0391] The test results are shown in Figures 1 to 5b. Table 4 shows the IC50 determination results and maximum inhibition rates of the siRNAs. "NC" indicates a negative control. "NA" indicates not applicable. The decimal points or letters in the duplex numbers of the siRNA IDs represent different assay batches. Batches 1, 2, and 3 all involved human INHBE, and the remaining batches also involved human INHBE. Table 5a shows the relative activity obtained after normalization by IC50 values ​​using IN-Ref8 as the standard. Table 5b shows the inhibition rates of IN-Ref8 and IN-Ref8m at different concentrations.

[0392] Table 4

[0393] Table 5a

[0394] Table 5b

[0395] 2.2 Reporter gene method

[0396] SK-Hep-1-PsiCheck-INHBE cells contain a tandem expression system for the full-length INHBE mRNA sequence and the luciferase gene. They were cultured in MEM medium (Gibco) supplemented with 10% FBS (Gibco), 1 μg / ml puromycin, and double-antibody (Gibco) in an atmosphere of 5% CO2 at 37°C. Once the cells had grown to nearly completely cover the culture flask, they were trypsinized and resuspended. The resuspended cells were adjusted to a density of 1.5×10 4 Cells were seeded into 96-well plates at 1 μL / well and siRNA transfection complexes were added. The siRNA transfection complexes consisted of Opti-MEM (Gibco) containing 0.3 μL / well Lipofectamine RNAi Max (Thermo) and siRNA in a 1:1 ratio. After incubation for a specified period, cells were lysed and Renilla substrate (Vazyme) was added for fluorescence activity detection.

[0397] The test results are shown in Figures 6 to 22, 23 and 24. Figures 9 and 16 were measured in mouse INHBE, and the rest were measured in human INHBE. Tables 6 and 8 show the IC50 determination results of siRNA (four-parameter fitting by Graphpad Prism) and the inhibition rate normalized to the blank control group, where the decimal places or letters in the duplex number of the siRNA ID represent different measurement batches. Among them, batches 4 and 5 both involve human INHBE, batch 6 involves mouse INHBE, and the remaining batches all involve human INHBE. Table 7 shows the IC50 values ​​obtained after standardization of the inhibition rate at the same concentration using IN-Ref8 as the standard. Table 9 shows the inhibition rates of IN-Ref8 and IN-Ref8m at different concentrations.

[0398] Table 6

[0399] Table 7

[0400] Table 8

[0401] Table 9

[0402] Example 3: siRNA in vitro stability detection method

[0403] 3.1. Stem-loop PCR

[0404] The stem-loop method has been widely used to detect the absolute concentration of siRNA and miRNA (Curr Protoc Mol Biol. 2011 Jul; Chapter 15: Unit 15.10.). Corresponding stem-loop primers were designed for different siRNAs, and after reverse transcription using these primers instead of oligo dT, the siRNA concentration was determined using fluorescent quantitative PCR and a standard curve conversion. Reverse transcription was performed using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme). The process was as follows: (1) Sample pretreatment: 1) 85°C, 5 minutes; 2) 60°C, 5 minutes; (2) Reverse transcription: Prepare the reaction system according to the conditions described in the kit and complete reverse transcription; (3) Prepare the reaction system using the conditions described in the SYBR green (TIANGEN) kit and complete fluorescent quantitative PCR. The reverse transcription process was completed by a BIO-RAD T100 Thermal Cycler PCR instrument, and the fluorescent quantitative PCR was completed by a Roche LC480 II.

[0405] 3.1.1 Serum stability

[0406] (1) Dilute siRNA to 2 μM, and use FBS (Gibco, fetal bovine serum) to dilute siRNA to 100 nM, with FBS accounting for ≥90%, and mark it as zero point; (2) Pipette 10 μL from zero point into a new sample tube, mark the corresponding time point, and incubate at 37°C for different time periods; (3) Take out the corresponding samples according to the incubation time, quickly freeze them in liquid nitrogen, and then store the samples at -80°C. Mix the samples before use, use the zero point group sample gradient dilution as the standard curve, and use the 3.1 Stem-loop PCR method for detection.

[0407] GraphPad Prism software was used to perform a linear fit of the Ct values ​​obtained by PCR with standards of varying concentrations to generate a standard curve corresponding to standard concentration and Ct value. The Ct values ​​of samples at each incubation time point were then back-calculated using the standard curve to determine the siRNA concentration in the sample. Sample concentrations at each time point were normalized to the concentration of the sample at 0 h, and the percentage of residual siRNA concentration in the sample at each time point relative to the 0 h concentration was calculated. Figure 25 shows the results of the siRNA serum stability test.

[0408] 3.1.2 Human Liver S9 Stability

[0409] (1) Dilute siRNA using human liver S9 (BioIVT); (2) Incubate at 37°C for varying periods of time. (3) Remove samples based on the incubation time and quickly freeze in liquid nitrogen. Store samples at -80°C until further use. Perform detection according to the Stem-loop PCR method in 3.1.

[0410] GraphPad Prism software was used to perform a linear fit of the Ct values ​​obtained by PCR with standards of varying concentrations to generate a standard curve corresponding to standard concentration and Ct value. The Ct values ​​of samples at each incubation time point were then back-calculated using this standard curve to determine the siRNA concentration in the sample. Sample concentrations at each time point were normalized to the concentration of the sample at 0 h, and the percentage of residual siRNA concentration at each time point relative to the 0 h concentration was calculated. Figure 26 shows the results of siRNA stability testing in human liver S9.

[0411] Example 4: Detection of siRNA drug concentration in the liver

[0412] After mice were subcutaneously administered with a dose of 3 mg / kg, liver tissue was taken at different times. The liver tissue was weighed, and PBS was added at 4°C and homogenized using a homogenizer (Shanghai Jingxin JXFSTPRP-64). The siRNA content in the liver homogenate was detected using the Stem-loop PCR method. The siRNA concentration in the liver tissue of the mice in the drug administration group was obtained after back calculation based on the standard curve. Figure 27 shows the changes in siRNA content in the liver of each group of mice (3 mice) after administration at different times. Figure 28 shows the percentage of different siRNA content in the mouse liver after standardization with the content of each RNAi in the liver tissue of mice (3 mice) 7 days after administration.

[0413] Example 5: In vivo efficacy of INHBE siRNA in healthy cynomolgus monkeys

[0414] To evaluate the in vivo activity of siRNA against INHBE, in vivo activity assays were performed using healthy cynomolgus monkeys. The siRNA conjugate (RNAi) was diluted with saline and subcutaneously injected at a dose of 9 mg / kg on day 1 according to the experimental design. Blank saline was used as a negative control. Liver biopsy samples were collected 1 day before administration and on days 14, 28, 42, 56, and 70 after administration. Total RNA was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit V2 (Vazyme). Fluorescence quantitative PCR was performed according to the manufacturer's instructions (HiScript II One Step RT-PCR Kit, Vazyme) to determine INHBE mRNA levels. Fluorescence quantitative PCR was performed using a LightCycler 480 II (Roche).

[0415] First, the test results were normalized using an internal reference gene (GAPDH) to obtain relative mRNA levels. Individual normalization was performed for each animal's INHBE mRNA levels. For individual normalization, the mean INHBE mRNA level for each animal at a time point was divided by the mean of the animal's pre-treatment expression level to determine the relative expression level "normalized to pre-treatment." Table 10 shows the mean relative expression levels for each group of animals after normalization using the mRNA level in each animal's own liver tissue before treatment. "Vehicle" represents the blank control.

[0416] Table 10

[0417] Although the present invention has been described in detail with reference to the embodiments of the present invention, these embodiments are provided to illustrate rather than limit the present invention. Other embodiments that can be obtained according to the principles of the present invention all fall within the scope defined by the claims of the present invention.

Claims

1. A dsRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ from any of the sequences shown in Table 1, Table 2a or Table 2b by 0, 1, 2 or 3 nucleotides, and the sense strand has at least 15, 16, 17, 18, 19, 20 or 21 nucleotides complementary to the antisense strand.

2. The dsRNA according to claim 1, characterized in that The antisense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ from any of the sequences shown in Table 1, Table 2a or Table 2b by 0, 1, 2 or 3 nucleotides.

3. The dsRNA according to claim 1 or 2, characterized in that The length of the antisense strand and the sense strand are each independently 17-25 nucleotides.

4. The dsRNA according to any one of claims 1 to 3, characterized in that It comprises any one of the antisense strand or sense strand sequences in Table 1, Table 2a or Table 2b.

5. The dsRNA according to any one of claims 1 to 4, characterized in that It comprises the antisense strand sequence and the sense strand sequence shown in the duplex sequence in Table 1, Table 2a or Table 2b.

6. The dsRNA according to any one of claims 1 to 5, characterized in that At least one nucleotide in the sense strand and the antisense strand is a modified nucleotide.

7. The dsRNA according to claim 6, characterized in that The modified nucleotides are selected from: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-fluoro, 2'-deoxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-F-arabino nucleotides, phosphorothioate modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides, inverted nucleotides and inosine base substituted nucleotides.

8. The dsRNA according to claim 1, wherein The antisense strand comprises a member selected from the group consisting of SEQ ID NOs: 86, 182, 212, 350, 376, 402, 440, 476, 478, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598 , 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 7 16, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832 , 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 970, 972, 974, 1766, 1770, 1772, 1774, 1776, 1778, 1786, 1802, 1806, 1812, 1814, 1816, 1818, 1820, 1840, 1842, 1844, 1846, 1848, 1850, 1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872, or 1874 of completely consecutive nucleotides.

9. The dsRNA according to claim 1, characterized in that The sense strand comprises a member selected from the group consisting of SEQ ID NOs: 85, 181, 211, 349, 375, 401, 439, 475, 477, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592 73, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 734 5. 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 969, 971, 973, 1765, 1769, 1771, 1773, 1775, 1 completely consecutive nucleotides of 777, 1785, 1801, 1805, 1811, 1813, 1815, 1817, 1819, 1839, 1841, 1843, 1845, 1847, 1849, 1851, 1853, 1855, 1857, 1859, 1861, 1863, 1865, 1867, 1869, 1871 or 1873.

10. The dsRNA according to claim 1, wherein It comprises a sense strand sequence and an antisense strand sequence selected from the following: SEQ ID NOs: 85 and 86; SEQ ID NOs: 181 and 182; SEQ ID NOs: 211 and 212; SEQ ID NOs: 349 and 350; SEQ ID NOs: 375 and 376; SEQ ID NOs: 401 and 402; SEQ ID NOs: 439 and 440; SEQ ID NOs: 475 and 476; SEQ ID NOs: 477 and 478; SEQ ID NOs: 501 and 502; SEQ ID NOs: 503 and 504; SEQ ID NOs: 505 and 506; SEQ ID NOs: 507 and 508; SEQ ID NOs: 509 and 510; SEQ ID NOs: 511 and 512; SEQ ID NOs: 513 and 514; SEQ ID NOs: 515 and 516; SEQ ID NOs: 517 and 518; SEQ ID NOs: 519 and 520; SEQ ID NOs: SEQ ID NOs: 521 and 522; SEQ ID NOs: 523 and 524; SEQ ID NOs: 525 and 526; SEQ ID NOs: 527 and 528; SEQ ID NOs: 529 and 530; SEQ ID NOs: 531 and 532; SEQ ID NOs: 533 and 534; SEQ ID NOs: 535 and 536; SEQ ID NOs: 537 and 538; SEQ ID NOs: 539 and 540; SEQ ID NOs: 541 and 542; SEQ ID NOs: 543 and 544; SEQ ID NOs: 545 and 546; SEQ ID NOs: 547 and 548; SEQ ID NOs: 549 and 550; SEQ ID NOs: 551 and 552; SEQ ID NOs: 553 and 554; SEQ ID NOs: 555 and 556; SEQ ID NOs: 557 and 558; SEQ ID NOs: 559 and 560; SEQ ID NOs: 561 and 562; SEQ ID NOs: 563 and 564; SEQ ID NOs: 565 and 566; SEQ ID NOs: 567 and 568; SEQ ID NOs: 569 and 570; SEQ ID NOs: 561 and 562; SEQ ID NOs: 563 and 564; SEQ ID NOs: 565 and 566; SEQ ID NOs: 567 and 568; SEQ ID NOs: 569 and 570; SEQ ID NOs: 571 and 572; SEQ ID NOs: 573 and 574; SEQ ID NOs: 575 and 576; SEQ ID NOs: 577 and 578; SEQ ID NOs: 579 and 580; SEQ ID NOs: 581 and 582; SEQ ID NOs: 583 and 584; SEQ ID NOs: 585 and 586; SEQ ID NOs: 587 and 588; SEQ ID NOs: 589 and 590;SEQ ID NOs: 591 and 592; SEQ ID NOs: 593 and 594; SEQ ID NOs: 595 and 596; SEQ ID NOs: 597 and 598; SEQ ID NOs: 599 and 600; SEQ ID NOs: 601 and 602; SEQ ID NOs: 603 and 604; SEQ ID NOs: 605 and 606; SEQ ID NOs: 607 and 608; SEQ ID NOs: 609 and 610; SEQ ID NOs: 611 and 612; SEQ ID NOs: 613 and 614; SEQ ID NOs: 615 and 616; SEQ ID NOs: 617 and 618; SEQ ID NOs: 619 and 620; SEQ ID NOs: 621 and 622; SEQ ID NOs: 623 and 624; SEQ ID NOs: 625 and 626; SEQ ID NOs: 627 and 628; SEQ ID NOs: 629 and 630; SEQ ID NOs: SEQ ID NOs: 631 and 632; SEQ ID NOs: 633 and 634; SEQ ID NOs: 635 and 636; SEQ ID NOs: 637 and 638; SEQ ID NOs: 639 and 640; SEQ ID NOs: 641 and 642; SEQ ID NOs: 643 and 644; SEQ ID NOs: 645 and 646; SEQ ID NOs: 647 and 648; SEQ ID NOs: 649 and 650; SEQ ID NOs: 651 and 652; SEQ ID NOs: 653 and 654; SEQ ID NOs: 655 and 656; SEQ ID NOs: 657 and 658; SEQ ID NOs: 659 and 660; SEQ ID NOs: 661 and 662; SEQ ID NOs: 663 and 664; SEQ ID NOs: 665 and 666; SEQ ID NOs: 667 and 668; SEQ ID NOs: 669 and 670; SEQ ID NOs: 671 and 672; SEQ ID NOs: 671 and 672; SEQ ID NOs: 673 and 674; SEQ ID NOs: 675 and 676; SEQ ID NOs: 677 and 678; SEQ ID NOs: 679 and 680; SEQ ID NOs: 681 and 682; SEQ ID NOs: 683 and 684; SEQ ID NOs: 685 and 686; SEQ ID NOs: 687 and 688; SEQ ID NOs: 689 and 690; SEQ ID NOs: 691 and 692; SEQ ID NOs: 693 and 694; SEQ ID NOs: 695 and 696; SEQ ID NOs: 697 and 698; SEQ ID NOs: 699 and 700;SEQ ID NOs: 701 and 702; SEQ ID NOs: 703 and 704; SEQ ID NOs: 705 and 706; SEQ ID NOs: 707 and 708; SEQ ID NOs: 709 and 710; SEQ ID NOs: 711 and 712; SEQ ID NOs: 713 and 714; SEQ ID NOs: 715 and 716; SEQ ID NOs: 717 and 718; SEQ ID NOs: 719 and 720; SEQ ID NOs: 721 and 722; SEQ ID NOs: 723 and 724; SEQ ID NOs: 725 and 726; SEQ ID NOs: 727 and 728; SEQ ID NOs: 729 and 730; SEQ ID NOs: 731 and 732; SEQ ID NOs: 733 and 734; SEQ ID NOs: 735 and 736; SEQ ID NOs: 737 and 738; SEQ ID NOs: 739 and 740; SEQ ID NOs: 741 and 742; SEQ ID NOs: 741 and 742; SEQ ID NOs: 743 and 744; SEQ ID NOs: 745 and 746; SEQ ID NOs: 747 and 748; SEQ ID NOs: 749 and 750; SEQ ID NOs: 751 and 752; SEQ ID NOs: 753 and 754; SEQ ID NOs: 755 and 756; SEQ ID NOs: 757 and 758; SEQ ID NOs: 759 and 760; SEQ ID NOs: 761 and 762; SEQ ID NOs: 763 and 764; SEQ ID NOs: 765 and 766; SEQ ID NOs: 767 and 768; SEQ ID NOs: 769 and 770; SEQ ID NOs: 771 and 772; SEQ ID NOs: 773 and 774; SEQ ID NOs: 775 and 776; SEQ ID NOs: 777 and 778; SEQ ID NOs: 779 and 780; SEQ ID NOs: 781 and 782; SEQ ID NOs: 781 and 782; SEQ ID NOs: 783 and 784; SEQ ID NOs: 785 and 786; SEQ ID NOs: 787 and 788; SEQ ID NOs: 789 and 790; SEQ ID NOs: 791 and 792; SEQ ID NOs: 793 and 794; SEQ ID NOs: 795 and 796; SEQ ID NOs: 797 and 798; SEQ ID NOs: 799 and 800; SEQ ID NOs: 801 and 802; SEQ ID NOs: 803 and 804; SEQ ID NOs: 805 and 806; SEQ ID NOs: 807 and 808; SEQ ID NOs: 809 and 810;SEQ ID NOs: 811 and 812; SEQ ID NOs: 813 and 814; SEQ ID NOs: 815 and 816; SEQ ID NOs: 817 and 818; SEQ ID NOs: 819 and 820; SEQ ID NOs: 821 and 822; SEQ ID NOs: 823 and 824; SEQ ID NOs: 825 and 826; SEQ ID NOs: 827 and 828; SEQ ID NOs: 829 and 830; SEQ ID NOs: 831 and 832; SEQ ID NOs: 833 and 834; SEQ ID NOs: 835 and 836; SEQ ID NOs: 837 and 838; SEQ ID NOs: 839 and 840; SEQ ID NOs: 841 and 842; SEQ ID NOs: 843 and 844; SEQ ID NOs: 845 and 846; SEQ ID NOs: 847 and 848; SEQ ID NOs: 849 and 850; SEQ ID NOs: 851 and 852; SEQ ID NOs: 853 and 854; SEQ ID NOs: 855 and 856; SEQ ID NOs: 857 and 858; SEQ ID NOs: 859 and 860; SEQ ID NOs: 851 and 852; SEQ ID NOs: 853 and 854; SEQ ID NOs: 855 and 856; SEQ ID NOs: 857 and 858; SEQ ID NOs: 969 and 970; SEQ ID NOs: 971 and 972; SEQ ID NOs: 973 and 974; SEQ ID NOs: 1765 and 1766; SEQ ID NOs: 1769 and 1770; SEQ ID NOs: 1771 and 1772; SEQ ID NOs: 1773 and 1774; SEQ ID NOs: 1775 and 1776; SEQ ID NOs: 1777 and 1778; SEQ ID NOs: 1785 and 1786; SEQ ID NOs: 1801 and 1802; SEQ ID NOs: 1805 and 1806; SEQ ID NOs: 1811 and 1812; SEQ ID NOs: 1813 and 1814; SEQ ID NOs: 1815 and 1816; SEQ ID NOs: 1815 and 1816; SEQ ID NOs: 1817 and 1818; SEQ ID NOs: 1819 and 1820; SEQ ID NOs: 1839 and 1840; SEQ ID NOs: 1841 and 1842; SEQ ID NOs: 1843 and 1844; SEQ ID NOs: 1845 and 1846; SEQ ID NOs: 1847 and 1848; SEQ ID NOs: 1849 and 1850; SEQ ID NOs: 1851 and 1852; SEQ ID NOs: 1853 and 1854; SEQ ID NOs: 1855 and 1856; SEQ ID NOs: 1857 and 1858; SEQ ID NOs: 1859 and 1860;SEQ ID NOs: 1861 and 1862; SEQ ID NOs: 1863 and 1864; SEQ ID NOs: 1865 and 1866; SEQ ID NOs: 1867 and 1868; SEQ ID NOs: 1869 and 1870; SEQ ID NOs: 1871 and 1872; or, SEQ ID NOs: 1873 and 1874. ; 11. A double-stranded RNAi agent comprising the dsRNA according to any one of claims 1 to 10, and optionally comprising a targeting ligand.

12. The double-stranded RNAi agent according to claim 11, characterized in that The double-stranded RNAi agent comprises the dsRNA of any one of claims 1 to 10, and at least one targeting ligand, wherein the sense strand of the dsRNA is conjugated to the targeting ligand.

13. The double-stranded RNAi agent according to claim 11, characterized in that The targeting ligand comprises N-acetyl-galactosamine (GalNAc), or the targeting ligand is a GalNAc derivative.

14. The double-stranded RNAi agent according to claim 11, characterized in that The structure of the double-stranded RNAi agent is selected from Table A1, wherein R 2 The dsRNA according to any one of claims 1 to 10.

15. A double-stranded ribonucleic acid interference (dsRNAi) agent that inhibits the expression of inhibin subunit beta E (INHBE) in a cell, wherein the dsRNAi agent comprises a sense strand and an antisense strand that form a double-stranded RNA (dsRNA) region, the antisense strand comprising a region complementary to INHBE mRNA, wherein the complementary region comprises at least 15 consecutive nucleotides, wherein the dsRNAi agent comprises at least one non-canonical base-pairing nucleotide.

16. The dsRNAi agent according to claim 15, wherein The dsRNAi agent comprises at least one non-canonical base-pairing nucleotide in positions 1-11 in the direction from the 5' end to the 3' end; and / or comprises at least one non-canonical base-pairing nucleotide in positions 12-21.

17. The dsRNAi agent according to claim 15 or 16, wherein The dsRNAi agent comprises one, two, three, four, five or more non-canonical base-pairing nucleotides.

18. The dsRNAi agent according to any one of claims 15 to 17, wherein For the sequence fragment N1N2G0N3N4 in positions 1-11 or positions 12-21 in the direction from the 5' end to the 3' end, wherein N1, N2, N3 and N4 are each independently a nucleotide containing adenine (A), cytosine (C), guanine (G), thymine (T) or uracil (U) as a base, G0 is a nucleotide containing guanine as a base, when at least three bases among N1, N2, N3 and N4 are adenine or uracil, the guanine of G0 in the sequence is replaced by hypoxanthine (I).

19. The dsRNAi agent according to claim 18, wherein At least one of N1, N2, N3, N4 and G0 further has a modified sugar group and / or a modified internucleotide linkage.

20. The dsRNAi agent according to any one of claims 15 to 19, wherein The dsRNAi agent or RNA comprises at least one non-canonical base-pairing nucleotide and / or modified nucleotide in the 6th, 7th or 8th position in the direction from the 5' end to the 3' end of the oligonucleotide chain.

21. The dsRNAi agent according to any one of claims 15 to 20, wherein When at least four nucleotides in positions 2-8 in the direction from the 5' end to the 3' end are A or U, and at least one nucleotide in positions 6-8 is G, the G at positions 6, 7 and / or 8 is replaced by a non-canonical base pairing nucleotide.

22. The dsRNAi agent according to any one of claims 15 to 21, wherein The bases in the non-canonical base pairing nucleotides include bases selected from inosine (I), xanthosine (X), 7-methylguanosine (m7G), N6-methyladenosine (m6A), dihydrouridine, 5-methylcytosine (m5C), pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ).

23. A pharmaceutical composition, characterized in that It comprises the double-stranded RNAi agent or a pharmaceutically acceptable salt thereof according to any one of claims 11 to 22, and a pharmaceutically acceptable carrier.

24. The pharmaceutical composition according to claim 23, characterized in that The pharmaceutical composition is formulated for subcutaneous or intravenous administration.

25. The pharmaceutical composition according to claim 23 or 24, characterized in that The carrier is a non-buffered solution or a buffered solution.

26. A method for inhibiting INHBE expression in a cell, characterized in that: The cell is contacted with the double-stranded RNAi agent according to any one of claims 11 to 22, so that the mRNA transcript of the INHBE gene is degraded, thereby inhibiting the expression of the INHBE gene in the cell.

27. The method of claim 26, wherein: The cells are hepatocytes.

28. The method according to claim 26 or 27, characterized in that INHBE expression is inhibited by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

29. Use of a double-stranded RNAi agent as described in any one of claims 11-22 for the preparation of a medicament for treating a subject having a disorder mediated by INHBE expression.

30. The use according to claim 29, characterized in that The subject suffers from a metabolic disorder.

31. The use according to claim 30, characterized in that The metabolic disorder is metabolic syndrome, type 2 diabetes, obesity, hypertriglyceridemia, dyslipidemia, liver inflammation, fatty liver, hypercholesterolemia, liver disease associated with elevated liver enzymes, non-alcoholic steatohepatitis, cardiovascular disease, kidney disease; optionally, the metabolic syndrome includes one or more of abdominal obesity, insulin resistance, dyslipidemia and hypertension.

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