siRNA targeting inhibin βE, siRNA complexes and pharmaceutical uses thereof

Abstract

The present disclosure relates to siRNAs, siRNA complexes, and pharmaceutical uses thereof that target inhibin βE, particularly to siRNAs, siRNA complexes, compositions, and pharmaceutical uses thereof that target inhibin βE. The present disclosure also relates to pharmaceutical compositions, cells, or reagent kits containing the siRNAs, and methods of using the siRNAs and siRNA complexes to treat and / or prevent subjects suffering from related disorders.

Description

[Technical Field]

[0001] This disclosure claims priority to Chinese Patent Application No. 202310194029.7 filed on March 2, 2023, Chinese Patent Application No. 202310420703.9 filed on April 19, 2023, Chinese Patent Application No. 202311271773.9 filed on September 28, 2023, and Chinese Patent Application No. 202410023838.6 filed on January 8, 2024, the contents of which are all incorporated herein by reference.

[0002] The present disclosure belongs to the field of biomedicine, and specifically relates to siRNAs, complexes, compositions targeting the inhibin βE gene (INHBE), and pharmaceutical uses thereof. [Background technology]

[0003] The inhibin βE gene (INHBE) is a member of the TGF-β (transforming growth factor-β) protein superfamily and is involved in the regulation of many cellular processes, including cell proliferation, apoptosis, immune responses, and hormone secretion. Inhibin plays a role in inhibiting the secretion of follicle-stimulating hormone by the pituitary gland and may be involved in the regulation of many different functions, such as hypothalamic and pituitary hormone secretion, gonadal hormone secretion, germ cell development and maturation, erythroid differentiation, insulin secretion, neuronal survival, embryonic axis development, or skeletal growth. Furthermore, INHBE can be upregulated under conditions of endoplasmic reticulum stress, which may inhibit cell proliferation and growth in the pancreas and liver.

[0004] Body fat distribution is an important criterion for determining cardiovascular and metabolic diseases. Among various body fat distributions, there are specific types of body fat distribution that are prone to serious cardiovascular or metabolic diseases, characterized by a relatively high distribution of fat around the waist and / or a relatively low distribution of fat around the buttocks, resulting in a relatively high waist-to-hip ratio (WHR). Metabolic diseases caused by abnormal body fat distribution include type 2 diabetes, hyperlipidemia, dyslipidemia (elevated or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very-low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B, or other lipid moieties), obesity (especially abdominal obesity), dyslipidemia, insulin resistance, nonalcoholic steatohepatitis, elevated blood pressure and / or hypertension, hyperglycemia, metabolic syndrome, coronary artery disease, and other atherosclerotic diseases.

[0005] Currently, the methods for treating metabolic diseases mainly involve lifestyle modification, dietary adjustment, exercise fitness, and the use of lipid-lowering drugs (such as statins), but these methods are relatively ineffective, and the use of lipid-lowering drugs causes some side effects.Interfering RNAi (siRNA) is an effective method for silencing gene expression, and can specifically degrade target gene mRNAs such as INHBE associated with metabolic diseases through post-transcriptional control, thereby effectively inhibiting the expression of the target gene and achieving the goal of effectively treating such diseases. Summary of the Invention

[0006] WO2022028462A relates to modified siRNAs for suppressing off-targets, and the present disclosure incorporates the above patent application in its entirety.

[0007] WO2023274395A relates to nucleic acid ligands and conjugates thereof, and the present disclosure is incorporated by reference in its entirety.

[0008] The present disclosure provides siRNAs that target INHBE.

[0009] In some embodiments, the present disclosure provides siRNAs comprising a sense strand and an antisense strand that form a double-stranded region.

[0010] the sense strand comprises at least 15 (e.g., 16, 17, 18, 19, 20, or 21) consecutive nucleotides and differs from any one of SEQ ID NOs: 1 to 9 by 3 or less (e.g., 0, 1, 2, or 3) nucleotides; The antisense strand contains at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, 23) consecutive nucleotides and differs from any one of the nucleotide sequences of SEQ ID NO:10 to SEQ ID NO:18 by 3 or less (e.g., 0, 1, 2, 3) nucleotides.

[0011] In some embodiments, antisense strand is at least partially reverse complementary to target sequence, so as to mediate RNA interference.In some embodiments, there are 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less mismatches between antisense strand and target sequence.In some embodiments, antisense strand is completely reverse complementary to target sequence.

[0012] In some embodiments, the sense strand is at least partially reverse-complementary to the antisense strand to form a double-stranded region. In some embodiments, there are 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less mismatches between the sense strand and the antisense strand. In some embodiments, the sense strand is completely reverse-complementary to the antisense strand.

[0013] In some embodiments, the siRNA of the present disclosure comprises one or two blunt ends.

[0014] In some specific embodiments, each strand of the siRNA independently contains 1 to 2 unpaired nucleotides.

[0015] In some embodiments, the siRNA of the present disclosure comprises an overhang located at the 3' end of the antisense strand.

[0016] In some embodiments, the sense strand and the antisense strand each independently have 16 to 35, 16 to 34, 17 to 34, 17 to 33, 18 to 33, 18 to 32, 18 to 31, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 19 to 25, 19 to 24, or 19 to 23 nucleotides (e.g., 19, 20, 21, 22, or 23 nucleotides).

[0017] In some embodiments, the sense strand and the antisense strand are the same or different in length, and the sense strand is 19 to 23 nucleotides in length and the antisense strand is 19 to 26 nucleotides in length. The length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure may be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 19, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25, or 23 / 26.

[0018] In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 21, 21 / 21, 21 / 23, or 23 / 25. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 21, 21 / 21, or 21 / 23. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 21.

[0019] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides and differs by no more than two nucleotides from the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 9, SEQ ID NO: 19 to SEQ ID NO: 122. In some embodiments, the sense strand differs by no more than one nucleotide, and in some embodiments, by one nucleotide. In some embodiments, the antisense strand comprises a sequence of at least 15 consecutive nucleotides and differs by no more than two nucleotides from any one of the nucleotide sequences of SEQ ID NO: 10 to SEQ ID NO: 18, SEQ ID NO: 123 to SEQ ID NO: 226, and in some embodiments, differs by no more than one nucleotide from the nucleotide sequence, and in some embodiments, differs by one nucleotide.

[0020] In some embodiments, the sense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of any one of SEQ ID NOs: 1 to 9, 19 to 122. In some embodiments, the sense strand comprises at least 16 contiguous nucleotides of the nucleotide sequence of any one of SEQ ID NOs: 1 to 9, 19 to 122. In some embodiments, the sense strand comprises at least 17 contiguous nucleotides of the nucleotide sequence of any one of SEQ ID NOs: 1 to 9, 19 to 122. In some embodiments, the sense strand comprises at least 19 contiguous nucleotides of the nucleotide sequence of any one of SEQ ID NOs: 1 to 9, 19 to 122.

[0021] In some embodiments, the antisense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of any one of SEQ ID NOs: 10 to 18, 123 to 226. In some embodiments, the antisense strand comprises at least 17 contiguous nucleotides of the nucleotide sequence of any one of SEQ ID NOs: 10 to 18, 123 to 226. In some embodiments, the antisense strand comprises at least 19 contiguous nucleotides of the nucleotide sequence of any one of SEQ ID NOs: 10 to 18, 123 to 226. In some embodiments, the antisense strand comprises at least 20 contiguous nucleotides of the nucleotide sequence of any one of SEQ ID NOs: 10 to 18, 123 to 226. In some embodiments, the antisense strand comprises at least 21 contiguous nucleotides of the nucleotide sequence of any one of SEQ ID NOs: 10 to 18, 123 to 226.

[0022] In some embodiments, the sense strand comprises or is selected from the nucleotide sequence of any one of SEQ ID NO:1 to SEQ ID NO:9, SEQ ID NO:19 to SEQ ID NO:122.

[0023] In some embodiments, the antisense strand comprises or is selected from the nucleotide sequence of any one of SEQ ID NO: 10 to SEQ ID NO: 18, and SEQ ID NO: 123 to SEQ ID NO: 226.

[0024] In some embodiments, the present specification provides an siRNA targeting inhibin β E, comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand and the antisense strand are 1) a sense strand shown in SEQ ID NO: 1 and an antisense strand shown in SEQ ID NO: 10; 2) the sense strand shown in SEQ ID NO: 2 and the antisense strand shown in SEQ ID NO: 11; 3) the sense strand shown in SEQ ID NO: 3 and the antisense strand shown in SEQ ID NO: 12; 4) the sense strand shown in SEQ ID NO: 4 and the antisense strand shown in SEQ ID NO: 13; 5) the sense strand shown in SEQ ID NO: 5 and the antisense strand shown in SEQ ID NO: 14; 6) the sense strand shown in SEQ ID NO: 6 and the antisense strand shown in SEQ ID NO: 15; 7) the sense strand shown in SEQ ID NO: 7 and the antisense strand shown in SEQ ID NO: 16; 8) the sense strand shown in SEQ ID NO: 8 and the antisense strand shown in SEQ ID NO: 17; 9) the sense strand shown in SEQ ID NO: 9 and the antisense strand shown in SEQ ID NO: 18; 10) the sense strand shown in SEQ ID NO: 19 and the antisense strand shown in SEQ ID NO: 123; 11) the sense strand shown in SEQ ID NO: 20 and the antisense strand shown in SEQ ID NO: 124; 12) the sense strand shown in SEQ ID NO: 21 and the antisense strand shown in SEQ ID NO: 125; 13) the sense strand shown in SEQ ID NO: 22 and the antisense strand shown in SEQ ID NO: 126; 14) the sense strand shown in SEQ ID NO: 23 and the antisense strand shown in SEQ ID NO: 127; 15) the sense strand shown in SEQ ID NO: 24 and the antisense strand shown in SEQ ID NO: 128; 16) the sense strand shown in SEQ ID NO: 25 and the antisense strand shown in SEQ ID NO: 129; 17) A sense strand shown in SEQ ID NO: 26 and an antisense strand shown in SEQ ID NO: 130; 18) the sense strand shown in SEQ ID NO: 27 and the antisense strand shown in SEQ ID NO: 131; 19) The sense strand shown in SEQ ID NO: 28 and the antisense strand shown in SEQ ID NO: 132; 20) the sense strand shown in SEQ ID NO: 29 and the antisense strand shown in SEQ ID NO: 133; 21) A sense strand shown in SEQ ID NO: 30 and an antisense strand shown in SEQ ID NO: 134; 22) the sense strand shown in SEQ ID NO: 31 and the antisense strand shown in SEQ ID NO: 135; 23) the sense strand shown in SEQ ID NO: 32 and the antisense strand shown in SEQ ID NO: 136; 24) The sense strand shown in SEQ ID NO: 33 and the antisense strand shown in SEQ ID NO: 137; 25) the sense strand shown in SEQ ID NO: 34 and the antisense strand shown in SEQ ID NO: 138; 26) The sense strand shown in SEQ ID NO: 35 and the antisense strand shown in SEQ ID NO: 139; 27) The sense strand shown in SEQ ID NO: 36 and the antisense strand shown in SEQ ID NO: 140; 28) The sense strand shown in SEQ ID NO: 37 and the antisense strand shown in SEQ ID NO: 141; 29) The sense strand shown in SEQ ID NO: 38 and the antisense strand shown in SEQ ID NO: 142; 30) The sense strand shown in SEQ ID NO: 39 and the antisense strand shown in SEQ ID NO: 143; 31) The sense strand shown in SEQ ID NO: 40 and the antisense strand shown in SEQ ID NO: 144; 32) The sense strand shown in SEQ ID NO: 41 and the antisense strand shown in SEQ ID NO: 145; 33) the sense strand shown in SEQ ID NO: 42 and the antisense strand shown in SEQ ID NO: 146; 34) The sense strand shown in SEQ ID NO: 43 and the antisense strand shown in SEQ ID NO: 147; 35) The sense strand shown in SEQ ID NO: 44 and the antisense strand shown in SEQ ID NO: 148; 36) The sense strand shown in SEQ ID NO: 45 and the antisense strand shown in SEQ ID NO: 149; 37) The sense strand shown in SEQ ID NO: 46 and the antisense strand shown in SEQ ID NO: 150; 38) The sense strand shown in SEQ ID NO: 47 and the antisense strand shown in SEQ ID NO: 151; 39) The sense strand shown in SEQ ID NO: 48 and the antisense strand shown in SEQ ID NO: 152; 40) The sense strand shown in SEQ ID NO: 49 and the antisense strand shown in SEQ ID NO: 153; 41) The sense strand shown in SEQ ID NO: 50 and the antisense strand shown in SEQ ID NO: 154; 42) The sense strand shown in SEQ ID NO: 51 and the antisense strand shown in SEQ ID NO: 155; 43) The sense strand shown in SEQ ID NO: 52 and the antisense strand shown in SEQ ID NO: 156; 44) The sense strand shown in SEQ ID NO: 53 and the antisense strand shown in SEQ ID NO: 157; 45) The sense strand shown in SEQ ID NO: 54 and the antisense strand shown in SEQ ID NO: 158; 46) The sense strand shown in SEQ ID NO: 55 and the antisense strand shown in SEQ ID NO: 159; 47) The sense strand shown in SEQ ID NO: 56 and the antisense strand shown in SEQ ID NO: 160; 48) The sense strand shown in SEQ ID NO: 57 and the antisense strand shown in SEQ ID NO: 161; 49) The sense strand shown in SEQ ID NO: 58 and the antisense strand shown in SEQ ID NO: 162; 50) The sense strand shown in SEQ ID NO: 59 and the antisense strand shown in SEQ ID NO: 163; 51) The sense strand shown in SEQ ID NO: 60 and the antisense strand shown in SEQ ID NO: 164; 52) The sense strand shown in SEQ ID NO: 61 and the antisense strand shown in SEQ ID NO: 165; 53) The sense strand shown in SEQ ID NO: 62 and the antisense strand shown in SEQ ID NO: 166; 54) The sense strand shown in SEQ ID NO: 63 and the antisense strand shown in SEQ ID NO: 167; 55) The sense strand shown in SEQ ID NO: 64 and the antisense strand shown in SEQ ID NO: 168; 56) The sense strand shown in SEQ ID NO: 65 and the antisense strand shown in SEQ ID NO: 169; 57) The sense strand shown in SEQ ID NO: 66 and the antisense strand shown in SEQ ID NO: 170; 58) The sense strand shown in SEQ ID NO: 67 and the antisense strand shown in SEQ ID NO: 171; 59) The sense strand shown in SEQ ID NO: 68 and the antisense strand shown in SEQ ID NO: 172; 60) The sense strand shown in SEQ ID NO: 69 and the antisense strand shown in SEQ ID NO: 173; 61) The sense strand shown in SEQ ID NO: 70 and the antisense strand shown in SEQ ID NO: 174; 62) The sense strand shown in SEQ ID NO: 71 and the antisense strand shown in SEQ ID NO: 175; 63) The sense strand shown in SEQ ID NO: 72 and the antisense strand shown in SEQ ID NO: 176; 64) The sense strand shown in SEQ ID NO: 73 and the antisense strand shown in SEQ ID NO: 177; 65) The sense strand shown in SEQ ID NO: 74 and the antisense strand shown in SEQ ID NO: 178; 66) The sense strand shown in SEQ ID NO: 75 and the antisense strand shown in SEQ ID NO: 179; 67) The sense strand shown in SEQ ID NO: 76 and the antisense strand shown in SEQ ID NO: 180; 68) The sense strand shown in SEQ ID NO: 77 and the antisense strand shown in SEQ ID NO: 181; 69) The sense strand shown in SEQ ID NO: 78 and the antisense strand shown in SEQ ID NO: 182; 70) The sense strand shown in SEQ ID NO: 79 and the antisense strand shown in SEQ ID NO: 183; 71) The sense strand shown in SEQ ID NO: 80 and the antisense strand shown in SEQ ID NO: 184; 72) The sense strand shown in SEQ ID NO: 81 and the antisense strand shown in SEQ ID NO: 185; 73) The sense strand shown in SEQ ID NO: 82 and the antisense strand shown in SEQ ID NO: 186; 74) The sense strand shown in SEQ ID NO: 83 and the antisense strand shown in SEQ ID NO: 187; 75) The sense strand shown in SEQ ID NO: 84 and the antisense strand shown in SEQ ID NO: 188; 76) The sense strand shown in SEQ ID NO: 85 and the antisense strand shown in SEQ ID NO: 189; 77) The sense strand shown in SEQ ID NO: 86 and the antisense strand shown in SEQ ID NO: 190; 78) The sense strand shown in SEQ ID NO: 87 and the antisense strand shown in SEQ ID NO: 191; 79) The sense strand shown in SEQ ID NO: 88 and the antisense strand shown in SEQ ID NO: 192; 80) The sense strand shown in SEQ ID NO: 89 and the antisense strand shown in SEQ ID NO: 193; 81) The sense strand shown in SEQ ID NO: 90 and the antisense strand shown in SEQ ID NO: 194; 82) The sense strand shown in SEQ ID NO: 91 and the antisense strand shown in SEQ ID NO: 195; 83) The sense strand shown in SEQ ID NO: 92 and the antisense strand shown in SEQ ID NO: 196; 84) The sense strand shown in SEQ ID NO: 93 and the antisense strand shown in SEQ ID NO: 197; 85) The sense strand shown in SEQ ID NO: 94 and the antisense strand shown in SEQ ID NO: 198; 86) The sense strand shown in SEQ ID NO: 95 and the antisense strand shown in SEQ ID NO: 199; 87) The sense strand shown in SEQ ID NO: 96 and the antisense strand shown in SEQ ID NO: 200; 88) The sense strand shown in SEQ ID NO: 97 and the antisense strand shown in SEQ ID NO: 201; 89) The sense strand shown in SEQ ID NO: 98 and the antisense strand shown in SEQ ID NO: 202; 90) The sense strand shown in SEQ ID NO: 99 and the antisense strand shown in SEQ ID NO: 203; 91) The sense strand shown in SEQ ID NO: 100 and the antisense strand shown in SEQ ID NO: 204; 92) The sense strand shown in SEQ ID NO: 101 and the antisense strand shown in SEQ ID NO: 205; 93) The sense strand shown in SEQ ID NO: 102 and the antisense strand shown in SEQ ID NO: 206; 94) The sense strand shown in SEQ ID NO: 103 and the antisense strand shown in SEQ ID NO: 207; 95) The sense strand shown in SEQ ID NO: 104 and the antisense strand shown in SEQ ID NO: 208; 96) The sense strand shown in SEQ ID NO: 105 and the antisense strand shown in SEQ ID NO: 209; 97) The sense strand shown in SEQ ID NO: 106 and the antisense strand shown in SEQ ID NO: 210; 98) The sense strand shown in SEQ ID NO: 107 and the antisense strand shown in SEQ ID NO: 211; 99) The sense strand shown in SEQ ID NO: 108 and the antisense strand shown in SEQ ID NO: 212; 100) The sense strand shown in SEQ ID NO: 109 and the antisense strand shown in SEQ ID NO: 213; 101) The sense strand shown in SEQ ID NO: 110 and the antisense strand shown in SEQ ID NO: 214; 102) The sense strand shown in SEQ ID NO: 111 and the antisense strand shown in SEQ ID NO: 215; 103) The sense strand shown in SEQ ID NO: 112 and the antisense strand shown in SEQ ID NO: 216; 104) The sense strand shown in SEQ ID NO: 113 and the antisense strand shown in SEQ ID NO: 217; 105) The sense strand shown in SEQ ID NO: 114 and the antisense strand shown in SEQ ID NO: 218; 106) The sense strand shown in SEQ ID NO: 115 and the antisense strand shown in SEQ ID NO: 219; 107) The sense strand shown in SEQ ID NO: 116 and the antisense strand shown in SEQ ID NO: 220; 108) The sense strand shown in SEQ ID NO: 117 and the antisense strand shown in SEQ ID NO: 221; 109) The sense strand shown in SEQ ID NO: 118 and the antisense strand shown in SEQ ID NO: 222; 110) The sense strand shown in SEQ ID NO: 119 and the antisense strand shown in SEQ ID NO: 223; 111) The sense strand shown in SEQ ID NO: 120 and the antisense strand shown in SEQ ID NO: 224; 112) the sense strand set forth in SEQ ID NO: 121 and the antisense strand set forth in SEQ ID NO: 225, and 113) The sense strand shown in SEQ ID NO: 122 and the antisense strand shown in SEQ ID NO: 226; The present invention may include or be selected from the following combinations:

[0025] In some embodiments, at least one nucleotide in the sense strand and / or the antisense strand is a modified nucleotide.

[0026] In some embodiments, all nucleotides are modified nucleotides.

[0027] In some embodiments, the modified nucleotide is a 2'-methoxy modified nucleotide or a 2'-fluoro modified nucleotide.

[0028] In some embodiments, three consecutive nucleotides in the sense strand of the siRNA are 2'-fluoro modified nucleotides.

[0029] In some embodiments, in the sense strand of the siRNA, three consecutive nucleotides located at positions 7 to 9 of the 5' end are 2'-fluoro-modified nucleotides.

[0030] In some embodiments, in the sense strand of the siRNA, three consecutive nucleotides located at positions 7 to 9 of the 5' end are 2'-fluoro-modified nucleotides, and the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides. In some embodiments, in the sense strand, four nucleotides located at positions 5 and 7 to 9 of the 5' end are each independently 2'-fluoro-modified nucleotides, and the remaining positions of the sense strand are all 2'-methoxy-modified nucleotides.

[0031] In some embodiments, from the 5' to the 3' direction, the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand are each independently modified with a 2'-fluoro group, and the remaining nucleotides are modified with a 2'-methoxy group. In some embodiments, from the 5' to the 3' direction, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, and 18 of the antisense strand are each independently modified with a 2'-fluoro group, and the remaining nucleotides are modified with a 2'-methoxy group. In some embodiments, from the 5' to the 3' direction, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are each independently modified with a 2'-fluoro group, and the remaining nucleotides are modified with a 2'-methoxy group.

[0032] In some embodiments, at least one phosphodiester group in the sense strand and / or antisense strand is a phosphodiester group having a modified group. This modified group allows the siRNA to have improved stability in biological samples or environments. In some embodiments, the phosphodiester group having a modified group is a thiophosphodiester group.

[0033] In some embodiments, the thiophosphodiester group is Between the first and second nucleotides of the 5'-terminal end of the sense strand, Between the second and third nucleotides at the 5'-end of the sense strand, Between the first and second nucleotides at the 3'-end of the sense strand, Between the second and third nucleotides at the 3'-end of the sense strand, Between the first and second nucleotides at the 5' end of the antisense strand, Between the second and third nucleotides at the 5' end of the antisense strand, Between the first and second nucleotides at the 3'-terminal end of the antisense strand, and Between the second and third nucleotides at the 3' end of the antisense strand, It is in at least one of the positions selected from the above.

[0034] In some embodiments, the sense strand and / or the antisense strand comprises a plurality of thiophosphodiester groups, the thiophosphodiester groups being: Between the first and second nucleotides of the 5'-terminal end of the sense strand, Between the second and third nucleotides at the 5'-end of the sense strand, Between the first and second nucleotides at the 5' end of the antisense strand, Between the second and third nucleotides at the 5' end of the antisense strand, Between the first and second nucleotides at the 3'-terminal end of the antisense strand, and Between the second and third nucleotides at the 3' end of the antisense strand, is located.

[0035] The present disclosure further provides an siRNA complex comprising any one of the above siRNAs and a targeting ligand linked to the end of the siRNA.

[0036] In some embodiments, the siRNA is covalently or non-covalently linked to the targeting ligand.

[0037] In some embodiments, the targeting ligand targets the liver; in some embodiments, the targeting ligand binds to the asialoglycoprotein receptor (ASGPR); and in some embodiments, the targeting ligand comprises a galactose cluster or a galactose derivative cluster, and the galactose derivative is selected from N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, Nn-butyrylgalactosamine, or N-isobutyrylgalactosamine.

[0038] In some embodiments, the targeting ligand comprises one or more of N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, Nn-butyrylgalactosamine, or N-isobutyrylgalactosamine, and preferably comprises N-acetyl-galactosamine. In some embodiments, the targeting ligand is linked to the 3' end of the sense strand of the siRNA.

[0039] In some embodiments, the targeting ligand is linked to the 3' or 5' end of the sense or antisense strand in the siRNA via a phosphodiester, thiophosphodiester, or phosphonate group.

[0040] In some embodiments, the targeting ligand is linked to the siRNA terminus via a phosphodiester group.

[0041] In some embodiments, the targeting ligand is indirectly linked to the siRNA terminus via a phosphodiester, thiophosphodiester, or phosphonate group, hi some embodiments, the targeting ligand is indirectly linked to the siRNA terminus via a phosphodiester group.

[0042] In some embodiments, the targeting ligand is linked directly to the siRNA terminus via a phosphodiester group, a thiophosphodiester group, or a phosphonate group, and in some embodiments, directly to the siRNA terminus via a phosphodiester group.

[0043] In some embodiments, the targeting ligand is linked directly to the 3' end of the siRNA sense strand via a phosphodiester or thiophosphodiester group, and in some embodiments, is linked directly to the 3' end of the siRNA sense strand via a phosphodiester group.

[0044] In some embodiments, the targeting moiety of the targeting ligand comprises one or more targeting groups or moieties that cooperate to direct the delivery of the therapeutic reagent linked to it to a desired target location. In some cases, the targeting moiety can bind to a cell or cell receptor and trigger endocytosis to facilitate entry of the therapeutic reagent into the cell. The targeting moiety can include a compound with affinity for a cell receptor or cell surface molecule or an antibody. Various targeting ligands, including targeting moieties, can be linked to therapeutic reagents and other compounds to target the reagent to a cell and a specific cell receptor.

[0045] In some embodiments, types of targeting moieties include carbohydrates, cholesterol and cholesteryl groups, or steroids. Targeting moieties capable of binding to cell receptors include sugars such as galactose, galactose derivatives (e.g., N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, Nn-butyrylgalactosamine, N-isobutyrylgalactosamine), mannose, and mannose derivatives.

[0046] It has been shown that targeting moieties that bind to the asialoglycoprotein receptor (ASGPR) can be used to target the delivery of oligomeric compounds to the liver. The asialoglycoprotein receptor is abundantly expressed in liver cells (hepatocytes). Cellular receptor targeting moieties that target ASGPR include galactose and galactose derivatives. Specifically, clusters of galactose derivatives, including clusters of two, three, four, or more N-acetyl-galactosamine (GalNAc or NAG), can promote the uptake of certain compounds in hepatocytes. The GalNAc clusters coupled to oligomeric compounds target the compositions to the liver, where the N-acetyl-galactosamine sugars can bind to the asialoglycoprotein receptor on the surface of liver cells. Binding to the asialoglycoprotein receptor is thought to trigger receptor-mediated endocytosis, thereby promoting the entry of the compounds into the cells.

[0047] In some embodiments, the targeting ligand may comprise two, three, four, or more than four targeting moieties. In some embodiments, the targeting ligands disclosed herein may comprise one, two, three, four, or more than four targeting moieties linked to a branching group via L2.

[0048] In some embodiments, each targeting moiety comprises a galactosamine derivative, which is N-acetyl-galactosamine. Other sugars that can be used as targeting moieties and have affinity for the asialoglycoprotein receptor may be selected from galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, Nn-butyryl-galactosamine, and N-isobutyryl-galactosamine, etc.

[0049] In some embodiments, the targeting ligand of the present disclosure comprises N-acetylgalactosamine as a targeting moiety. [ka] .

[0050] In some embodiments, the targeting ligand comprises three terminal galactosamines or galactosamine derivatives (e.g., N-acetyl-galactosamine), each of which has affinity for a sialoglycoprotein receptor. In some embodiments, the targeting ligand comprises three terminal N-acetyl-galactosamines (GalNAc or NAG) as targeting moieties.

[0051] In some embodiments, the targeting ligand comprises four terminal galactosamines or galactosamine derivatives (e.g., N-acetyl-galactosamine), each of which has affinity for the asialoglycoprotein receptor. In some embodiments, the targeting ligand comprises four terminal N-acetyl-galactosamines (GalNAc or NAG) as targeting moieties.

[0052] The term commonly used in the art when referring to three terminal N-acetyl-galactosamines is tri-antennary. Includes antennary, trivalent and trimer.

[0053] The term commonly used in the art when referring to four terminal N-acetyl-galactosamines is tetraantennary. Antennary, tetravalent and tetramer.

[0054] In some embodiments, the targeting ligand provided by the present disclosure is a compound of Formula (II) or Formula (III) or a pharmaceutically acceptable salt thereof:

[0055] [ka] .

[0056] In some embodiments, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, Nn-butyrylgalactosamine, or N-isobutyrylgalactosamine can be substituted for the N-acetyl-galactosamine moiety in the targeting ligand.

[0057] In some embodiments, the siRNA and / or siRNA complex of the present disclosure has a nucleotide sequence set forth in any pair of sense and antisense strands in Tables 1a, 1b or 2a, 2b.

[0058] In some embodiments, the siRNA and / or siRNA complex of the present disclosure comprises or is selected from any one of SEQ ID NOs: 275 to 383.

[0059] In some embodiments, the sense strand in the siRNA and / or siRNA complex of the present disclosure comprises or is selected from the nucleotide sequence set forth in any one of SEQ ID NOs: 227 to 242, 243 to 250, and 275 to 383, and / or the antisense strand comprises or is selected from the nucleotide sequence set forth in any one of SEQ ID NOs: 251 to 266, 267 to 274, and 384 to 492.

[0060] In some embodiments, wherein the sense strand and the antisense strand are: 1) a sense strand shown in SEQ ID NO: 227 and an antisense strand shown in SEQ ID NO: 251; 2) the sense strand shown in SEQ ID NO: 228 and the antisense strand shown in SEQ ID NO: 252; 3) the sense strand shown in SEQ ID NO: 229 and the antisense strand shown in SEQ ID NO: 253; 4) the sense strand shown in SEQ ID NO: 230 and the antisense strand shown in SEQ ID NO: 254; 5) the sense strand shown in SEQ ID NO: 231 and the antisense strand shown in SEQ ID NO: 255; 6) the sense strand shown in SEQ ID NO: 232 and the antisense strand shown in SEQ ID NO: 256; 7) the sense strand shown in SEQ ID NO: 233 and the antisense strand shown in SEQ ID NO: 257; 8) the sense strand shown in SEQ ID NO: 234 and the antisense strand shown in SEQ ID NO: 258; 9) the sense strand shown in SEQ ID NO: 235 and the antisense strand shown in SEQ ID NO: 259; 10) the sense strand shown in SEQ ID NO: 236 and the antisense strand shown in SEQ ID NO: 260; 11) the sense strand shown in SEQ ID NO: 237 and the antisense strand shown in SEQ ID NO: 261; 12) the sense strand shown in SEQ ID NO: 238 and the antisense strand shown in SEQ ID NO: 262; 13) the sense strand shown in SEQ ID NO: 239 and the antisense strand shown in SEQ ID NO: 263; 14) the sense strand shown in SEQ ID NO: 240 and the antisense strand shown in SEQ ID NO: 264; 15) the sense strand shown in SEQ ID NO: 241 and the antisense strand shown in SEQ ID NO: 265; 16) the sense strand shown in SEQ ID NO: 242 and the antisense strand shown in SEQ ID NO: 266; 17) the sense strand shown in SEQ ID NO: 243 and the antisense strand shown in SEQ ID NO: 267; 18) the sense strand shown in SEQ ID NO: 244 and the antisense strand shown in SEQ ID NO: 268; 19) the sense strand shown in SEQ ID NO: 245 and the antisense strand shown in SEQ ID NO: 269; 20) the sense strand shown in SEQ ID NO: 246 and the antisense strand shown in SEQ ID NO: 270; 21) the sense strand shown in SEQ ID NO: 247 and the antisense strand shown in SEQ ID NO: 271; 22) the sense strand shown in SEQ ID NO: 248 and the antisense strand shown in SEQ ID NO: 272; 23) the sense strand set forth in SEQ ID NO: 249 and the antisense strand set forth in SEQ ID NO: 273, and 24) The sense strand shown in SEQ ID NO: 250 and the antisense strand shown in SEQ ID NO: 274; The present invention may include or be selected from the following combinations:

[0061] In another aspect, the present disclosure provides a composition comprising an siRNA described in the present disclosure, an siRNA complex, and one or more pharmaceutically acceptable excipients, such as a vehicle, carrier, diluent, and / or delivery polymer.

[0062] A variety of drug delivery systems are known and applicable to the siRNA or siRNA complexes of the present disclosure, such as packaging in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the siRNA or siRNA complexes, receptor-mediated cellular endocytosis, and construction of nucleic acids as part of reverse transcribing viruses or other vectors.

[0063] In another aspect, the present disclosure provides use of the above-described siRNA, siRNA complex, or composition in the preparation of a medicament for treating a disease in a subject, which in some embodiments is selected from a liver-derived disease.

[0064] In another aspect, the present disclosure provides a method of treating a disease in a subject, comprising administering to the subject an siRNA, siRNA complex, or composition described above.

[0065] In another aspect, the present disclosure provides a method of inhibiting mRNA expression in a subject, comprising administering to the subject an siRNA, siRNA complex, or composition described above.

[0066] In another aspect, the present disclosure provides a method for delivering an expression-inhibitory oligomeric compound to the liver in vivo, comprising administering to a subject the above-described siRNA, siRNA complex, or composition.

[0067] The siRNA, siRNA complex or composition and method disclosed herein can reduce the level of a target mRNA in a cell, a group of cells, a tissue or a subject, and comprises administering to a subject a therapeutically effective amount of a siRNA, siRNA complex or composition described in the present disclosure, wherein the siRNA is linked to a targeting ligand, thereby inhibiting expression of the target mRNA in the subject.

[0068] In some embodiments, the subject has previously been identified as having a pathogenic upregulation of the target gene in the target cell or tissue.

[0069] A subject, as described in this disclosure, refers to a subject diagnosed as suffering from (or suspected of suffering from, or susceptible to) a disease or condition that would benefit from the reduction or inhibition of target mRNA expression.

[0070] Delivery may be by local administration (e.g., direct injection, implantation, or topical administration), systemic administration, or by subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.

[0071] In alternative embodiments, the pharmaceutical compositions provided by the present disclosure may be administered by injection, for example, intravenous, intramuscular, intracutaneous, subcutaneous, intraduodenal, or intraperitoneal injection.

[0072] In an alternative embodiment, after the targeting ligand is linked to the siRNA to form a complex, the complex may be packaged into a reagent kit.

[0073] In another aspect, the present disclosure further provides a pharmaceutical composition comprising the siRNA or siRNA complex of the present disclosure.

[0074] In some embodiments, the pharmaceutical composition may further include a pharmaceutically acceptable additive and / or adjuvant, which may be one or more of various agents or compounds commonly used in the art. For example, the pharmaceutically acceptable additive may include at least one of a pH buffering agent, a protective agent, and an osmotic pressure adjusting agent.

[0075] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg to 1000 mg.

[0076] In one embodiment, the pharmaceutical composition contains, based on the total weight of the composition, 0.01 to 99.99% of the compound, its medicinal salt, or its isotopic derivative. In one embodiment, the pharmaceutical composition contains 0.1 to 99.9% of the compound, its medicinal salt, or its isotopic derivative. In one embodiment, the pharmaceutical composition contains 0.5 to 99.5% of the compound, its medicinal salt, or its isotopic derivative. In one embodiment, the pharmaceutical composition contains 1 to 99% of the compound, its medicinal salt, or its isotopic derivative. In one embodiment, the pharmaceutical composition contains 2 to 98% of the compound, its medicinal salt, or its isotopic derivative.

[0077] In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% pharmaceutically acceptable excipients, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 1% to 99% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 2% to 98% pharmaceutically acceptable excipients.

[0078] In some embodiments, when the siRNA, siRNA complex, or pharmaceutical composition is contacted with cells expressing the target gene, the siRNA complex or pharmaceutical composition inhibits expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as by PCR, branched DNA (bDNA)-based methods, or protein-based methods, for example, immunofluorescence assays such as Western blots and flow cytometry.

[0079] In some embodiments, when the siRNA, siRNA complex, or pharmaceutical composition is contacted with cells expressing a target gene, the percentage of overexpression of target gene mRNA induced by the siRNA complex or pharmaceutical composition is 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, as measured by, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as by PCR, branched DNA (bDNA)-based methods, or protein-based methods, such as immunofluorescence analysis such as Western blotting or flow cytometry.

[0080] In some embodiments, when the siRNA, siRNA complex, or pharmaceutical composition is contacted with cells expressing the target gene, the siRNA complex retains on-target activity while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as by PCR or branched DNA (bDNA)-based methods, or protein-based methods, for example, immunofluorescence assays such as Western Blot or flow cytometry.

[0081] In some embodiments, when the siRNA, siRNA complex, or pharmaceutical composition is contacted with cells expressing the target gene, the siRNA complex reduces on-target activity by at most 20%, at most 19%, at most 15%, at most 10%, at most 5%, or more than 1%, while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as by PCR, branched DNA (bDNA)-based methods, or protein-based methods, such as immunofluorescence assays such as Western Blot and flow cytometry.

[0082] In some embodiments, when the siRNA, siRNA complex, or pharmaceutical composition is contacted with cells expressing the target gene, the siRNA complex enhances on-target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, while reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, as measured by, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as by PCR, branched DNA (bDNA)-based methods, or protein-based methods, for example, immunofluorescence assays such as Western blot and flow cytometry.

[0083] The present disclosure further provides a cell comprising an siRNA or siRNA complex of the present disclosure.

[0084] The present disclosure further provides a reagent kit comprising the siRNA, siRNA complex, or pharmaceutical composition of the present disclosure.

[0085] The present disclosure further provides a method for silencing a target gene or mRNA of a target gene in a cell, the method comprising the step of introducing into the cell an siRNA, siRNA complex and / or pharmaceutical composition according to the present disclosure.

[0086] The present disclosure further provides a method for silencing a target gene or mRNA of a target gene in a cell, either in vivo or in vitro, comprising introducing into the cell an siRNA, siRNA complex and / or pharmaceutical composition according to the present disclosure.

[0087] The present disclosure further provides a method for inhibiting expression of a target gene or mRNA of a target gene, comprising administering to a subject in need thereof an effective amount or dose of an siRNA, siRNA complex and / or pharmaceutical composition according to the present disclosure.

[0088] In some embodiments, administration is by modes of administration including intramuscular, intrabronchial, intrathoracic, intraperitoneal, intraarterial, intralymphatic, intravenous, subcutaneous, cerebrospinal, or a combination thereof.

[0089] In some embodiments, the effective amount or effective dose of the siRNA, siRNA complex, and / or pharmaceutical composition is about 0.001 mg / kg body weight to about 200 mg / kg body weight, about 0.01 mg / kg body weight to about 100 mg / kg body weight, or about 0.5 mg / kg body weight to about 50 mg / kg body weight.

[0090] In some embodiments, the target gene is inhibin βE (INHBE).

[0091] The present disclosure provides the above-mentioned siRNA and / or pharmaceutical composition and / or siRNA complex for treating and / or preventing a disease associated with inhibin βE (INHBE) gene expression in a subject, wherein in some embodiments, the disease is selected from metabolic disorder-related diseases, and in some embodiments, the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.

[0092] The present disclosure provides a method for treating and / or preventing a disease associated with inhibin βE (INHBE) gene expression in a subject, the method comprising administering to the subject an effective amount or dose of an siRNA, siRNA complex, or pharmaceutical composition described in the present disclosure, wherein in some embodiments, the disease is selected from metabolic disorder-related diseases, and in some embodiments, the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.

[0093] The present disclosure provides use of the above-mentioned siRNA and / or pharmaceutical composition and / or siRNA complex in the preparation of a medicament for treating and / or preventing a disease associated with inhibin βE (INHBE) gene expression, wherein in some embodiments, the disease is selected from metabolic disorder-related diseases, and in some embodiments, the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.

[0094] The present disclosure provides the use of the above-described siRNA and / or pharmaceutical composition and / or siRNA complex in the preparation of a medicament for inhibiting the expression of INHBE.

[0095] The present disclosure provides a method for inhibiting expression of INHBE, comprising administering to a subject an effective amount or dose of the above-described siRNA and / or pharmaceutical composition and / or siRNA complex.

[0096] The present disclosure provides the above-mentioned siRNA and / or pharmaceutical composition and / or siRNA complex for treating and / or preventing a disease, in some embodiments, the disease is selected from metabolic disorder-related diseases, and in some embodiments, the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.

[0097] The present disclosure provides a method for treating and / or preventing a disease, the method comprising administering to a subject an effective amount or dose of the above-described siRNA and / or pharmaceutical composition and / or siRNA complex, wherein in some embodiments, the disease is selected from metabolic disorder-related diseases, and in some embodiments, the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.

[0098] The present disclosure provides use of the above-described siRNA and / or pharmaceutical composition and / or siRNA complex in the preparation of a medicament for treating and / or preventing a disease, in some embodiments, the disease is selected from metabolic disorder-related diseases, and in some embodiments, the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease.

[0099] The present disclosure provides a method for delivering an siRNA to the liver that inhibits the expression and / or replication of INHBE in the body, the method comprising administering the siRNA and / or pharmaceutical composition and / or siRNA complex to a subject.

[0100] The present disclosure further provides a method for preparing an siRNA or an siRNA complex, the method comprising synthesizing the siRNA, siRNA complex described in this disclosure.

[0101] The present disclosure further provides an siRNA or siRNA complex characterized in that a T base replaces one or more, for example, 1, 2, 3, 5, 6, 7, 8, 9, 10, or all, of the U bases of any one of the siRNAs or siRNA complexes of the present disclosure.

[0102] Pharmaceutically acceptable salts of the compounds described in the present disclosure are selected from inorganic salts or organic salts, and the compounds described in the present disclosure can react with acidic or basic substances to produce the corresponding salts.

[0103] In another aspect, unless a configuration is explicitly stated, the compounds of the present disclosure may have specific geometric or stereoisomeric forms. The present disclosure includes cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, and all such compounds are intended to be within the scope of the present disclosure. Substituents, such as alkyl groups, may have additional asymmetric carbon atoms. All such isomers and mixtures thereof are within the scope of the present disclosure.

[0104] Additionally, unless a configuration is specified, the compounds and intermediates of the present disclosure may exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The terms "tautomer" or "tautomeric form" refer to structural isomers of different energies that are interconvertible via a low energy barrier.

[0105] The compounds of the present disclosure may be asymmetric, e.g., have one or more stereoisomers. Unless otherwise specified, all stereoisomers include, e.g., enantiomers and diastereomers. Compounds of the present disclosure that contain asymmetric carbon atoms can be isolated in optically pure form or racemic form. Optically pure forms can be resolved from racemic mixtures or synthesized using chiral raw materials or chiral reagents.

[0106] Optically active (R)- and (S)-isomers and D- and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. Single enantiomers of certain compounds of the present disclosure can be prepared by asymmetric synthesis or derivatization with chiral auxiliaries, where the resulting diastereomeric mixture is isolated and cleavage of the groups assists in providing the desired enantiomer in pure form. Alternatively, if the molecule contains a basic (e.g., amino) or acidic (e.g., carboxy) functional group, diastereomeric salts can be formed with an appropriate optically active acid or base, followed by diastereomeric resolution using conventional methods well known in the art, followed by recovery to provide the enantiomerically pure product. Separation of enantiomers and diastereomers is typically accomplished by chromatography, using a chiral stationary phase and optionally combined with chemical derivatization (e.g., carbamates from amines).

[0107] The present disclosure further includes some isotopically labeled compounds of the present disclosure that are the same as those described herein, except that one or more atoms have been replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Illustrative isotopes that can be attached to compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, for example, 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0108] Unless otherwise specified, when a position is specifically designated as deuterium (D), it should be understood that the position is deuterium (i.e., at least 10% deuterium incorporated) with an abundance at least 1000 times greater than the natural abundance of deuterium (which is 0.015%). For example, a compound having an abundance greater than the natural abundance of deuterium may be at least 1000 times more abundant, at least 2000 times more abundant, at least 3000 times more abundant, at least 4000 times more abundant, at least 5000 times more abundant, at least 6000 times more abundant, or even greater. The present disclosure further includes various deuterated forms of the compounds of Formula I and Formula II. Each available hydrogen atom connected to a carbon atom may be independently replaced with a deuterium atom. Those skilled in the art can synthesize deuterated forms of the compounds of Formula I and Formula II by referring to the relevant literature. Deuterated forms of the compounds of Formula I and Formula II, when prepared, may use commercially available deuterated starting materials or may be synthesized by conventional techniques using deuterated reagents, including, but not limited to, deuterated borane, tritiated borane in tetrahydrofuran, lithium aluminum deuterated hydride, deuterated iodoethane, deuterated iodomethane, and the like.

[0109] Unless otherwise specified, in the chemical structures of the compounds described in this disclosure: [ka] The bond "" indicates that the configuration is not specified, i.e., if chiral isomers exist in the chemical structure, [ka] The bond [ka] or [ka] For convenience, all of the above structural formulas are depicted in one isomeric form, but the present disclosure also includes all isomers, such as tautomers, rotamers, geometric isomers, diastereomers, racemates, and enantiomers. In the chemical structures of the compounds described in the present disclosure, [ka] The bond is unspecified in configuration, i.e. [ka] The bond configuration may be E or Z, or may include both E and Z configurations simultaneously.

[0110] Terminology In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless expressly defined otherwise herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0111] Unless otherwise specified, in the context of the present disclosure, the terms "inhibin βE" and "INHBE" are used interchangeably in the present disclosure. INHBE includes, but is not limited to, human INHBE, cynomolgus monkey INHBE, mouse INHBE, and rat INHBE, and their amino acid sequences and complete coding sequences and mRNA sequences can be easily obtained using previously disclosed databases such as GenBank, UniProt, OMIM, and the Macaca Genome Project site.

[0112] The term "INHBE" also refers to naturally occurring DNA sequence variations of the INHBE gene, such as single nucleotide polymorphisms (SNPs) in the INHBE gene. Exemplary SNPs can be found in the dbSNP database.

[0113] The term "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during INHBE transcription, including the RNA processing product mRNA as the primary transcription product. The targeted portion of the target sequence must be long enough to serve as a substrate for iRNA-directed cleavage. In one embodiment, the target sequence is within the protein-coding region of INHBE.

[0114] As used herein, in the context of RNA-mediated gene silencing, the sense strand (also referred to as SS, SS strand, or significant strand) refers to the strand that contains the same or essentially the same sequence as the target mRNA sequence, and the antisense strand (also referred to as AS or AS strand) refers to the strand that has a sequence complementary to the target mRNA sequence.

[0115] In the context of describing the siRNA sense strand described herein, the term "a sequence that differs by no more than three nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 1 to 106, and that comprises at least 15 consecutive nucleotides" is intended to indicate that the siRNA sense strand described herein comprises at least 15 consecutive nucleotides of the sense strand of any one of SEQ ID NOs: 1 to 106, or a sequence that differs by no more than three nucleotides from at least 15 consecutive nucleotides of the sense strand of any one of SEQ ID NOs: 1 to 106 (optionally, a sequence that differs by no more than two nucleotides, optionally, a sequence that differs by one nucleotide).

[0116] In the present disclosure, the "5' region" of the sense strand or antisense strand, i.e., the "5' end" and "5' terminus" are used interchangeably. For example, nucleotides 2 to 8 of the 5' region of the antisense strand may be substituted with nucleotides 2 to 8 of the 5' terminus of the antisense strand. Similarly, the "3' region," "3' terminus," and "3' terminus" of the sense strand or antisense strand are also used interchangeably.

[0117] Unless otherwise specified, in the context of the present disclosure, "G," "C," "A," "T," and "U" represent nucleotides containing the bases guanine, cytosine, adenine, thymidine, and uracil, respectively. It is well known to those skilled in the art that the substitution of the bases T and U does not significantly affect the properties of the RNAi agent sequence, and U can be arbitrarily substituted with T in the sequences of the present disclosure, and the sequences obtained after the substitution are also within the scope of protection of the present disclosure. In the sequences of the present disclosure, the direction from the 5' end to the 3' end of the same nucleic acid strand is from left to right, and a lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleoside, a lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluoro-modified nucleoside, and a lowercase letter s indicates that the two nucleosides adjacent to the letter s are linked by a thiophosphodiester group. Unless otherwise specified, the terms "RNAi agent," "nucleotide," "compound," "chemical modification," "oligonucleotide," "double-stranded RNAi inhibitor molecule," "siRNA," "siRNA complex," "dsRNA," "nucleic acid," and "RNAi" of the present disclosure may each independently exist in the form of a salt, mixed salt, or non-salt (e.g., free acid or free base). When present in the form of a salt or mixed salt, it may be a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salts" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. When they exist in the form of salts, some groups can be ionized to form anions / cations, for example, phosphodiester groups and thiophosphodiester groups can exist in the form of anions. The salt structures corresponding to the following structures are also within the scope of protection of the present disclosure. Unless otherwise specified, the 3'-position of the first nucleotide at the 3'-end of each chain is a hydroxy group, and the 5'-position of the first nucleotide at the 5'-end of each chain is a hydroxy group.

[0118] The above modifications and linking groups each have the structure shown in the table below, where Base represents a base: [Table 1]

[0119] As used in this disclosure, the term "2'-fluoro-modified nucleotide" refers to a nucleotide formed by replacing the hydroxy group at the 2' position of the ribosyl group of a nucleotide with a fluoro group, and "non-2'-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the hydroxy group at the 2' position of the ribosyl group of a nucleotide with a non-fluoro group.

[0120] As used in this disclosure, the term "2'-methoxy modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxy group of a ribosyl group with a methoxy group.

[0121] As used herein, the terms "complementary" and "reverse complementary" can be used interchangeably and have the meaning known to those skilled in the art that in a double-stranded nucleic acid molecule, bases in one strand pair with bases in another strand in a complementary manner. In DNA, the purine base adenine always pairs with the pyrimidine base thymine (or uracil in RNA), and the purine base guanine always pairs with the pyrimidine base cytosine. Each base pair contains one purine and one pyrimidine. When adenine in one strand always pairs with thymine (or uracil) in the other strand and guanine always pairs with cytosine, the two strands are said to be complementary to each other, and the sequence of the strand can be deduced from the sequence of the complementary strand. Accordingly, "mismatch," as used in the art, means that bases at corresponding positions in a double-stranded nucleic acid are not present in a complementary pairing.

[0122] As used herein, the term "inhibition" is used interchangeably with "reduction," "silencing," "downregulation," "suppression," and other similar terms, and includes any level of inhibition. Inhibition can be assessed by an absolute or relative reduction in the level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-administration baseline level or a level determined from an untreated or control (e.g., buffer control or inactive agent control only)-treated subject, cell, or sample. For example, the degree of inhibition of target gene expression by siRNA can be expressed by the amount of mRNA overexpression, for example, the amount of mRNA overexpression can be 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less. The inhibition rate of target gene expression can be detected using the Dual-Glo (registered trademark) Luciferase Assay System. The firefly chemiluminescence value (Fir) and the Renilla chemiluminescence value (Ren) are read, and the relative value Ratio = Ren / Fir is calculated. In the present disclosure, the excess mRNA expression ratio (or excess activity %) = Ratio (siRNA-treated group) / Ratio (siRNA-free control group), and the inhibition rate (%) = 100% - excess mRNA expression (%).

[0123] Unless otherwise specified, the terms "compound," "ligand," "nucleic acid-ligand complex," "siRNA complex," "nucleic acid," "complex," "chemical modification," "targeting ligand," "dsRNA," and "RNAi" of the present disclosure can each independently exist in the form of a salt, mixed salt, or non-salt (e.g., free acid or free base). When present in the form of a salt or mixed salt, it may be a pharmaceutically acceptable salt.

[0124] The term "pharmaceutically acceptable salts" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0125] "Pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic or organic acid that is capable of retaining the bioavailability of the free base without other adverse effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc., and organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, caproate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, mesylate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalenedisulfonate, etc. These salts can be prepared by methods known in the art.

[0126] "Pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that can retain the bioavailability of the free acid without other adverse effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts, with sodium salts being preferred. Salts derived from organic bases include, but are not limited to, salts of primary amines, secondary amines, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, glycine betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamide resins, and the like. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0127] An "effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition required to achieve any one or more beneficial or desired therapeutic results. With respect to prophylactic use, beneficial or desired results include elimination or reduction of the risk, reduction in severity, or delay in the onset of a condition, including biochemical, histological, and / or behavioral manifestations of the condition, its complications, and intermediate pathological phenotypes manifested in the progression of the condition. With respect to therapeutic use, beneficial or desired results include clinical results, such as a reduction in the incidence of or amelioration of one or more symptoms of various target gene-, target mRNA-, or target protein-related conditions of the present disclosure, a reduction in the dosage of other drugs required to treat the condition, an improvement in the therapeutic efficacy of another drug, and / or a delay in the progression of a target gene-, target mRNA-, or target protein-related condition of the present disclosure in a patient.

[0128] As used herein, the terms "patient," "subject," or "individual" can be used interchangeably and include a human or a non-human animal, such as a mammal, for example, a human or a monkey.

[0129] The siRNA provided by the present disclosure can be obtained by conventional preparation methods in the art (for example, solid-phase synthesis and liquid-phase synthesis). Here, commercial customization services for solid-phase synthesis already exist. Modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.

[0130] The term "chemical modification" or "modification" includes any change to a nucleotide by chemical means, such as adding or removing a chemical moiety, or substituting one chemical moiety for another.

[0131] The term "base" includes any known DNA and RNA base and base analogs, such as purines and pyrimidines, as well as the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogs.

[0132] The terms "flat-ended" and "blunt-ended" can be used interchangeably and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of an siRNA, i.e., the absence of overhanging nucleotides. In many cases, an siRNA with both blunt ends is double-stranded throughout its entire length.

[0133] The term "about" or "approximately" refers to a numerical value that is within an acceptable error range of a specific value as determined by one of ordinary skill in the art, as determined by how the numerical portion is measured (i.e., the limitations of the measurement system). For example, "about" may mean within or more than a standard deviation of 1. Alternatively, "about" or "essentially including" may mean a variation within a range of up to 20%, e.g., between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, or between 0.5% and 1%. In this disclosure, the term "about" preceding a number or numerical range includes, in each case, the specified number of embodiments. Unless otherwise specified, when specific values ​​appear in this application and claims, the meaning of "about" or "essentially including" should be assumed to be within an acceptable error range of the specific value.

[0134] Unless otherwise specified, "optionally," "optionally," "selectively," or "selectively" means that the subsequently described event or circumstance may occur, but may not occur, and this description includes cases where the event or circumstance occurs and cases where it does not occur. For example, "optionally, R1 and R2 are directly linked to form a ring" means that R1 and R2 are directly linked to form a ring may occur, but is not necessarily required to exist, and this description includes cases where R1 and R2 are directly linked to form a ring and cases where R1 and R2 do not form a ring.

[0135] In the chemical structures of the present disclosure: [ka] can be linked to any one or more groups in accordance with the scope of the invention described herein.

[0136] In the context of this disclosure: [ka] In the base [ka] can be substituted with any group capable of linking to an adjacent nucleotide.

[0137] The term "linked," when referring to a connection between two molecules, refers to a covalent bond between the two molecules or a non-covalent bond (e.g., a hydrogen bond or an ionic bond) between the two molecules, and includes a direct link and an indirect link.

[0138] The term "directly linked" means that a first compound or group is connected to a second compound or group without any intervening atoms or groups of atoms.

[0139] The term "indirectly linked" refers to a first compound or group being linked to a second compound or group through an intermediate group, compound, or molecule (eg, a linking group).

[0140] The term "substituted" refers to the replacement of any one or more hydrogen atoms on a specified atom (usually a carbon, oxygen, or nitrogen atom) with any group defined herein, provided that the replacement does not exceed the normal valence of the specified atom and results in the formation of a stable compound. Non-limiting examples of substituents include a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a cyano group, a hydroxy group, an oxo group, a carboxy group, a cycloalkyl group, a cycloalkenyl group, a heterocyclyl group, a heteroaryl group, an aryl group, a ketone, an alkoxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group, or a halogen (e.g., F, Cl, Br, I). When the substituent is a ketone or oxo (i.e., =0), two (2) hydrogens on the atom are replaced.

[0141] "Substituted with one or more..." means that the group may be substituted with a single or multiple substituents. When the group is substituted with multiple substituents, the group may be multiple of the same substituents, or may be a combination of one or multiple different substituents. DETAILED DESCRIPTION OF THE INVENTION

[0142] The present disclosure will be further explained below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples of the present disclosure generally follow standard conditions, such as those found in Cold Spring Harbor's Antibody Technology Laboratory Manual and Molecular Cloning Manual, or conditions suggested by raw material or product manufacturers. Reagents for which specific sources are not specified are standard commercially available reagents.

[0143] Example 1. Design of human INHBE siRNA We designed 19 / 21nt siRNAs targeting the human INHBE gene (NM_031479.5) to satisfy the general rules for active siRNAs. The sequences of the unmodified sense and antisense strands are shown in Tables 1a and 1b, and the sequences of the sense and antisense strands modified with 2'-fluoro, 2'-methoxy, etc. are shown in Tables 2a and 2b.

[0144] [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7]

[0145] In Table 2 above, the sequences are arranged from left to right in the direction from the 5' end to the 3' end, the lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide, the lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide, the lowercase letter s indicates that the link between the two adjacent nucleosides or linked nucleosides on the left and right of the letter s and the delivery group NAG0052' or L96' is a thiophosphodiester linkage, and unless otherwise specified, the two adjacent nucleosides are linked via a phosphodiester group, and unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each chain is a hydroxy group, and the 5' position of the first nucleotide at the 5' end of each chain is a hydroxy group.

[0146] The structures of the above 2'-methoxy modified nucleotide, 2'-fluoro modified nucleotide, thiophosphodiester group, phosphodiester group, NAG0052' and L96' are shown in the table below, and when the siRNA or siRNA complex of the present disclosure exists in a salt form, for example, a sodium salt form, the salt form structures corresponding to the structures in Table 3 below also fall within the scope of protection of the present disclosure.

[0147] [Table 6-1] [Table 6-2] Here, Base represents a base.

[0148] Example 2. Synthesis of siRNA and siRNA complexes The synthesis of siRNA is no different from conventional phosphoramidite solid-phase synthesis. The synthesis process is briefly described below. Starting with a general-purpose CPG support, nucleoside phosphoramidite monomers were linked one by one using a synthesis program on a Dr. Oligo48 synthesizer (Biolytic). Nucleoside phosphoramidite monomers, such as 2'-F RNA and 2'-O-methyl RNA, were purchased from Shanghai Jiaowei or Suzhou Jima. 5-Ethylthio-1H-tetrazole (ETT) was used as the activating agent (0.6 M acetonitrile solution), a solution of 0.22 M PADS in a 1:1 volume ratio of acetonitrile and trimethylpyridine (Suzhou Colema) was used as the sulfurizing reagent, and iodopyridine / water solution (Colema) was used as the oxidizing agent.

[0149] After solid-phase synthesis was completed, the oligoribonucleotides were dissolved from the solid support and soaked in a 3:1 solution of 28% aqueous ammonia and ethanol at 50°C for 16 hours. After centrifugation, the supernatant was transferred to a separate centrifuge tube, concentrated, and evaporated to dryness. The resulting oligonucleotides were purified by C18 reverse-phase chromatography using a mobile phase of 0.1 M TEAA and acetonitrile, followed by 3% trifluoroacetic acid to remove DMTr. The target oligonucleotides were collected, lyophilized, and identified as the desired products by LC-MS and quantified by UV (260 nm).

[0150] The resulting single-stranded oligonucleotides were complementarily paired and annealed in an equimolar ratio, and the resulting double-stranded siRNA was dissolved in 1x PBS and adjusted to the concentration required for the experiment before use.

[0151] An siRNA complex containing NAG0052' could be prepared according to the same method as described in the examples of WO2023138663A1, and an siRNA complex containing L96' was prepared according to the same method as described in the examples of WO2014025805A1.

[0152] Example 3. Verification of psiCHECK on-target levels In vitro molecular level simulations and on-target activity screening were performed on HEK293A cells using nine concentration gradients for the siRNAs listed in Table 2.

[0153] The psi-CHECK plasmid used in this example was purchased from Shanghai Bioengineering Co., Ltd. An on-target sequence corresponding to the siRNA was constructed in the INHBE gene and inserted into the psiCHECK-2 plasmid, i.e., the GSCM on-target plasmid. This plasmid contains the Renilla luciferase gene and the firefly luciferase gene. As a dual reporter gene system, the siRNA target sequence was inserted into the 3'UTR region of the Renilla luciferase gene. The activity of the siRNA against the target sequence was reflected by detecting the expression of Renilla luciferase calibrated with firefly luciferase. Detection was performed using the Dual-Luciferase Reporter Assay System (Promega, E2940).

[0154] HEK293A cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum and incubated at 37°C in 5% CO. 24 h before transfection, HEK293A cells were seeded into 96-well plates at a seeding density of 8 × 10 3 Cells / well, medium 100 μL / well.

[0155] Cells were co-transfected with siRNA and the corresponding plasmid using Lipofectamine 2000 (ThermoFisher, 11668019) according to the manufacturer's instructions. 0.2 μL of Lipofectamine 2000 was used per well. For the on-target sequence plasmid, the transfection amount of plasmid was 20 ng per well. The siRNA concentration was set at 20 nM at the highest concentration point, followed by a 4-fold gradient dilution of 20 nM, followed by 5 nM, 1.25 nM, 0.3125 nM, 0.0781 nM, 0.0195 nM, 0.0049 nM, 0.0012 nM, and 0.0003 nM. 24 h after transfection, on-target levels were detected using the Dual-Luciferase Reporter Assay System (Promega, E2940).

[0156] The experimental samples and equipment used in this example are detailed in Tables 4 and 5. The dilution scheme used in this example is shown in Table 6. The experimental results are shown in Table 7.

[0157] [Table 7] [Table 8-1] [Table 8-2] [Table 9] [Table 10-1] [Table 10-2]

[0158] The results in Table 7 demonstrate that the siRNAs of the present disclosure have good on-target inhibitory activity against INHBE.

[0159] Example 4. Multi-concentration inhibitory activity of INHBE in human hepatoma cell line (Hep3B) 24 h prior to transfection, Hep3B cells were seeded in a 96-well plate at approximately 20,000 cells / well in 100 μL of medium. Following the manufacturer's instructions, samples were transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150). The final transfection concentrations were 20, 4, 0.8, 0.16, 0.032, 0.0064, and 0.00128 nM. After 24 hours of treatment, total RNA was extracted from the cells using a high-throughput cell RNA extraction reagent kit (FG0417-L / FG0418-XL, magnetic bead method), followed by RNA reverse transcription (Takara, RR037A) and real-time quantitative PCR (Thermo, 4444557) to measure human INHBE mRNA levels. Human INHBE mRNA levels were normalized based on the GAPDH internal reference gene level.

[0160] The equipment used in this experiment is shown in Table 8.

[0161] [Table 11]

[0162] Here, the real-time quantitative PCR detection was performed using a probe Q-PCR detection experiment, and the primer information is shown in Table 9. [Table 12]

[0163] Results analysis method After the Q-PCR detection experiment is completed, the system automatically obtains the corresponding Ct value according to the set threshold. The expression of a gene can be relatively quantified by comparing the Ct values. Comparing the Ct values ​​means calculating the difference in gene expression based on the difference between the Ct value of the internal reference gene, also known as 2-△△Ct, where △△Ct = [(Ct of the target gene in the experimental group - Ct of the internal reference group) - (Ct of the target gene in the control group - Ct of the internal reference group)]. Inhibition rate (%) = (1 - excess target gene expression) × 100%.

[0164] The results are expressed as the percentage excess of human INHBE mRNA expression in siRNA-treated cells, where IC 50 The values ​​correspond to the concentration of siRNA used when the excess percentage of INHBE mRNA expression is 50%.

[0165] The data results in Table 10 show that the siRNAs of the present disclosure have high levels of on-target inhibitory activity against the INHBE gene in Hep3B cells.

[0166] [Table 13]

[0167] Example 5. Multi-point inhibitory activity of INHBE in primary human hepatocytes (PHH) 24 h prior to transfection, PHH cells were seeded into 96-well plates at approximately 30,000 cells / well in 100 μL of medium. Following the manufacturer's instructions, samples were transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150). The final transfection concentrations were 20, 4, 0.8, 0.16, 0.032, 0.0064, and 0.00128 nM. After 24 hours of treatment, total RNA was extracted from the cells using a high-throughput cell RNA extraction reagent kit (FG0417-L / FG0418-XL, magnetic bead method), followed by RNA reverse transcription (Takara, RR037A) and real-time quantitative PCR (Thermo, 4444557) to measure human INHBE mRNA levels. Human INHBE mRNA levels were normalized based on the GAPDH internal reference gene level.

[0168] The equipment, primer information, result analysis method, and calculation method for the overexpression percentage of target gene mRNA used in this experiment were the same as those in Example 4.

[0169] The data results in Table 11 show that the siRNAs of the present disclosure have high levels of on-target inhibitory activity against the INHBE gene in PHH cells.

[0170] [Table 14]

[0171] Example 6. Verification of psiCHECK on-target levels In vitro molecular level simulation and on-target activity screening of siRNAs were performed in HEK293A cells using two concentration gradients. The experimental procedures, materials and equipment used, dilution procedures, and analysis methods were the same as in Example 3.

[0172] As can be seen from the results in Table 12, the siRNAs of the present disclosure have a good inhibitory effect on INHBE.

[0173] [Table 15-1] [Table 15-2]

[0174] Example 7. psiCHECK on-target level verification In vitro molecular level simulation and on-target activity screening of siRNAs were performed in HEK293A cells using nine concentration gradients. The experimental procedures, materials and equipment used, dilution procedures, and analysis methods were all the same as in Example 3.

[0175] As can be seen from the results in Table 13, in the results >20, even under the condition of the maximum concentration of 20 nM in this example, the inhibition rate could not reach 50%, and the siRNA of the present disclosure has a good inhibitory effect on INHBE.

[0176] [Table 16]

[0177] Example 8. Multi-concentration inhibitory activity of INHBE in human hepatoma cell line (Huh7) 24 h prior to transfection, Huh7 cells were seeded into a 96-well plate at approximately 15,000 cells / well in 100 μL of medium. Following the manufacturer's instructions, samples were transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150). The final transfection concentrations for the samples were 20, 4, 0.8, 0.16, 0.032, 0.0064, and 0.00128 nM. After 24 hours of treatment, total RNA was extracted from the cells using a high-throughput cell RNA extraction reagent kit (FG0417-L / FG0418-XL, magnetic bead method), followed by RNA reverse transcription (Takara, RR037A) and real-time quantitative PCR (Thermo, 4444557) to measure human INHBE mRNA levels. Human INHBE mRNA levels were normalized based on the GAPDH internal reference gene level.

[0178] The equipment, primer information, and result analysis method used in this experiment were as described in Example 4.

[0179] The data results in Table 14 show that all siRNAs and their complexes have high levels of on-target inhibitory activity against the INHBE gene in Huh7 cells.

[0180] [Table 17]

[0181] Example 9. Multi-concentration inhibitory activity of INHBE in primary monkey hepatocytes (PCH) 24 h prior to transfection, PCH cells were seeded into 96-well plates at approximately 30,000 cells / well in 100 μL of medium. Following the manufacturer's instructions, samples were transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150) at gradient final concentrations of 20, 4, 0.8, 0.16, 0.032, 0.0064, and 0.00128 nM. After 24 hours of treatment, total RNA was extracted from the cells using a high-throughput cell RNA extraction reagent kit (FG0417-L / FG0418-XL, magnetic bead method), followed by RNA reverse transcription (Takara, RR037A) and real-time quantitative PCR (Thermo, 4444557) to measure monkey INHBE mRNA levels. The monkey INHBE mRNA levels were corrected based on the GAPDH internal reference gene level.

[0182] The equipment used in this experiment is as shown in Table 7 of Example 4.

[0183] Here, the real-time quantitative PCR detection was performed using a probe Q-PCR detection experiment, and the primer information is shown in Table 15.

[0184] [Table 18]

[0185] The methods for analyzing the results and calculating the percentage of overexpression of target mRNA were the same as in Example 4.

[0186] The data results in Table 16 show that all siRNA complexes have high levels of on-target inhibitory activity against the INHBE gene in PCH cells.

[0187] [Table 19]

[0188] Example 10. psiCHECK on-target level verification In vitro molecular level simulation and on-target activity screening of siRNAs were performed in HEK293A cells using nine concentration gradients. The experimental procedures, materials and equipment used, and dilution procedures were the same as in Example 3.

[0189] The data results in Table 17 show that the siRNAs of the present disclosure have higher levels of on-target inhibitory activity compared to the control sequences.

[0190] [Table 20]

[0191] Example 11. psiCHECK on-target level verification In vitro molecular level simulation and on-target activity screening of siRNAs were performed in HEK293A cells using nine concentration gradients. The experimental process, experimental materials and equipment used, dilution process, and result analysis method were all the same as in Example 3. The data results in Table 18 indicate that the siRNAs of the present disclosure have high levels of on-target inhibitory activity.

[0192] [Table 21]

Claims

1. 1. An siRNA comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprises at least 15 consecutive nucleotides and differs by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9; and the antisense strand comprises at least 15 consecutive nucleotides and differs by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16, 17, and 18; siRNA.

2. the sense strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 1 to 9, and the antisense strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 10 to 18; Preferably, the sense strand comprises at least 19 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 1-9, preferably by no more than 1 nucleotide; and / or The antisense strand comprises at least 21 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs: 10 to 18, and preferably by no more than 1 nucleotide. The siRNA described in claim 1.

3. Group 1: the sense strand shown in SEQ ID NO: 1 and the antisense strand shown in SEQ ID NO: 10 Group 2: the sense strand shown in SEQ ID NO: 2 and the antisense strand shown in SEQ ID NO: 11 Group 3) of the sense strand shown in SEQ ID NO: 3 and the antisense strand shown in SEQ ID NO: 12 Group 4) of the sense strand shown in SEQ ID NO: 4 and the antisense strand shown in SEQ ID NO: 13 Group 5: the sense strand shown in SEQ ID NO: 5 and the antisense strand shown in SEQ ID NO: 14 Group 6: the sense strand shown in SEQ ID NO: 6 and the antisense strand shown in SEQ ID NO: 15 Group 7, the sense strand shown in SEQ ID NO: 7 and the antisense strand shown in SEQ ID NO: 16 Group 8) of the sense strand shown in SEQ ID NO: 8 and the antisense strand shown in SEQ ID NO: 17, and Group 9: a sense strand represented by SEQ ID NO: 9 and an antisense strand represented by SEQ ID NO: 18; 3. The siRNA of claim 1 or 2.

4. At least one nucleotide of the sense strand and / or the antisense strand is a modified nucleotide.

4. The siRNA according to claim 1.

5. Three consecutive nucleotides in the sense strand are 2'-fluoro modified nucleotides, preferably, in the 5' to 3' direction, the nucleotides at positions 7, 8, and 9 of the sense strand are each independently a 2'-fluoro modified nucleotide, and the nucleotides at positions 2, 6, 12, and 14 of the antisense strand are each independently a 2'-fluoro modified nucleotide, in the 5' to 3' direction. The siRNA described in claim 4.

6. At least one phosphodiester group in the sense strand and / or the antisense strand is a phosphodiester group having a modifying group, preferably a thiophosphodiester group.

6. The siRNA according to claim 1 .

7. The thiophosphodiester group is Between the first and second nucleotides at the 5'-terminal end of the sense strand, Between the second and third nucleotides at the 5'-terminal end of the sense strand, Between the first and second nucleotides at the 3'-terminal end of the sense strand, Between the second and third nucleotides at the 3'-terminal end of the sense strand, Between the first and second nucleotides at the 5'-end of the antisense strand, Between the second and third nucleotides at the 5'-end of the antisense strand, Between the first and second nucleotides at the 3'-terminal end of the antisense strand, and Between the second and third nucleotides at the 3'-end of the antisense strand, at least one of the positions selected from Preferably, the sense strand and / or the antisense strand comprises a plurality of thiophosphodiester groups. The siRNA described in claim 6.

8. the sense strand comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 227 to 242; And / or the antisense strand comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 251 to 266; 8. The siRNA according to claim 1 .

9. An siRNA complex comprising the siRNA according to any one of claims 1 to 8 and a targeting ligand linked to the end of the siRNA, Preferably, the targeting ligand targets the liver; More preferably, the targeting ligand binds to the asialoglycoprotein receptor (ASGPR) and is selected from one or more of N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, or N-isobutyrylgalactosamine; More preferably, the targeting ligand is 【Chemistry 1】 It has the structure: siRNA complex.

10. The targeting ligand is linked to the 3' or 5' end of the sense strand or antisense strand of the siRNA via a thiophosphodiester group or a phosphodiester group, and preferably, the targeting ligand is linked to the 3' end of the sense strand of the siRNA. The siRNA complex of claim 9.

11. The sense strand comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 243 to 250, and / or the antisense strand comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 267 to 274; The siRNA complex of claim 10.

12. A composition comprising the siRNA according to any one of claims 1 to 8 or the siRNA complex according to any one of claims 9 to 11, and a pharmaceutically acceptable carrier. Pharmaceutical compositions.

13. In preparing a drug for reducing the expression of inhibin βE (INHBE) using the siRNA according to any one of claims 1 to 8 or the siRNA complex according to any one of claims 9 to 11, use.

14. 13. A method for reducing expression of inhibin βE (INHBE) in a subject in need thereof, comprising administering to said subject an effective amount or effective dose of the siRNA of any one of claims 1 to 8, or the siRNA conjugate of any one of claims 9 to 11, or the pharmaceutical composition of claim 12. method.

15. A method for treating and / or preventing a disease in a subject in need thereof, comprising administering to the subject an effective amount or effective dose of the siRNA according to any one of claims 1 to 8, or the siRNA conjugate according to any one of claims 9 to 11, or the pharmaceutical composition according to claim 12, wherein preferably the disease is selected from metabolic disorder-related diseases, more preferably the disease is selected from metabolic syndrome (MS), cardiovascular disease, obesity, hepatitis, and kidney disease. method.

16. A method for delivering siRNA to the liver that inhibits the expression and / or replication of INHBE in the body in a subject in need thereof, comprising administering to the subject the siRNA of any one of claims 1 to 8, or the siRNA conjugate of any one of claims 9 to 11, or the pharmaceutical composition of claim 12. method.

17. A method for preparing an siRNA or an siRNA complex, comprising synthesizing the siRNA according to any one of claims 1 to 8, or the siRNA complex according to any one of claims 9 to 11, or the pharmaceutical composition according to claim 12. method.

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