Modified dsrna, double-stranded rnai agent, and use thereof

Abstract

A modified dsRNA, a double-stranded RNAi agent, and a use thereof. The modified dsRNA and the double-stranded RNAi agent can reduce non-specific binding, thereby reducing off-target toxicity, and improving the inhibitory activity on each target.

Description

Modified dsRNA, double-stranded RNAi agents and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present disclosure claims priority to Chinese patent application number 202311337926.5, filed on October 16, 2023, entitled “Modified dsRNA, double-stranded RNAi agents and uses thereof” and Chinese patent application number 202311337910.4, filed on October 16, 2023, entitled “A dsRNA for inhibiting LPA gene expression and uses thereof”, the entire contents of the above patent applications are hereby incorporated into the present disclosure by reference. Technical Field

[0003] The present disclosure relates to a modified dsRNA, a double-stranded RNAi agent, and uses thereof. Background Art

[0004] RNA interference (RNAi) is a process that effectively silences or inhibits the expression of a target gene by selectively inactivating the corresponding mRNA of the target gene through double-stranded RNA (dsRNA). RNA interference is activated by double-stranded RNA that is transported into the cell cytoplasm. The silencing mechanism can result in the degradation of the target mRNA induced by small interfering RNA (siRNA) or short hairpin RNA (shRNA), or the inhibition of translation of specific mRNAs induced by microRNAs (miRNAs).

[0005] In the early development of RNAi projects, most of them used completely unmodified or slightly modified compounds. However, because unmodified dsRNA can induce innate immune responses, it has only shown limited clinical efficacy and unacceptable toxicity. In order to achieve clinical applications, people began to chemically modify siRNA. Generally, the modification sites of chemical modification can be different positions such as sugar groups, bases and phosphate backbones in the nucleotide sequence. Chemical modification of dsRNA improves its in vivo activity in various ways, such as increasing resistance to nucleases, improving cellular uptake, reducing immune stimulation and off-target effects, but it may also have a negative impact on the activity of dsRNA.

[0006] Numerous patents, including US6005087A, WO1991006556A1, WO2011139702A2, and WO2016028649A1, have disclosed most siRNA chemical modification methods. The most commonly used modifications are 2'-methoxy (2'-O-methyl) and 2'-fluoro (2'-F) modifications to the ribose group, as well as phosphorothioate (PS) modifications to replace phosphodiester bonds. These modifications can be used alone or in combination to prepare dsRNA. However, there is still a need to develop dsRNA with improved gene silencing efficacy and inhibitory activity.

[0007] Summary of the Invention

[0008] The present disclosure provides a modified dsRNA, a double-stranded RNAi agent, and its use in the preparation of a medicament for treating, inhibiting, or preventing related diseases. The modified dsRNA described in the present disclosure can be used to inhibit target gene expression, has reduced nonspecific binding, thereby reducing off-target toxicity, and improves inhibitory activity against various targets, with better efficacy than dsRNA without the specific modification.

[0009] RNAi agents typically comprise a sense strand and an antisense strand that form a duplex, double-stranded RNA (referred to herein as "dsRNA"). RNAi agents comprising dsRNA are also referred to herein as "dsRNAi" agents.

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

[0011] In one aspect, the present disclosure provides a modified dsRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a target gene mRNA, wherein at least one of the nucleotides at positions 2 to 11 in the antisense strand from the 5' end to the 3' end comprises hypoxanthine, and the nucleotide comprising hypoxanthine forms a non-canonical base pair (I:C pairing) with the cytosine at the corresponding position in the sense strand, and the modified dsRNA meets at least one of the following characteristics:

[0012] (1) In the continuous sequence N1N2I0N3N4 at positions 2 to 11 of the antisense strand, from the 5' end to the 3' end, at least three bases among N1, N2, N3 and N4 are adenine and / or uracil; wherein N1, N2, N3 and N4 are each independently a nucleotide containing adenine, cytosine, guanine, thymine or uracil as a base, and I0 is a nucleotide containing hypoxanthine as a base;

[0013] (2) the number of adenines and uracils in positions 2 to 8 of the antisense strand is 4 or more in the direction from the 5' end to the 3' end; and / or

[0014] (3) There is a difference in dissociation temperature (Tm) of at least 2°C between the modified dsRNA and a dsRNA with canonical base pairing (G:C pairing); wherein the G:C pairing dsRNA differs from the modified dsRNA only in that a guanine is present in place of a hypoxanthine (G:C pairing).

[0015] In some embodiments, the number of non-canonical base pairs is 1, 2, or 3 pairs.

[0016] In some embodiments, the base in at least one of the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th or 11th nucleotides from the 5' end to the 3' end of the antisense strand is hypoxanthine.

[0017] In some embodiments, in the modified dsRNA, the base of at least one nucleotide from position 2 to position 8 in the antisense strand from the 5' end to the 3' end is hypoxanthine.

[0018] In some embodiments, in the modified dsRNA, the base of at least one nucleotide from position 6 to position 8 in the antisense strand from the 5' end to the 3' end is hypoxanthine, for example, position 6, 7 and / or 8.

[0019] In some embodiments, there is no guanine nucleotide in the consecutive sequence N1N2I0N3N4 at positions 2-11 of the antisense strand.

[0020] In some embodiments, in the consecutive sequence N1N2I0N3N4 at positions 2-11 of the antisense strand, the bases N1, N2, N3 and N4 are each independently adenine and / or uracil.

[0021] In some such embodiments, at least one nucleotide of N1, N2, N3, N4, and I0 further has a modified sugar group and / or a modified internucleotide linkage.

[0022] In some embodiments, the modified dsRNA of the invention has a Tm change relative to the G:C paired dsRNA of at least 2° C., e.g., 2° C., more than 2° C., 3° ​​C., 4° C., 5° C., or more. In some such embodiments, the Tm is calculated using a formula or algorithm described herein.

[0023] In some embodiments, the length of the sense strand and the antisense strand is each independently 17-25 nucleotides, for example, each independently 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides; preferably, the length of the sense strand and the antisense strand is each independently 19-23 nucleotides; more preferably, the length of the sense strand and the antisense strand is each independently 19-21 nucleotides.

[0024] In some embodiments, the other nucleotides in the sense and antisense strands are unmodified nucleotides.

[0025] In some embodiments, substantially all of the nucleotides in the sense strand and the antisense strand are modified nucleotides.

[0026] In some embodiments, all nucleotides in the sense strand and the antisense strand are modified nucleotides.

[0027] In some embodiments, the modified dsRNA of the present disclosure further comprises at least one modified nucleotide selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-F-arabinonucleotides, phosphorothioate modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleic acids, inverted nucleotides, and non-canonical base modified nucleotides. Exemplary non-canonical base modified nucleotides include, but are not limited to, bases selected from inosine (I), xanthosine (X), 7-methylguanosine (m7G), N6-methyladenosine (m6A), dihydrouridine, 5-methylcytosine (m5C), pseudouridine (Ψ), and N1-methylpseudouridine (m1Ψ).

[0028] In some embodiments, the modified dsRNA further comprises at least one modified nucleotide selected from the group consisting of 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-F-arabino nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides, and inverted nucleotides. In some such embodiments, the inverted nucleotide is selected from the group consisting of inverted A nucleotides, inverted dA nucleotides, inverted dT nucleotides, inverted C nucleotides, and inverted U nucleotides.

[0029] In some embodiments, the modified dsRNA further meets at least one of the following characteristics:

[0030] 1) the antisense strand comprises 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8) 2'-fluoro modified nucleotides;

[0031] 2) the antisense strand comprises 1-4 (e.g., 1, 2, 3, 4) phosphorothioate internucleotide linkages;

[0032] 3) the antisense strand comprises 12-19 (e.g., 12, 13, 14, 15, 16, 17, 18, 19) 2'-O-methyl modified nucleotides;

[0033] 4) the sense strand comprises 2-4 (e.g., 2, 3, 4) 2'-fluoro modified nucleotides;

[0034] 5) the sense strand comprises 1-4 (e.g., 1, 2, 3, 4) phosphorothioate internucleotide linkages;

[0035] 6) The sense strand comprises 14-17 (eg, 14, 15, 16, 17) 2'-O-methyl modified nucleotides.

[0036] In some embodiments, the target gene is LPA, and the modified dsRNA further meets at least one of the following characteristics:

[0037] 1) The antisense strand contains at least 5 or more 2'-fluoro modified nucleotides or 5 or more 2'-deoxy modified nucleotides, and the remaining sites are 2'-O-methyl modified nucleotides;

[0038] 2) In the antisense strand, positions 2, 5, 7, and 12 are 2'-deoxy modified nucleotides, position 14 is a 2'-fluoro modified nucleotide, positions 9 and 16 contain 0, 1, and 2 2'-fluoro modified nucleotides, respectively, and the remaining positions are 2'-O-methyl modified nucleotides;

[0039] 3) the first position of the antisense strand is a 5'-vinylphosphite modified nucleotide;

[0040] 4) the sense strand contains three or four 2'-fluoro modified nucleotides at positions 7, 8, 9, and 10;

[0041] 5) From the 5' end to the 3' end, at least one or two of the three nucleotides from the 5' end of the sense strand are phosphorothioate-modified nucleotides; and / or at least one or two of the three nucleotides from the 5' end and the 3' end of the antisense strand are phosphorothioate-modified nucleotides, respectively.

[0042] In some embodiments, the sense strand of the present disclosure is derived from the mRNA sequence of human LPA (e.g., Gene ID: 4018). Alternatively, the sense strand of the present disclosure is a fragment of the mRNA sequence of human LPA. In some embodiments, the sense strand of the present disclosure is derived from the mRNA sequence of cynomolgus monkey LPA (e.g., Gene ID: 101865897). Alternatively, the sense strand of the present disclosure is a fragment of the mRNA sequence of cynomolgus monkey LPA.

[0043] In some embodiments, the target gene is INHBE, and the modified dsRNA further meets at least one of the following characteristics:

[0044] 1) From the 5' end to the 3' end, the nucleotides at positions 2, 4, 12, and 14 of the antisense strand are 2'-fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides;

[0045] 2) From the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of the sense strand are 2'-fluorine-modified nucleotides.

[0046] In some embodiments, the sense strand of the present disclosure is derived from the mRNA sequence of human INHBE (e.g., Gene ID: 83729). Alternatively, the sense strand of the present disclosure is a fragment of the mRNA sequence of human INHBE. In some embodiments, the sense strand of the present disclosure is derived from the mRNA sequence of cynomolgus monkey INHBE (e.g., Gene ID: 102127493). Alternatively, the sense strand of the present disclosure is a fragment of the mRNA sequence of cynomolgus monkey INHBE.

[0047] In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides that differ from any of the sequences shown in Table 1 or Table 2 by 0, 1, 2, or 3 nucleotides, and the sense strand has at least 15, 16, 17, 18, 19, 20, or 21 nucleotides complementary to the antisense strand.

[0048] In some embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides that differ from any of the sequences shown in Table 1 or Table 2 by 0, 1, 2, or 3 nucleotides.

[0049] In some embodiments, the sense strand sequence is at least substantially complementary to the antisense strand sequence. In some preferred embodiments, the sense strand sequence is fully complementary to the antisense strand sequence (ie, 100% complementary).

[0050] In some embodiments, the modified dsRNA comprises any one of the antisense or sense strand sequences in Table 1 or Table 2. In some embodiments, the modified dsRNA comprises any one of the sense or antisense strand sequences shown in SEQ ID NOs: 1-1154.

[0051] In some embodiments, the modified dsRNA comprises an antisense strand sequence and a sense strand sequence as shown in the duplex sequences in Table 1 or Table 2. In some embodiments, the modified dsRNA comprises a duplex selected from LPAI-001 to LPAI-239. In some embodiments, the modified dsRNA comprises a duplex selected from INI-001 to INI-308.

[0052] In some embodiments, the antisense strand comprises completely consecutive nucleotides selected from SEQ ID NO: 1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, or 1152.

[0053] In some embodiments, the antisense strand comprises a member selected from the group consisting of SEQ ID NOs: 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1208 9, 1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261 or 1263 completely consecutive nucleotides.

[0054] In some embodiments, the sense strand comprises completely consecutive nucleotides selected from SEQ ID NO: 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149, or 1151.

[0055] In some embodiments, the modified dsRNA comprises a sense strand sequence and an antisense strand sequence selected from the group consisting of SEQ ID NOs: 1093 and 1094; SEQ ID NOs: 1095 and 1096; SEQ ID NOs: 1097 and 1098; SEQ ID NOs: 1099 and 1100; SEQ ID NOs: 1101 and 1102; SEQ ID NOs: 1103 and 1104; SEQ ID NOs: 1105 and 1106; SEQ ID NOs: 1107 and 1108; SEQ ID NOs: 1109 and 1110; SEQ ID NOs: 1111 and 1112; SEQ ID NOs: 1113 and 1114; SEQ ID NOs: 1115 and 1116; SEQ ID NOs: 1117 and 1118; SEQ ID NOs: 1119 and 1120; SEQ ID NOs: 1121 and 1122; SEQ ID NOs: 1123 and 1124. NO: 1123 and 1124; SEQ ID NO: 1125 and 1126; SEQ ID NO: 1127 and 1128; SEQ ID NO: 1129 and 1130; SEQ ID NO: 1131 and 1132; SEQ ID NO: 1133 and 1134; SEQ ID NO: 1135 and 1136; or, SEQ ID NO: 1137 and 1138; SEQ ID NO: 1139 and 1140; SEQ ID NO: 1141 and 1142; SEQ ID NO: 1143 and 1144; SEQ ID NO: 1145 and 1146; SEQ ID NO: 1147 and 1148; SEQ ID NO: 1149 and 1150; or, SEQ ID NO: 1151 and 1152.

[0056] In some embodiments, the sense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 121 0, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262 or 1264 of completely consecutive nucleotides.

[0057] In some embodiments, the modified dsRNA comprises an antisense strand sequence and a sense strand sequence selected from the group consisting of SEQ ID NOs: 1153 and 1154, SEQ ID NOs: 1155 and 1156, SEQ ID NOs: 1157 and 1158, SEQ ID NOs: 1159 and 1160, SEQ ID NOs: 1161 and 1162, SEQ ID NOs: 1163 and 1164, SEQ ID NOs: 1165 and 1166, SEQ ID NOs: 1167 and 1168, SEQ ID NOs: 1169 and 1170, SEQ ID NOs: 1171 and 1172, SEQ ID NOs: 1173 and 1174, SEQ ID NOs: 1174 and 1176, SEQ ID NOs: 1177 and 1178, SEQ ID NOs: 1179 and 1180, SEQ ID NOs: 1181 and 1182, SEQ ID NOs: 1183 and 1184 SEQ ID NOs: 1209 and 1210, SEQ ID NOs: 1211 and 1212, SEQ ID NOs: 1213 and 1214, SEQ ID NOs: 1215 and 1216, SEQ ID NOs: 1217 and 1218, SEQ ID NOs: 1219 and 1220, SEQ ID NOs: 1221 and 1222, SEQ ID NOs: 1223 and 1224, SEQ ID NOs: 1225 and 1226, SEQ ID NOs: 1227 and 1228, SEQ ID NOs: 1229 and 1230, SEQ ID NOs: 1231 and 1232, SEQ ID NOs: 1233 and 1234, SEQ ID NOs: 1235 and 1236, SEQ ID NOs: 1237 and 1238 SEQ ID NOs: 1217 and 1218, SEQ ID NOs: 1219 and 1220, SEQ ID NOs: 1221 and 1222, SEQ ID NOs: 1223 and 1224, SEQ ID NOs: 1225 and 1226, SEQ ID NOs: 1227 and 1228, SEQ ID NOs: 1229 and 1230, SEQ ID NOs: 1231 and 1232, SEQ ID NOs: 1233 and 1234, SEQ ID NOs: 1235 and 1236, SEQ ID NOs: 1237 and 1238, SEQ ID NOs: 1239 and 1240, SEQ ID NOs: 1241 and 1242, SEQ ID NOs: 1243 and 1244, SEQ ID NOs: 1245 and 1246, SEQ ID NOs: 1247 and 1248NO: 1247 and 1248, SEQ ID NO: 1249 and 1250, SEQ ID NO: 1251 and 1252, SEQ ID NO: 1253 and 1254, SEQ ID NO: 1255 and 1256, SEQ ID NO: 1257 and 1258, SEQ ID NO: 1259 and 1260, SEQ ID NO: 1261 and 1262, SEQ ID NO: 1263 and 1264.

[0058] In some embodiments, the modified dsRNA comprises an LPA selected from the group consisting of LPA-318, LPA-319, LPA-324, LPA-370, LPA-558, LPA-560, LPA-407, LPA-449, LPA-564, LPA-566, LPA-487, LPA-518, LPA-562, LPA-568, LPA-816, LPA-817, LPA-818, LPA-819, LPA -820, LPA-821, LPA-822, LPA-823, LPA-824, LPA-825, LPA-826, LPA-827, LPA-828, LPA-829, LPA-830, LPA-831, LPA-832, LPA-833, LPA-834, LPA-835, LPA-836, LPA-837, LPA-838, LPA-880, LPA-867, LPA-86 8. LPA-869, LPA-870, LPA-871, LPA-873, LPA-874, LPA-875, LPA-876, LPA-877, LPA-878, LPA-879, LPA -872, LPA-864, LPA-865, LPA-866, IN-267, IN-268, IN-301, IN-360, IN-361, IN-389, IN-417, IN-418, I N-439, IN-440, IN-441, IN-442, IN-443, IN-464, IN-465, IN-488, IN-489, IN-503, IN-504, IN-518, IN -Duplexes of -519, IN-520, IN-521, IN-522, IN-523, IN-524, IN-525, IN-526, IN-527, IN-528, IN-538 or IN-539.

[0059] In the present invention, in the modified dsRNA, the antisense strand has sufficient complementarity with the target sequence to mediate RNA interference. In some embodiments, the target sequence is a sequence selected from the following targets: PCSK9 (proprotein convertase subtilisin 9), ANGPTL3 (angiopoietin-like protein 3), LPA (apolipoprotein a), INHBE (inhibin subunit βE), ACVR1C (activin A receptor type 1C), PLIN1 (lipoprotein 1), PDE3B (phosphodiesterase 3B), INHBC (inhibin subunit βC), GDF-8 (MSTN) (myostatin), SOD1 (superoxide dismutase 1), APP (amyloid precursor protein), C3 (complement protein C3), C5 (complement protein C5), HTT (huntingtin protein), DMPK (myotonic dystrophy protein kinase), HSD17B13 (17β β-Hydroxysteroid dehydrogenase 13), PNPLA3 (patatin-like phospholipase domain-containing protein 3), XDH (xanthine dehydrogenase), AGT (angiotensin), AKT (protein kinase B), Kras (Kirsten rat sarcoma viral oncogene homolog), SHP2 (protein tyrosine phosphatase 2 containing SrC homology 2), TGF-β (transforming growth factor beta receptor 2), IFN-α (interferon α / β receptor α chain), IL-13 (interleukin-13), IL-6 (interleukin 6), Myc (myelocytokine viral oncogene), IL-4 (interleukin 4), IL-17 (interleukin 17), TERT (telomerase reverse transcriptase), KHK (fructokinase), Factor VII (coagulation factor VII), Factor X (coagulation factor X), FactorXI (coagulation factor XI), thrombin (Thrombin), TPX2 (Xklp2 targeting protein), apoB (apolipoprotein B), SAA (amyloid A), TTR (transthyretin), RSV (respiratory syncytial virus), PDGFβ (platelet-derived growth factor receptor β)), Erb-B (tyrosine kinase receptor), Src gene, CRK gene, GRB2 (growth factor receptor binding protein 2), MEKK (mitogen-activated protein kinase kinase), JNK (c-Jun N-terminal kinase), RAF gene, Erk1 / 2 (extracellular regulated protein kinase 1 / 2), MYB gene, JUN gene, FOS gene, BCL-2 gene, hepciden (hepcidin), PC (activated protein C), CCND (cyclin D), VEGF (vascular endothelial growth factor), EGFR (epidermal growth factor receptor), CCNA (cyclin A), CCNE (cyclin E), WNT-1 gene, β-catenin gene, c-MET gene, PKC (protein kinase C), NFKB (nuclear factor kappa-light-chain-enhancer of activated B cells), STAT3 (signal transducer and activator of transcription 3), survivin, TOP1 (topoisomerase I), TOP2A (topoisomerase IIα).

[0060] In some embodiments, the antisense oligonucleotides of the present disclosure are substantially complementary to the target sequence (e.g., LPA mRNA, INHBE mRNA) and comprise a contiguous nucleotide sequence that is at least about 85% complementary to any one of the sense strand oligonucleotides or portions of the sense strand oligonucleotides provided herein, based on the entire length, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary.

[0061] In some embodiments, the antisense oligonucleotides of the present disclosure are substantially complementary to any of the sense strand oligonucleotides provided herein and comprise a contiguous nucleotide sequence that is at least about 85% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary, to any of the sense strand oligonucleotides or portions of the sense strand oligonucleotides provided herein, based on the entire length.

[0062] In some embodiments, the dsRNA of the present disclosure comprises a sense strand that is substantially complementary to an antisense oligonucleotide that is complementary to a target sequence, e.g., LPA mRNA, INHBE mRNA, wherein the sense strand is at least about 85% complementary, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary to any one of the antisense oligonucleotides or portions of the antisense oligonucleotides based on their entire length.

[0063] In some embodiments, the double-stranded region of the dsRNA is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotide pairs in length.

[0064] In some embodiments, the antisense strand of the dsRNA is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length.

[0065] In some embodiments, the sense strand of the dsRNA is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length.

[0066] In some embodiments, the sense and antisense strands of the dsRNA are independently 15 to 30 nucleotides in length.

[0067] In some embodiments, the sense and antisense strands of the dsRNA are independently 19 to 25 nucleotides in length.

[0068] In some embodiments, the sense and antisense strands of the dsRNA are independently 21 to 23 nucleotides in length.

[0069] In some embodiments, the sense strand of the dsRNA is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, wherein the sense and antisense strands form a double-stranded region of 21 consecutive base pairs with a 2-nucleotide-long single-stranded overhang at the 3'-end.

[0070] In another aspect, the present disclosure provides a double-stranded RNAi agent comprising any of the aforementioned modified dsRNAs and optionally comprising a targeting ligand. The targeting ligand is typically conjugated to the dsRNA and serves to target the RNAi agent to cells.

[0071] In some embodiments, the double-stranded RNAi agent comprises any of the aforementioned modified dsRNAs and further comprises at least one targeting ligand, the sense strand of the dsRNA being conjugated to the targeting ligand. In some preferred embodiments, the 3' end of the sense strand is conjugated to the targeting ligand.

[0072] In some embodiments, the targeting ligand of the present disclosure is specifically targeted to the asialoglycoprotein receptors (ASGPR) on the surface of hepatocytes. Preferably, the targeting ligand comprises N-acetyl-galactosamine (GalNAc), or the targeting ligand is a GalNAc derivative. More preferably, the targeting ligand is any targeting ligand disclosed in WO2022266753A1 (targeting moiety). Unless otherwise clearly contradictory, WO2022266753A1 is incorporated herein by reference in its entirety.

[0073] In some embodiments, the structure of the double-stranded RNAi agent is selected from Formula 1 to Formula 33, wherein R 2 is the modified dsRNA disclosed herein. According to common knowledge in the art, R 2 The dsRNA agent is formed by conjugating the 3' end or 5' end of the sense strand to a targeting ligand; preferably, the 3' end of the sense strand is conjugated to the targeting ligand.

[0074] Table A. Structure of the double-stranded RNAi agent of the present application

[0075] In another aspect, the present disclosure also provides cells, vectors, and host cells comprising the modified dsRNA of the present invention.

[0076] In another aspect, the present disclosure also provides a cell, a vector, a host cell, and a pharmaceutical composition comprising the double-stranded RNAi agent of the present invention.

[0077] In some embodiments, the pharmaceutical composition comprises any of the aforementioned double-stranded RNAi agents or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present disclosure can be used to prevent and / or treat various corresponding diseases, conditions, or symptoms, as further described below.

[0078] In some embodiments, the pharmaceutical composition is formulated for administration by injection or infusion, e.g., for intravenous, subcutaneous, intraperitoneal, or intramuscular administration. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0079] In some embodiments, the carrier of the pharmaceutical composition is a non-buffered solution or a buffered solution. Typical non-buffered solutions are saline or water, and buffered solutions include one or more of acetate, citrate, prolamin, carbonate, and phosphate. A preferred buffered solution is phosphate-buffered saline (PBS).

[0080] In another aspect of the present disclosure, methods for inhibiting target gene expression in cells are also provided. These methods include contacting the cells with a double-stranded RNAi agent or dsRNA of the present invention to degrade the mRNA transcript of the target gene, thereby inhibiting the expression of the target gene in the cells.

[0081] In some embodiments, the target gene is selected from any one of the following groups: PCSK9, ANGPTL3, LPA, INHBE, ACVR1C, PLIN1, PDE3B, INHBC, GDF-8 (MSTN), SOD1, APP, C3, C5, HTT, DMPK, HSD17B13, PNPLA3, XDH, AGT, AKT, Kras, SHP2, TGF-β, IFN-α, IL-13, IL-6, Myc, IL-4, IL-17, TERT, KHK, Factor VII, Factor X, Factor XI, Thrombin, TPX2, apoB, SAA, TTR, RSV, PDGFβ, Erb-B, Src, CRK, GRB2, MEKK, JNK, RAF, Erk1 / 2, MYB, JUN, FOS, BCL- 2. Hepciden, PC, CCND, VEGF, EGFR, CCNA, CCNE, WNT-1, β-catenin, c-MET, PKC, NFKB, STAT3, survivin, TOP1 and TOP2A.

[0082] In some embodiments, the target gene is the LPA gene or the INHBE gene.

[0083] In some embodiments, the cell is in a subject. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is an adipocyte. In some embodiments, the subject is a human.

[0084] In some embodiments, target gene expression is inhibited by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0085] In another aspect, the present disclosure also provides a method for treating a subject having a disease mediated by target gene expression, wherein the method comprises administering to the subject a therapeutically effective amount of a double-stranded RNAi agent of the present invention, thereby inhibiting the expression of the target gene in cells.

[0086] In some embodiments, the target gene is selected from the mRNA genes involved in the following targets: PCSK9, ANGPTL3, LPA, INHBE, ACVR1C, PLIN1, PDE3B, INHBC, GDF-8 (MSTN), SOD1, APP, C3, C5, HTT, DMPK, HSD17B13, PNPLA3, XDH, AGT, AKT, Kras, SHP2, TGF-β, IFN-α, IL-13, IL-6, Myc, IL-4, IL-17, TERT, KHK, Factor VII, Factor X, Factor XI, Thrombin, TPX2, apoB, SAA, TTR, RSV, PDGFβ, Erb-B, Src, CRK, GRB2, MEKK, JNK, RAF, Erk1 / 2, MYB, JUN, FOS, BCL- 2. Hepciden, PC, CCND, VEGF, EGFR, CCNA, CCNE, WNT-1, β-catenin, c-MET, PKC, NFKB, STAT3, survivin, TOP1 and TOP2A.

[0087] In some embodiments, the target gene is the LPA gene or the INHBE gene.

[0088] In some embodiments, the subject is a human.

[0089] In some embodiments, the subject has an LPA-associated disease or condition or an INHBE-associated disease or condition.

[0090] In some embodiments, the LPA-associated disease or condition is one or more of Berger's disease, metabolic syndrome, aortic regurgitation, aortic dissection, retinal artery occlusion, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, heterozygous or homozygous familial hypercholesterolemia, hyperlipoproteinemia, cerebrovascular atherosclerosis, venous thrombosis, congestive heart failure, ischemic heart disease, carotid artery disease, myocardial infarction, stroke, atrial fibrillation, heart failure, hyperlipidemia, type 2 diabetes, and non-alcoholic fatty liver disease.

[0091] In some embodiments, the INHBE-related disease or condition is one or more of a metabolic disorder, metabolic syndrome, type 2 diabetes, obesity, prediabetes, hypertriglyceridemia, lipodystrophy, liver inflammation, fatty liver, hypercholesterolemia, a disease associated with elevated liver enzymes, non-alcoholic steatohepatitis, cardiovascular disease, kidney disease, abdominal obesity, insulin resistance, hypertension, dyslipidemia, cardiometabolic disorder, and cancer associated with INHBE expression. The metabolic syndrome includes, but is not limited to, one or more of abdominal obesity, insulin resistance, hypertension, dyslipidemia, and hyperlipidemia.

[0092] In some embodiments, the expression of the target gene in the cell is inhibited so that the protein level of the target gene in the serum of the subject is reduced by at least 50%, 60%, 70%, 80%, 90% or 95% compared to before administration of the dsRNAi agent.

[0093] In some embodiments, the double-stranded RNAi agent is administered to a subject at a dose of about 0.10 mg / kg to about 50 mg / kg, for example, at a dose of about 0.01 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 50 mg / kg, about 10 mg / kg to about 30 mg / kg, about 10 mg / kg to about 20 mg / kg, about 15 mg / kg to about 20 mg / kg, about 15 mg / kg to about 25 mg / kg, about 15 mg / kg to about 30 mg / kg, or about 20 mg / kg to about 30 mg / kg.

[0094] In some embodiments, the method further comprises determining the target gene expression level in a sample from the subject. In some embodiments, the target gene expression level in a sample from the subject is determined before, during, and / or after administration of the dsRNAi agent to the subject. Any suitable sample can be used, such as, but not limited to, a blood sample, a serum sample, or a liver tissue sample.

[0095] In some embodiments, the method further comprises administering to the subject an additional therapeutic agent to treat the metabolic disorder, including but not limited to insulin, glucagon-like peptide 1 (GLP-1) agonists, sulfonylureas, seglininides, biguanides, thiazolidinediones, α-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, statins, and combinations of any of the foregoing.

[0096] In some embodiments, the double-stranded RNAi agent of the present disclosure can be administered simultaneously or sequentially with another therapeutic agent. In some embodiments, the double-stranded RNAi agent is administered before or after administration of another therapeutic agent, such as a standard therapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] FIG1A-FIG1B show the inhibitory activity of dsRNA against LPA.

[0098] 2A to 2D show the inhibitory activity of dsRNA against LPA (full curves). DETAILED DESCRIPTION

[0099] The present disclosure provides RNAi agents and compositions that trigger RNA-induced silencing complex (RISC)-mediated cutting of RNA transcripts of target genes. The gene can be in cells, such as adipocytes and / or hepatocytes (hepatocytes) or other liver cells (or liver cells), such as cells in a subject (e.g., a human). The use of these RNAi agents and compositions enables targeted degradation of target gene (e.g., LPA, INHBE, etc.) mRNA in mammals. As inhibitors of target gene expression, the RNAi agents and compositions of the present invention can be used to prevent, treat, and / or suppress target gene-related diseases or disorders.

[0100] Thus, the present disclosure provides methods for treating, preventing or inhibiting a target gene-associated disease or condition, such as, but not limited to, a metabolic disorder, e.g., metabolic syndrome; a carbohydrate disorder, e.g., type 2 diabetes, prediabetes; a lipid metabolism disorder, e.g., hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease; or, a weight disorder, e.g., obesity, overweight, using an RNAi composition that triggers RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the target gene.

[0101] The RNAi agents of the present disclosure comprise an antisense RNA strand having a region of up to about 30 nucleotides in length, e.g., 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20 -23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 21-22, 15, 15-16, 15-17, 15-20, 15-21, 15-22, 15-23, 15-24, or at least 15 nucleotides, said region being substantially complementary to at least a portion of a transcript mRNA of a target gene.

[0102] In certain embodiments, one or both strands of a double-stranded RNAi agent of the present disclosure are up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides, with a region of at least 15 consecutive nucleotides that is substantially complementary to at least a portion of the transcript mRNA of the target gene. In some embodiments, RNAi agents with longer antisense strands can, for example, include a second RNA strand (sense strand) of 20-60 nucleotides in length, wherein the sense strand and antisense strand form a double-stranded region (duplex) of 15-30 consecutive nucleotides.

[0103] The use of RNAi agents of the present disclosure enables the target gene mRNA in mammals to be targeted for degradation. The present disclosure has confirmed that the RNAi agents of the present invention can trigger the cutting of the target gene RNA transcript mediated by the RNA-induced silencing complex (RISC), thereby resulting in significant inhibition of the expression of the target gene. In certain embodiments, the RNAi agents of the present disclosure are more effective (e.g., more potent) and / or more specific (e.g., safer, less off-target effects) than the RNAi agents of the previous targeting the same gene. In certain embodiments, by selecting a specific site in the RNAi agent targeting target gene mRNA, and / or including RNA modifications (e.g., non-canonical base pairing nucleotides, modified nucleotides, chemical modifications) to increase efficacy, effectiveness, specificity and / or safety. In some such embodiments, the RNAi agent includes at least one modified nucleotide, such as non-canonical base pairing nucleotides. The methods and compositions comprising these RNAi agents can be used to treat subjects suffering from target gene-related diseases or disorders such as metabolic disorders. Therefore, the present disclosure provides methods for treating, preventing or suppressing target gene-related diseases or disorders in subjects, and the subjects will benefit from using RNAi agents and compositions of the present disclosure to suppress or reduce target gene expression.

[0104] The present disclosure also provides methods for preventing at least one symptom in a subject suffering from a condition that benefits from inhibition or reduction of target gene expression. The following detailed description will disclose how to prepare and use RNAi agents and compositions thereof to inhibit target gene expression, as well as compositions, uses, and methods for treating subjects who would benefit from inhibition and / or reduction of target gene expression.

[0105] 1. Definition:

[0106] In order to provide a clear and consistent understanding of the terms used in the specification of the present invention, some definitions are provided below. In addition, unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0107] When used in conjunction with the term "comprising" in the claims and / or the specification, the use of the word "a" can mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." Similarly, the word "another" can mean at least a second or more.

[0108] The term "or" is used herein to mean the term "and / or" and can be used interchangeably with the term "and / or" unless the context clearly indicates otherwise. For example, "sense strand or antisense strand" is understood to mean "sense strand or antisense strand, or sense strand and antisense strand."

[0109] As used in this specification and claims, the words "comprising" (and any forms of including, such as "comprises" and "including"), "having" (and any forms of having, such as "have" and "having"), "including" (and any forms of including, such as "including" and "including"), and "comprising" (and any forms of including, such as "containing" and "including"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0110] The term "about" or "approximately" is used to indicate that the value includes the error introduced by the instruments and methods used in determining the value. The term "about" when used in conjunction with a numerical value is intended to encompass numerical values ​​within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as such variations are appropriate for performing the disclosed methods. When "about" precedes a series of numbers or a range, it should be understood that "about" can modify each number in the series or range.

[0111] The terms "at least," "not less than," or "or more" preceding a number or a range of numbers should be understood to include the number adjacent to the term "at least," as well as all subsequent numbers or integers that can logically be included as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 15 nucleotides in a 17-nucleotide nucleic acid molecule" means 15, 16, or 17 nucleotides having the specified properties. When "at least" appears before a series of numbers or a range, it should be understood that "at least" can modify each number in the series or range.

[0112] As used herein, "not more than" or "or less" is understood to refer to the value adjacent to the phrase and logically lower values ​​or integers, as is logical from the context, to zero. For example, a duplex having an overhang of "not more than 2 nucleotides" has an overhang of 2, 1, or 0 nucleotides. When "not more than" appears before a series of numbers or a range, it is understood that "not more than" can modify each number in the series or range. As used herein, a range includes an upper limit and a lower limit.

[0113] The terms "LPA," "apolipoprotein(a)," and the abbreviation "apo(a)" refer to the apolipoprotein(a) polypeptide, a member of the apolipoprotein class of polypeptides that bind lipids to form lipoproteins. Apo(a) is a polymorphic glycoprotein encoded by the human LPA gene. LPA mRNA and apo(a) polypeptide are primarily expressed in the liver. The amino acid and nucleotide sequences of human LPA mRNA transcripts can be found, for example, in GenBank Accession No. NM_005577.4; the amino acid and nucleotide sequences of cynomolgus monkey LPA mRNA transcripts can be found in GenBank Accession No. XM_015448517.1. Further examples of LPA mRNA sequences are readily available using publicly available databases such as GenBank, UniProt, and OMIM.

[0114] The term "inhibin Beta E" and the term "INHBE" are used interchangeably, also referred to as "inhibin subunit Beta E chain," "inhibin Beta E," "inhibin βE," "activin βE," "activin Beta E," and "MGC4638." The sequence of human INHBE mRNA transcripts can be found, for example, in GenBank Accession No. NM_031479.5. The sequence of mouse INHBE mRNA can be found, for example, in GenBank Accession No. NM_008382.3 (SEQ ID NO: 2370). The predicted sequence of cynomolgus monkey INHBE mRNA can be found, for example, in GenBank Accession No. XM_005571319.3. Other examples of INHBE mRNA sequences are readily available through public databases, such as GenBank, UniProt, OMIM, and the Macaca Genome Project website. For more information on INHBE, for example, visit www.ncbi.nlm.nih.gov / gene / ?term=INHBE. As of the date of filing this application, each of the above-mentioned GenBank accession numbers and gene database numbers is incorporated herein by reference in its entirety.

[0115] The term "target sequence", "target gene mRNA" or "target mRNA" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a target gene, including mRNA that is a product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence is at least long enough to serve as a substrate for RNAi-directed cleavage at or near the nucleotide sequence portion of the mRNA molecule formed during transcription of the target gene. In one embodiment, the target sequence is located within the protein coding region of the target gene. In another embodiment, the target sequence is located within the 3'U region of the target gene. TR. The target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular condition or disease state. The target sequence can be about 19-36 nucleotides in length, for example, about 19-30 nucleotides in length, for example, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20- In some embodiments, the target sequence is 15-30 nucleotides in length, for example, 15-23 nucleotides in length, for example, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 17-30, 17-29, 1 In some embodiments, the target sequence has a length of 17-25 nucleotides, 19-21 nucleotides, 19-23 nucleotides, or 21-23 nucleotides. Ranges and lengths intermediate to the above ranges are also considered to be part of the present disclosure.

[0116] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by the sequence referred to using standard nucleotide nomenclature.

[0117] The terms "siRNA," "RNAi agent," "siRNA agent," and "RNA interference agent" are used interchangeably herein and refer to biologically active agents that contain RNA and mediate targeted cleavage of RNA transcripts through an RNA-induced silencing complex (RISC) pathway. RNAi agents direct the specific degradation of mRNA sequences through a process known as RNA interference. RNAi agents modulate (e.g., inhibit) the expression of genes in cells, such as cells in a subject (e.g., a mammalian subject, such as a human). In some embodiments, the RNAi agent used in the compositions, uses, and methods of the invention comprises a double-stranded RNA (dsRNA) or duplex of the invention and may be referred to herein as a "double-stranded RNAi agent," "dsRNAi agent," or "dsRNA agent."

[0118] In certain embodiments, the dsRNAi agent of the present invention includes a double-stranded RNA agent, which, when introduced into a cell, is processed into short interfering RNA by a nuclease called Dicer. Short interfering RNA is integrated into RISC, and one or more helicases unwind the RNA duplex, allowing the complementary antisense strand to guide target recognition. After binding to the target mRNA, one or more nucleases in RISC will cut the target mRNA to induce silencing. Therefore, in other embodiments, the siRNA agent relates to single-stranded RNA produced in the cell and promotes the formation of the RISC complex to achieve silencing of the target gene. In some such embodiments, the RNAi agent is a single-stranded siRNA (ssRNAi), which can be introduced into a cell or organism to inhibit the target mRNA. The single-stranded RNAi agent binds to the RISC nuclease, Argonaute2, and then cuts the target mRNA. The ssRNAi agent is generally 15-30 nucleotides in length and can be chemically modified. Any antisense oligonucleotide described herein can be used as the ssRNAi agent described herein. In some embodiments, the ssRNAi agent comprises at least one non-canonical base pairing nucleotide. In some embodiments, the ssRNAi agent comprises at least one modified nucleotide.

[0119] The term "double-stranded RNA" or "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands, with "sense" (or "justice") and "antisense" orientations relative to the target RNA. In some embodiments of the present invention, dsRNA triggers the degradation of a target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi. Generally speaking, the majority of the nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Each strand of a dsRNA molecule may range in length from 12-40 nucleotides. For example, each strand can be between 14-40 nucleotides in length, 17-37 nucleotides in length, 25-37 nucleotides in length, 17-25 nucleotides in length, 17-22 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, or 21-23 nucleotides in length, and the sense and antisense strands can be equal or unequal lengths without limitation.

[0120] The term "antisense strand" refers to a strand of an iRNA (e.g., dsRNA) that includes a region that is substantially complementary to a target sequence (e.g., INHBE mRNA). As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence. In cases where the complementary region is not completely complementary to the target sequence, there may be mismatches within the interior or terminal regions of the molecule. Typically, the most tolerated mismatches are present within the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides at the 5'- and / or 3'-ends of the dsRNA. The antisense and sense strands of a dsRNA may have the same or different lengths, all of which are known in the art.

[0121] When a first sequence is referred to as being "substantially complementary" to a second sequence, the two sequences can be fully complementary (i.e., complementary over the entire length of one or both nucleotide sequences), or they can form one or more, but generally no more than 5, 4, 3, or 2, mismatched base pairs upon hybridization over a distance of up to 30 base pairs, while retaining the ability to hybridize under appropriate conditions (conditions relevant to their application, such as inhibition of gene expression, such as physiological conditions). It should be noted that when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches for purposes of determining complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, wherein the longer oligonucleotide comprises a 21-nucleotide sequence that is fully complementary to the shorter oligonucleotide, can be referred to as "fully complementary" for the purposes described herein.

[0122] The term "sense strand" refers to the strand of a dsRNA that comprises a region that is substantially complementary to a region of the antisense strand.

[0123] As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence relative to a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions. Such conditions may, for example, be stringent conditions, wherein stringent conditions may include: 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, 40°C to 70°C for 12-16 hours. Other conditions, such as physiologically relevant conditions that may be encountered in an organism, may also be applicable. For example, complementary sequences are sufficient to enable the relevant function of the nucleic acid, such as RNAi. A skilled person can determine the set of conditions that is most suitable for testing the complementarity of two sequences based on the ultimate use of the hybridizing nucleotides.

[0124] The terms "complementary," "fully complementary," and "substantially complementary" as used herein may be used to describe base matching between the sense and antisense strands of a dsRNA, or between two oligonucleotides or polynucleotides, such as the antisense strand of a dsRNA agent and a target sequence, and their meanings will be understood from the context in which the terms are used.

[0125] The term "melting temperature" or "Tm" is used herein to refer to the point at which 50% of double-stranded RNA (dsRNA) molecules open or denature (i.e., 50% of the double-stranded RNA molecules are separated into single strands and 50% of the complementary oligonucleotide chains do not hybridize to each other). For a given oligonucleotide, its corresponding Tm value can be calculated using any acceptable formula or software known in the art. For example, but not limited to, Tm can be calculated using the OligoAnalyzer™ tool from Integrated DNA Technologies (IDT) (Coralville, Iowa, USA); using the Tm calculation tool on the website http: / / insilico.ehu.es / tm.php?formula=basic, etc. The term "ΔTm" refers to the difference in Tm (e.g., calculated Tm) between two different oligonucleotides (e.g., an unmodified oligonucleotide and an oligonucleotide having the same sequence that includes one or more modified nucleotides). In certain embodiments, ΔTm is used to refer to the difference in melting temperature between two dsRNA regions or duplexes of the present invention, wherein one of the dsRNA regions or duplexes comprises a nucleotide substitution by at least one modified nucleotide (e.g., a non-canonical base-paired nucleotide). In some embodiments, ΔTm is used to refer to the difference in melting temperature between two oligonucleotides (e.g., two antisense strands, two sense strands), wherein one of the oligonucleotides comprises at least one nucleotide substituted by a non-canonical base-paired nucleotide. In some embodiments, "ΔTm" refers to the difference in melting temperature between a dsRNA in which there is no non-canonical base pairing (i.e., G:C canonical pairing) and a dsRNA of the present invention in which there is a 1:C non-canonical base pairing. According to common knowledge in the art, the melting temperature of a dsRNA with a G:C canonical pairing is generally higher than that of a dsRNA with a 1:C non-canonical base pair.

[0126] "G", "C", "A", "T" and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymine (also known as 5-methyluracil) and uracil as bases, respectively. However, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in further detail below. G, C, A, T and U are referred to as "canonical" nucleotides in this article, and the definition of canonical nucleotides is shown in Table B. Accordingly, guanine, cytosine, adenine, thymine and uracil are referred to as "canonical" bases in this article, which are the common bases for RNA or DNA construction. In this article, when only stating or describing for base groups or base parts and without ambiguity, "G" can also be used to represent guanine, "C" to represent cytosine, "A" to represent adenine, "T" to represent thymine and "U" to represent uracil. For canonical bases or nucleotides, in most cases, A pairs with U (T in DNA) and G pairs with C, following the Watson-Crick base pairing rules (referred to herein as "canonical base pairing"). Herein, bases other than canonical bases are considered "non-canonical bases." "Non-canonical base pairs" refer to base pairs that do not follow the Watson-Crick base pairing rules, such as base pairs formed by non-canonical bases and canonical bases (the I:C pairing formed by hypoxanthine and cytosine), G:U wobble base pairing, etc.

[0127] Table B. Definitions of canonical nucleotides

[0128] It should be understood that non-canonical base pairing nucleotides (e.g., modified nucleotides having base pairing characteristics that differ from the canonical nucleotides they replace) can differ not only in the base pairs they form, but also in the strength or stability of the base pairing. Non-canonical base pairing may be stronger or weaker than canonical base pairing. For example, m1Ψ (modified from U) pairs more strongly with A than U pairs with A, and m1Ψ-G pairs even more strongly than m1Ψ-A pairs. Thus, by replacing canonical nucleotides with non-canonical base pairing nucleotides, the base pairing strength can be altered, thereby altering the Tm of the antisense strand duplex and / or the Tm of hybridization between the antisense strand and the target RNA (e.g., LPA mRNA, INHBE mRNA). Thus, in some embodiments, canonical nucleotides are replaced with non-canonical base pairing nucleotides, thereby altering the Tm of the oligonucleotide (e.g., altering the calculated Tm of the oligonucleotide, altering the Tm of the resulting double-stranded RNA molecule, such as the Tm of the duplex formed by the antisense strand hybridized with the target mRNA and / or the sense strand). Without wishing to be bound by theory, by changing the Tm of the oligonucleotide by replacing at least one canonical nucleotide with non-canonical base pairing nucleotides, the efficacy, effectiveness, specificity, safety and / or off-target effects of dsRNAi can be regulated and controlled. For example, but not limited to, the efficacy or effectiveness of the dsRNAi agent can be increased by increasing the pairing strength with the desired target mRNA and / or reducing the pairing strength with off-target mRNA. Similarly, the undesirable off-target effects can be reduced by increasing the pairing strength with the desired target mRNA and / or reducing the pairing strength with off-target mRNA. Therefore, in some embodiments, the RNAi agent of the present invention has improved efficacy, effectiveness, specificity and / or safety compared to similar RNAi agents that do not comprise at least one non-canonical base pairing nucleotides.

[0129] In certain embodiments of the dsRNAi agents, antisense strands, and sense strands of the present invention, at least one canonical nucleotide is replaced by a non-canonical base pairing nucleotide. In some such embodiments, one nucleotide is replaced by a non-canonical base pairing nucleotide, i.e., the dsRNAi agent, antisense strand, or sense strand comprises one non-canonical base pairing nucleotide. In some embodiments, two, three, four, five, or more nucleotides are replaced by non-canonical base pairing nucleotides, i.e., the dsRNAi agent, antisense strand, or sense strand comprises two, three, four, five, or more non-canonical base pairing nucleotides. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more nucleotides in an oligonucleotide or dsRNAi agent are modified nucleotides, such as non-canonical base pairing nucleotides. In some embodiments, all nucleotides in a dsRNAi agent, e.g., in the sense strand and / or antisense strand, are modified nucleotides, e.g., non-canonical base-pairing nucleotides. In some embodiments, at least one nucleotide in a dsRNAi agent, e.g., in the sense strand and / or antisense strand, is a modified nucleotide, e.g., non-canonical base-pairing nucleotide.

[0130] Therefore, in certain embodiments, the RNAi agent of the present invention comprises at least one non-canonical base pairing nucleotide, that is, at least one nucleotide in antisense strand and / or sense strand is replaced by non-canonical base pairing nucleotide.In some such embodiments, at least one non-canonical base pairing nucleotide is present on the antisense strand.In some such embodiments, at least one non-canonical base pairing nucleotide is present on the sense strand.In some such embodiments, at least one non-canonical base pairing nucleotide is present on the antisense strand and the sense strand simultaneously.In some embodiments, at least one non-canonical base pairing nucleotide is present in the complementarity region, that is, the region substantially complementary to the target sequence in the oligonucleotide, for example, the region complementary to the target sequence (for example, LPA mRNA, INHBE mRNA) in the antisense strand of the present invention.

[0131] In certain embodiments, replacing at least one canonical nucleotide with a non-canonical base pairing nucleotide changes the melting temperature (Tm) of the oligonucleotide or dsRNA duplex. In some embodiments, the Tm changes by at least 2°C (i.e., the ΔTm is at least about 2°C). In some embodiments, the ΔTm is about 2°C. In some embodiments, the ΔTm exceeds 2°C. In some embodiments, the ΔTm is about 2.5°C, 3°C, 3.5°C, 4°C, 4.5°C, or 5°C.

[0132] In certain embodiments, at least one non-canonical base pairing nucleotide is present at positions 1-11, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and / or 11, from the 5' to the 3' direction of the antisense strand.

[0133] In certain embodiments, at least one non-canonical base pairing nucleotide is present at positions 12-21, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, and / or 21, of the antisense strand in the 5' to 3' direction.

[0134] In certain embodiments of the oligonucleotides and dsRNAi agents of the invention, in the 5' to 3' direction, if at least four nucleotides in positions 2-8 of the antisense strand are A or U and at least one of the nucleotides in positions 6-8 is G, then the G at positions 6, 7 and / or 8 is replaced by a non-canonical base pairing nucleotide.

[0135] In certain embodiments of the oligonucleotides and dsRNAi agents of the present invention, in the sequence 5′-N1N2G0N3N4-3′, when at least three bases among N1, N2, N3 and N4 are adenine or uracil, the guanine in G0 is replaced by a non-canonical base, such as hypoxanthine, wherein N1, N2, N3 and N4 are each independently a nucleotide comprising adenine (A), cytosine (C), guanine (G) or uracil (U), and G0 is a nucleotide comprising guanine.

[0136] It should be understood that all non-canonical base pairing nucleotides or non-canonical bases disclosed herein or known in the art are suitable for use in the RNAi agents, compositions and methods of the present invention. Non-limiting examples of non-canonical base pairing nucleotides (or nucleosides) or non-canonical bases are defined as shown in Table C. In some embodiments, the non-canonical base pairing nucleosides (or nucleotides) or non-canonical bases are the modifying groups shown in Table C. In some embodiments, the non-canonical base is hypoxanthine. In some embodiments, the non-canonical base pairing nucleoside is inosine. In some embodiments, the non-canonical base pairing nucleotide is a canonical nucleotide capable of wobble pairing. The aforementioned combinations are also encompassed.

[0137] Table C. Examples of non-canonical base-pairing nucleosides (or nucleotides), non-canonical bases according to certain embodiments

[0138] The term "modified nucleotide" refers to any nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage and / or a modified nucleobase. Thus, the term "modified nucleotide" encompasses substitution, addition or removal of, for example, a functional group or atom of an internucleoside linkage, a sugar moiety or a nucleobase. Modifications suitable for use in the present disclosure include all types of modifications disclosed herein or known in the art. In some embodiments, the modified nucleotide is a non-canonical base pairing nucleotide as defined herein, i.e., a nucleotide having a pairing characteristic that is different from the canonical nucleotide that it replaces. In other embodiments, the modified nucleotide may not have different pairing characteristics, but may still have desired or advantageous features of the RNAi agents, compositions and methods of the present invention, such as stability, resistance to degradation (e.g., nuclease resistance), manufacturability, etc. In certain embodiments, the RNAi agent of the present invention comprises at least one modified nucleotide in addition to at least one non-canonical base pairing nucleotide, i.e., in addition to comprising at least one non-canonical base pairing nucleotide, at least one additional nucleotide is replaced by an additional modified nucleotide (which may or may not be a non-canonical base pairing nucleotide) in the antisense strand and / or the sense strand. In some such embodiments, at least one additional modified nucleotide is present on the antisense strand. In some such embodiments, at least one additional modified nucleotide is present on the sense strand. In some such embodiments, at least one additional modified nucleotide is present on both the antisense strand and the sense strand. In some such embodiments, at least one additional modified nucleotide is present on the same strand as the at least one non-canonical base pairing nucleotide. In other embodiments, at least one additional modified nucleotide is not present on the same strand as the at least one non-canonical base pairing nucleotide, i.e., is present on another strand. In some embodiments, at least one additional modified nucleotide is present in a complementary region, i.e., a region in an oligonucleotide that is substantially complementary to a target sequence, e.g., a region in an antisense strand of the present invention that is complementary to a target sequence mRNA (e.g., INHBE mRNA).

[0139] In the present invention, non-limiting examples of common modified nucleotides and related moieties are defined as shown in Table D. In some embodiments, the modified nucleotide is a modified nucleotide shown in Table C. In some embodiments, at least one additional modified nucleotide of the RNAi agent of the present invention is a modified nucleotide shown in Table C.

[0140] Table D Definitions of modified nucleotides in some embodiments.

[0141] Among them, the modification pattern of inverted nucleotides is also called inverted bases in some literature, which refers to those bases with linkages reversed from the normal 5' to 3' linkages (ie, 5' to 5' linkages or 3' to 3' linkages).

[0142] In some embodiments, the inclusion of deoxynucleotides may be considered to constitute modified nucleotides.

[0143] It is understood that all types of modifications disclosed herein or known in the art are applicable to the RNAi agents, compositions and methods of the present disclosure.

[0144] The term "derivative" as used in the present disclosure should be understood as another compound that is similar in structure but different in some minor structures.

[0145] The terms "inhibit" and the like refer to reducing or effectively stopping, and can be used interchangeably with "reduce", "silence", "downregulate", "suppress" and other similar terms, and include any level of inhibition. As a non-limiting example, "inhibit" herein refers to reducing or effectively reducing the onset or progression of a metabolic disorder or related disease in a subject, including a reduction in one or more aspects of the disease (e.g., symptoms, tissue characteristics, cell activity, inflammatory activity or immune activity, etc.), or no detectable worsening.

[0146] As used herein, "inhibited expression of a gene (eg, LPA, INHBE)" refers to a decrease in the amount or level of an RNA transcript (eg, LPA mRNA, INHBE mRNA) or protein encoded by the gene and / or a decrease in the amount or level of activity of the gene in a cell, cell population, sample, or subject, as compared to an appropriate reference (eg, a reference cell, cell population, sample, or subject). As used herein, "inhibiting target gene expression" refers to a decrease in the amount or level of mRNA for a target gene in a cell, cell population, sample, or subject, such as an inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, compared to an appropriate reference (e.g., a reference cell, cell population, sample, or subject).

[0147] As used herein, the phrase "contacting cells with an RNAi agent" (e.g., dsRNAi agent) includes contacting cells by any possible means. Contacting cells with an RNAi agent includes contacting cells with an RNAi agent in vitro or contacting cells with an RNAi agent in vivo. The contact can be carried out directly or indirectly. Therefore, for example, the RNAi agent can be brought into physical contact with the cell, or alternatively, the RNAi agent can be placed in a situation where it will allow or cause it to subsequently contact the cell. Contacting cells in vitro can be carried out by, for example, incubating the cell with the RNAi agent. Contacting cells in vivo can be carried out, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another region (e.g., bloodstream or subcutaneous space) so that the RNAi agent subsequently reaches the tissue where the cell is located. For example, the RNAi agent can contain or be coupled to a targeting ligand, such as GalNAc, which guides the RNAi agent to a site of interest, such as the liver. In other embodiments, the RNAi agent can contain or be coupled to one or more C22 hydrocarbon chains and one or more GalNAc derivatives. In other embodiments, the RNAi agent contains or is coupled to one or more C22 hydrocarbon chains and does not contain or is not coupled to one or more GalNAc derivatives. A combination of in vitro and in vivo contact methods is also possible. For example, cells can also be contacted with the RNAi agent of the present disclosure in vitro and subsequently transplanted into a subject. In certain embodiments, contacting cells with the RNAi agent includes promoting or influencing the uptake or absorption of cells. The absorption or uptake of the RNAi agent can occur by unassisted diffusion or active cell processes, or by adjuvants or devices. The introduction of RNAi agents into cells can be carried out in vitro or in vivo. For example, for in vivo introduction, the RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction of cells includes methods known in the art, such as electroporation and lipofection.

[0148] "Subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. In certain embodiments, the subject is a human.

[0149] In some embodiments, the "treatment" of any disease or condition refers to improving at least one disease or condition. In certain embodiments, "treatment" refers to improving at least one physical parameter, which may or may not be perceived by the patient. In certain embodiments, "treatment" refers to physically (e.g., stabilization of obvious symptoms), physiologically (e.g., stabilization of physical parameters), or both to suppressing a disease or condition. In some embodiments, "treatment" refers to improving the quality of life of a subject in need or reducing the symptoms or side effects of a disease. A "therapeutically effective amount" refers to the amount of a dsRNA or dsRNAi agent sufficient to achieve the treatment or prevention of a disease when administered to a cell, tissue, or subject alone or in combination with other therapeutic drugs. A "therapeutically effective amount" will vary according to the compound or RNAi agent, the disease and its severity, and the age, weight, etc. of the subject suffering from the disease to be treated or prevented. The term "therapeutically effective amount" as used herein refers to the amount of a compound or composition sufficient to prevent, treat, suppress, reduce, improve, or eliminate one or more causes, symptoms, or complications of a disease or condition, such as metabolic syndrome. The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein.

[0150] In some embodiments, "prevention" or "prevention" of any disease or condition refers to at least reducing the likelihood of the risk (or susceptibility) of acquiring the disease or condition (i.e., so that at least one clinical symptom of the disease does not appear in a patient who may be exposed to or susceptible to the disease but does not yet experience or display symptoms of the disease).

[0151] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0152] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., a lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material (involved in carrying or transporting a compound or dsRNAi agent from one organ or part of the body to another organ or part of the body).

[0153] 2. RNA Interference

[0154] The present disclosure provides RNA (siRNA or RNAi) agents that suppress the expression of metabolic disease-related target genes by RNA interference (RNAi) processes. In some embodiments, RNAi agents include double-stranded ribonucleic acid (dsRNA) molecules for suppressing target gene expression in cells (e.g., adipocytes and / or liver cells, e.g., hepatocytes). siRNA or RNA agents comprising dsRNA molecules are also referred to herein as "dsRNAi" agents. In certain embodiments, the cells are present in a subject. In some embodiments, the subject is a mammal, e.g., a human. In some embodiments, the target gene is INHBE, and the subject has already suffered from or is susceptible to metabolic diseases (e.g., metabolic syndrome), carbohydrate disorders (e.g., type 2 diabetes, prediabetes), lipid metabolism disorders (e.g., hyperlipidemia, hypertension, lipodystrophy), kidney disease, cardiovascular disease, and / or weight disorders (e.g., obesity, overweight). dsRNAi agents include antisense strands with complementary regions that are complementary to at least a portion of the mRNA formed in the expression of the target gene. In some embodiments, the length of the complementary region is about 19-30 nucleotides (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length). In some embodiments, the length of the complementary region is about 15-30 nucleotides (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 nucleotides in length). In certain embodiments, the dsRNAi agent comprises at least one modified nucleotide, as described herein. In certain embodiments, the dsRNAi agent comprises at least one non-canonical base pairing nucleotide, as described herein.

[0155] According to the method disclosed herein, cells expressing target genes (e.g., human, primate, non-primate or mouse LPA gene or INHBE gene) are contacted with siRNA agents that inhibit target gene expression. In some embodiments, the expression of the target gene is suppressed by at least about 50%. The suppression of target gene expression can be determined using any suitable method, such as, but not limited to, by a method based on PCR or branched DNA (bDNA), or by a protein-based method, such as by immunofluorescence analysis, using, for example, Western blotting or flow cytometry techniques. In certain embodiments, the suppression of expression is determined by the rt-PCR method provided in the examples herein (e.g., as described in the examples below, using, for example, 10 nM concentration of siRNA in a suitable organism cell line). In certain embodiments, an animal model is used to determine the suppression of expression in vivo, such as by knocking down the human gene in a rodent expressing a human gene (e.g., a mouse expressing the LPA gene or INHBE gene). In some such embodiments, siRNA is administered to a subject (e.g., an animal model) in a single dose (e.g., 3 mg / kg, 6 mg / kg, or 9 mg / kg).

[0156] DsRNA comprises two RNA chains, and these two RNA chains are complementary and hybridize to form a duplex structure under the conditions that dsRNA will be used (for example, under physiological conditions).One chain (antisense strand) of dsRNA comprises a complementary region that is substantially complementary to the target sequence and is usually completely complementary.The target sequence can be derived from the mRNA sequence formed during the expression of the target gene.Another chain (sense strand) comprises a region complementary to the antisense strand so that when the two chains are combined under suitable conditions, they will hybridize and form a duplex structure.As described elsewhere herein and known in the art, the complementary sequence of dsRNA can be contained in the self-complementary region of a single nucleic acid molecule, rather than on a separate oligonucleotide. Generally, the duplex structure is 15 to 30 base pairs in length, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30 , 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22 base pairs. In certain embodiments, the length of the duplex structure is 17 to 25 base pairs, for example, 17-23, 17-25, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24, or 24-25 base pairs in length, for example, 19-21 base pairs in length. Ranges and lengths intermediate to the above ranges and lengths are also considered part of this disclosure.

[0157] Similarly, the region complementary to the target sequence is 15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-29. 23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths intermediate to the above-recited ranges and lengths are also considered to be part of this disclosure.

[0158] In some embodiments, the duplex structure is 19 to 30 base pairs in length. Similarly, the region complementary to the target sequence is 19 to 30 nucleotides in length.

[0159] In some embodiments, the duplex structure is 15 to 23 base pairs in length. Similarly, the region complementary to the target sequence is 15 to 23 nucleotides in length.

[0160] In some embodiments, the dsRNA is about 19 to about 23 nucleotides in length, or about 25 to about 30 nucleotides in length. In some embodiments, the dsRNA is about 15 to about 23 nucleotides in length, or about 17 to about 23 nucleotides in length, or about 17 to about 25 nucleotides in length, or about 19 to about 21 nucleotides in length.

[0161] The duplex region is the major functional portion of the dsRNA, e.g., a duplex region of about 15 to about 30 base pairs, or about 17 to about 30 base pairs, or about 19 to about 30 base pairs, e.g., about 15-23, 15-25, 17-25, 17-23, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, to the extent that the dsRNA can be processed into a functional duplex (e.g., 15-30 base pairs or at least 15 base pairs) that can target the desired RNA for cleavage, the dsRNA can be an RNA molecule or RNA molecule complex having a duplex region of greater than 30 base pairs. Thus, in one embodiment, the dsRNA is a miRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA.In another embodiment, the siRNA agent useful for targeting target gene expression is not produced in the target cell by cleavage of a larger dsRNA.

[0162] DsRNA as described herein can also include one or more single-stranded nucleotide overhangs, for example 1-4, 2-4, 1-3, 2-3, 1, 2, 3 or 4 nucleotides. The dsRNA with at least one nucleotide overhang has excellent inhibitory properties relative to its blunt-ended counterpart. The nucleotide overhangs can comprise or consist of nucleotides / nucleoside analogs comprising deoxynucleotides / nucleosides. The overhangs can be located on sense strand, antisense strand or its combination in any. In addition, the nucleotides of the overhangs can be present in the 5'-end, 3'-end or both ends of the antisense strand or sense strand of the dsRNA.

[0163] "Flat" or "blunt-ended" means that there are no unpaired nucleotides at the ends of the dsRNA, i.e., there are no nucleotide overhangs. A "blunt-ended" dsRNA is double-stranded over its entire length, i.e., there are no nucleotide overhangs at either end of the molecule. The dsRNAi agents of the present disclosure include dsRNAs that have no nucleotide overhangs at one end (i.e., agents having one overhang and one blunt end) or dsRNAs that have no nucleotide overhangs at either end. In some embodiments, such oligonucleotides are double-stranded over their entire length.

[0164] dsRNA can be synthesized by standard methods known in the art. Double-stranded RNAi compounds of the present disclosure can be prepared using a two-step procedure. First, each chain of the double-stranded RNA molecule is prepared separately and then annealed. Each chain of the siRNA compound can be prepared using solution phase or solid phase organic synthesis or both. The advantage of organic synthesis is that oligonucleotide chains containing non-natural or modified nucleotides can be easily prepared. Similarly, single-stranded oligonucleotides of the present disclosure can be prepared using solution phase or solid phase organic synthesis or both.

[0165] In one aspect, the dsRNA of the present disclosure comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. In some embodiments, the sense strand is selected from the sequences provided in any one of Tables 1 and 2, and the corresponding antisense strand of the sense strand is selected from the sequences provided in any one of Tables 1 and 2. In this aspect, one of the two sequences is complementary to the other of the two sequences, wherein one of the sequences is substantially complementary to the mRNA sequence produced during expression of the relevant target gene. Thus, in this aspect, the dsRNA will comprise two oligonucleotides, wherein one oligonucleotide is described as the sense strand of any one of Tables 1 and 2, and the second oligonucleotide is described as the corresponding antisense strand of any one of the sense strands in Tables 1 and 2.

[0166] It is well known to those skilled in the art that dsRNAs having a duplex structure of about 20 to 23 base pairs (e.g., 21 base pairs) are considered particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20: 6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14: 1714-1719; Kim et al. (2005) Nat Biotech 23: 222-226). In some embodiments, the dsRNA can include at least one strand that is at least 21 nucleotides in length. It is reasonable to expect that shorter duplexes of the dsRNAs in Tables 1 and 2, with only a few nucleotides removed from one or both ends, can also have similar effects compared to the above-mentioned dsRNAs. Thus, dsRNAs having a sequence of at least 12, 13, 14, 15, 19, 20, or more contiguous nucleotides derived from any one of Tables 1 and 2, and whose ability to inhibit target gene expression does not differ by more than about 5%, 10%, 15%, 20%, 25%, or 30% from a dsRNA comprising the entire sequence, are considered to be within the scope of the present disclosure.

[0167] 3. Modification of RNAi Agents

[0168] The RNA of the iRNA (e.g., dsRNA) of the present disclosure is chemically modified to enhance stability or other beneficial properties. In certain embodiments of the present disclosure, the iRNA, e.g., dsRNA of the present disclosure, at least conforms to the modification pattern as described above, i.e., comprises a specific hypoxanthine modification pattern. Specifically, in the modified dsRNA of the present disclosure, at least one of the nucleotides from position 2 to position 11 in the direction from the 5' end to the 3' end of the antisense strand comprises hypoxanthine, and the modified dsRNA conforms to at least one of the following characteristics:

[0169] (1) In the continuous sequence N1N2I0N3N4 at positions 2 to 11 of the antisense strand, from the 5' end to the 3' end, at least three bases among N1, N2, N3 and N4 are adenine and / or uracil; wherein N1, N2, N3 and N4 are each independently a nucleotide containing adenine, cytosine, guanine, thymine or uracil as a base, and I0 is a nucleotide containing hypoxanthine as a base;

[0170] (2) the number of adenines and uracils in positions 2 to 8 of the antisense strand is 4 or more in the direction from the 5' end to the 3' end; and / or

[0171] (3) The modified dsRNA has a dissociation temperature (Tm) that is at least 2°C different from that of a GC-paired dsRNA; wherein the GC-paired dsRNA differs from the modified dsRNA only in that a guanine is present in place of a hypoxanthine (G:C pairing).

[0172] In some embodiments, the number of non-canonical base pairs is 1, 2, or 3 pairs.

[0173] In some embodiments, the base in at least one of the nucleotides at position 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 in the antisense strand from the 5' end to the 3' end is a hypoxanthine group.

[0174] In some embodiments, in the modified dsRNA, at least one of the nucleotides 2 to 8 in the antisense strand in the 5' to 3' direction comprises hypoxanthine.

[0175] In some embodiments, in the modified dsRNA, at least one of the nucleotides at positions 6 to 8 in the 5' to 3' direction of the antisense strand comprises hypoxanthine, such as positions 6, 7, and / or 8.

[0176] In some embodiments, the consecutive sequence N1N2I0N3N4 at positions 2-11 of the antisense strand does not contain guanine.

[0177] In some embodiments, in the consecutive sequence N1N2I0N3N4 at positions 2-11 of the antisense strand, the bases N1, N2, N3 and N4 are each independently adenine and / or uracil.

[0178] In some such embodiments, at least one nucleotide of N1, N2, N3, N4, and I0 further has a modified sugar group and / or a modified internucleotide linkage.

[0179] In some embodiments, the modified dsRNA of the present disclosure has a Tm change relative to the GC paired dsRNA of at least 2° C., such as 2° C., more than 2° C., 3° ​​C., 4° C., 5° C., or more. In some such embodiments, the Tm is calculated using a formula or algorithm described herein.

[0180] In some embodiments, the length of the sense strand and the antisense strand is each independently 17-25 nucleotides, for example, each independently 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides; preferably, the length of the sense strand and the antisense strand is each independently 19-23 nucleotides; more preferably, the length of the sense strand and the antisense strand is each independently 19-21 nucleotides.

[0181] In some embodiments, the other nucleotides in the sense and antisense strands are unmodified nucleotides.

[0182] In some embodiments, substantially all of the nucleotides in the sense strand and the antisense strand are modified nucleotides.

[0183] In some embodiments, all nucleotides in the sense strand and the antisense strand are modified nucleotides.

[0184] In some embodiments, the modified dsRNA of the present disclosure further comprises at least one modified nucleotide selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-F-arabinonucleotides, phosphorothioate modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleic acids, inverted nucleotides, and non-canonical base modified nucleotides. Exemplary non-canonical base modified nucleotides include, but are not limited to, bases selected from inosine (I), xanthosine (X), 7-methylguanosine (m7G), N6-methyladenosine (m6A), dihydrouridine, 5-methylcytosine (m5C), pseudouridine (Ψ), and N1-methylpseudouridine (m1Ψ).

[0185] In some embodiments, the modified dsRNA further comprises at least one modified nucleotide selected from the group consisting of 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-F-arabino nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides, and inverted nucleotides. In some such embodiments, the inverted nucleotide is selected from the group consisting of inverted A nucleotides, inverted dA nucleotides, inverted dT nucleotides, inverted C nucleotides, and inverted U nucleotides.

[0186] In some embodiments, the modified dsRNA further meets at least one of the following characteristics:

[0187] 1) the antisense strand comprises 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8) 2'-fluoro modified nucleotides;

[0188] 2) the antisense strand comprises 1-4 (e.g., 1, 2, 3, 4) phosphorothioate internucleotide linkages;

[0189] 3) the antisense strand comprises 12-19 (e.g., 12, 13, 14, 15, 16, 17, 18, 19) 2'-O-methyl modified nucleotides;

[0190] 4) the sense strand comprises 2-4 (e.g., 2, 3, 4) 2'-fluoro modified nucleotides;

[0191] 5) the sense strand comprises 1-4 (e.g., 1, 2, 3, 4) phosphorothioate internucleotide linkages;

[0192] 6) The sense strand comprises 14-17 (eg, 14, 15, 16, 17) 2'-O-methyl modified nucleotides.

[0193] In some embodiments, the target gene is LPA, and the modified dsRNA further meets at least one of the following characteristics:

[0194] 1) The antisense strand contains at least 5 or more 2'-fluoro modified nucleotides or 5 or more 2'-deoxy modified nucleotides, and the remaining sites are 2'-O-methyl modified nucleotides;

[0195] 2) In the antisense strand, positions 2, 5, 7, and 12 are 2'-deoxy modified nucleotides, position 14 is a 2'-fluoro modified nucleotide, positions 9 and 16 contain 0, 1, and 2 2'-fluoro modified nucleotides, respectively, and the remaining positions are 2'-O-methyl modified nucleotides;

[0196] 3) The first position of the antisense strand is a 5'-vinylphosphite modified nucleotide;

[0197] 4) the sense strand contains three or four 2'-fluoro modified nucleotides at positions 7, 8, 9, and 10;

[0198] 5) From the 5' end to the 3' end, at least one or two of the three nucleotides from the 5' end of the sense strand are phosphorothioate modified nucleotides; and / or at least one or two of the three nucleotides from the 5' end and the 3' end of the antisense strand are phosphorothioate modified nucleotides, respectively.

[0199] In some embodiments, the sense strand of the present disclosure is derived from the mRNA sequence of human LPA (e.g., Gene ID: 4018). Alternatively, the sense strand of the present disclosure is a fragment of the mRNA sequence of human LPA. In some embodiments, the sense strand of the present disclosure is derived from the mRNA sequence of cynomolgus monkey LPA (e.g., Gene ID: 101865897). Alternatively, the sense strand of the present disclosure is a fragment of the mRNA sequence of cynomolgus monkey LPA.

[0200] In some embodiments, the target gene is INHBE, and the modified dsRNA further meets at least one of the following characteristics:

[0201] 1) From the 5' end to the 3' end, the nucleotides at positions 2, 4, 12, and 14 of the antisense strand are 2'-fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides;

[0202] 2) From the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of the sense strand are 2'-fluorine-modified nucleotides.

[0203] In some embodiments, the sense strand of the present disclosure is derived from the mRNA sequence of human INHBE (e.g., Gene ID: 83729). Alternatively, the sense strand of the present disclosure is a fragment of the mRNA sequence of human INHBE. In some embodiments, the sense strand of the present disclosure is derived from the mRNA sequence of cynomolgus monkey INHBE (e.g., Gene ID: 102127493). Alternatively, the sense strand of the present disclosure is a fragment of the mRNA sequence of cynomolgus monkey INHBE.

[0204] In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides that differ from any of the sequences shown in Table 1 or Table 2 by 0, 1, 2, or 3 nucleotides, and the sense strand has at least 15, 16, 17, 18, 19, 20, or 21 nucleotides complementary to the antisense strand. In some embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides that differ from any of the sequences shown in Table 1 or Table 2 by 0, 1, 2, or 3 nucleotides. It should be understood that the "difference by 0, 1, 2, or 3 nucleotides" is not related to the specific hypoxanthine modification pattern disclosed herein.

[0205] In some embodiments, the dsRNA agents of the present disclosure comprise one or more targeting ligands, such as one or more GalNAc derivatives, and contain no additional chemical modifications known in the art and described herein in the remaining positions of the sense and antisense strands.

[0206] In some embodiments, the dsRNA agent of the present disclosure comprises one or more targeting ligands, such as one or more GalNAc derivatives, and comprises at least one additional nucleic acid modification described herein. For example, the dsRNAi agent may comprise at least one modification selected from the group consisting of modified internucleoside linkages, modified core bases, modified sugars, and any combination thereof. Without limitation, such modifications may be present anywhere in the dsRNA agent of the present disclosure. For example, the modification may be present in one of the RNA molecules. Modifications include, for example, terminal modifications, such as 5' end modifications (phosphorylation, conjugation, reverse connection) or 3' end modifications (conjugation, DNA nucleotides, reverse connection, etc.); base modifications, such as replacement of bases with stable bases, destabilizing bases, or bases paired with extended bases, removal of bases (absorptive nucleotides) or conjugation of bases; sugar modifications (e.g., at the 2'-position or 4'-position) or sugar replacement; or backbone modifications, including modification or replacement of phosphodiester bonds. Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or free of natural internucleoside linkages. RNAs with modified backbones include RNAs that do not have a phosphorus atom in the backbone, etc. In some embodiments, the backbone of the modified RNAi agent has a phosphorus atom in the internucleoside backbone.

[0207] In some embodiments, backbone modifications refer to internucleoside linkages or backbones including but not limited to phosphorothioate groups, chiral phosphorothioates, phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, chiral phosphonates, phosphinates, phosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters, morpholino linkages, wherein adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0208] In some embodiments, the antisense strand of the modified dsRNA comprises a nucleotide sequence (from 5' end → 3' end) of any antisense strand sequence in Table 1 or 2. In some embodiments, the sense strand of the modified dsRNA comprises a nucleotide sequence (from 5' end → 3' end) of any sense strand in Table 1 or 2. In some embodiments, the antisense strand of the modified dsRNA comprises a nucleotide sequence (from 5' end → 3' end) of any antisense strand in Table 1 or 2, and the sense strand comprises a nucleotide sequence (from 5' end → 3' end) of any sense strand in Table 1 or 2.

[0209] In some embodiments, the dsRNA agent of the present disclosure further comprises a phosphate or phosphate mimic located at the 5'-end of the antisense strand. In one embodiment, the phosphate mimic is 5'-vinyl phosphate (VP). In some embodiments, the 5'-end of the antisense strand of the dsRNA agent does not comprise 5'-vinyl phosphate (VP).

[0210] The end of the dsRNA reagent of the present disclosure can be modified, and this modification can be at one end or both ends.For example, the 3' and / or 5' end of dsRNA can be conjugated to other functional molecular entities, such as labeling moieties, such as fluorophores (such as pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based on, for example, sulfur, silicon, boron or ester). Functional molecular entities can be connected to sugar by phosphate groups and / or linkers. The terminal atom of the joint can connect or replace the phosphate group of sugar or the connecting atom of C-3', C-5', O, N, S or C groups. Alternatively, the joint can connect or replace the terminal atom of nucleotide substitutes (for example, PNA). When linker / phosphate functional molecular entity-linker / phosphate array is inserted between two chains of double-stranded oligomeric compounds, the array can replace the hairpin loop in hairpin type oligomeric compounds. In some embodiments, end modification can also be used for monitoring distribution, and in this case, the preferred group to be added includes fluorophores, such as fluorescein or Alexa dyes, such as Alexa 488. In some embodiments, terminal modifications may also be used to enhance uptake, and non-limiting modifications useful for this include targeting ligands.

[0211] The present disclosure also encompasses various salts, mixed salts, and free acid forms of the dsRNA agents. In some embodiments, the dsRNA agent is in free acid form. In some embodiments, the dsRNA agent is in salt form. In one embodiment, the dsRNA agent is in sodium salt form. According to common knowledge in the art, when the dsRNA agent of the present disclosure is in sodium salt form, sodium ions are present in the agent as counterions to the phosphodiester and / or phosphorothioate groups.

[0212] In certain embodiments, the dsRNA agent of the present disclosure is further modified by covalently linking one or more conjugate groups. Generally speaking, the conjugate group changes one or more properties of the dsRNA agent of the present disclosure connected, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cell distribution, cellular uptake, charge and removal. Conjugate groups are commonly used in the field of chemistry, and are directly or through optional linking moieties or linking groups connected to the parent compound. Conjugate groups preferably include but are not limited to polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, bile acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin and dyes.

[0213] In some embodiments, the targeting ligand of the present disclosure comprises N-acetyl-galactosamine (GalNAc), or a GalNAc derivative, such as L96 (see siRNA drug Inclisiran). In some embodiments, the targeting ligand is any targeting ligand disclosed in WO2022266753A1. Unless otherwise clearly contradictory, WO2022266753A1 is incorporated herein by reference in its entirety.

[0214] In some embodiments, the structure of the dsRNA agent is selected from Formula 1 to Formula 33, wherein R 2 According to common knowledge in this field, R 2 The dsRNA agent is formed by conjugating the 3' end or 5' end of the sense strand of the dsRNA to a targeting ligand.

[0215] IV. Delivery and Use of RNAi Agents

[0216] The RNAi agents of the present invention can be delivered to cells, such as cells in a subject (e.g., a subject with a metabolic disorder) in a variety of ways. For example, delivery can be carried out by contacting cells with the RNAi agents of the present invention in vitro or in vivo. In vivo delivery can also be carried out directly by administering a composition (e.g., a pharmaceutical composition) comprising an RNAi agent (e.g., a dsRNA agent) to the subject. Alternatively, in vivo delivery can be carried out indirectly by administering one or more vectors that encode and guide the expression of the RNAi agent.

[0217] In one embodiment, the cells are liver cells, such as hepatocytes. In one embodiment, the cells are adipocytes. In certain embodiments, the RNAi agent is taken up by one or more tissue or cell types present in an organ (e.g., liver, adipose tissue).

[0218] Another aspect of the present disclosure relates to a method for reducing the expression and / or activity of a target gene (e.g., LPA gene, INHBE gene) in a subject, comprising administering a dsRNAi agent of the present disclosure to the subject. In some embodiments, the method comprises administering a therapeutically effective amount of a dsRNAi agent of the present disclosure to the subject, thereby inhibiting the expression of the target gene in the cell. In some embodiments, the method comprises contacting the cell with a double-stranded RNAi agent of the present disclosure, such that the mRNA transcript of the target gene is degraded, thereby inhibiting the expression of the target gene in the cell.

[0219] In another aspect, the disclosure relates to a method of treating a subject having or at risk of a target gene-mediated disease, comprising administering to the subject a therapeutically effective amount of a dsRNAi agent of the disclosure, thereby treating the subject.

[0220] In another aspect, the present disclosure relates to a method of treating or preventing a metabolic disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a disclosed dsRNAi agent, such that the metabolic disorder is treated or prevented.

[0221] In another aspect, the present disclosure relates to a method of treating or preventing a disease or condition associated with LPA and / or INHBE in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a dsRNAi agent such that the disease or condition associated with LPA and / or INHBE is treated or prevented.

[0222] In some embodiments, the subject is a human.

[0223] In some embodiments, the target gene is selected from the mRNA genes involved in the following targets: PCSK9, ANGPTL3, LPA, INHBE, ACVR1C, PLIN1, PDE3B, INHBC, GDF-8 (MSTN), SOD1, APP, C3, C5, HTT, DMPK, HSD17B13, PNPLA3, XDH, AGT, AKT, Kras, SHP2, TGF-β, IFN-α, IL-13, IL-6, Myc, IL-4, IL-17, TERT, KHK, Factor VII, Factor X, Factor XI, Thrombin, TPX2, apoB, SAA, TTR, RSV, PDGFβ, Erb-B, Src, CRK, GRB2, MEKK, JNK, RAF, Erk1 / 2, MYB, JUN, FOS, BCL-2, hepciden, PC, CCND, VEGF, EGFR, CCNA, CCNE, WNT-1, β-catenin, c-MET, PKC, NFKB, STAT3, survivin, TOP1 and TOP2A. In some embodiments, the target gene is the LPA gene or the INHBE gene.

[0224] In some embodiments, the subject has a metabolic disorder.

[0225] In some embodiments, the metabolic disorder is one or more of metabolic syndrome, type 2 diabetes, obesity, prediabetes, hypertriglyceridemia, lipodystrophy, liver inflammation, fatty liver, hypercholesterolemia, diseases associated with elevated liver enzymes, non-alcoholic steatohepatitis, cardiovascular disease, and kidney disease. In some embodiments, the metabolic syndrome includes, but is not limited to, one or more of abdominal obesity, insulin resistance, hypertension, and dyslipidemia.

[0226] In some embodiments, the LPA-associated disease or condition is one or more of Berger's disease, metabolic syndrome, aortic regurgitation, aortic dissection, retinal artery occlusion, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, heterozygous or homozygous familial hypercholesterolemia, hyperlipoproteinemia, cerebrovascular atherosclerosis, venous thrombosis, congestive heart failure, ischemic heart disease, carotid artery disease, myocardial infarction, stroke, atrial fibrillation, heart failure, hyperlipidemia, type 2 diabetes, and non-alcoholic fatty liver disease.

[0227] In some embodiments, the INHBE-associated disease or condition is one or more of a metabolic disorder, metabolic syndrome, type 2 diabetes, obesity, prediabetes, hypertriglyceridemia, lipodystrophy, liver inflammation, fatty liver, hypercholesterolemia, a disease associated with elevated liver enzymes, nonalcoholic steatohepatitis, cardiovascular disease, kidney disease, abdominal obesity, insulin resistance, hypertension, dyslipidemia, a cardiometabolic disorder, and a cancer associated with INHBE expression.

[0228] Non-limiting examples of metabolic disorders include carbohydrate disorders, such as diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, galactosemia, hereditary fructose intolerance, fructose 1,6-bisphosphatase deficiency, glycogen storage diseases, inborn errors of glycosylation, insulin resistance, insulin insufficiency, hyperinsulinemia, impaired glucose tolerance (IGT), dysglycogen metabolism, amino acid metabolism disorders, such as maple syrup urine disease (MSUD), homocystinuria; organic acid metabolism disorders, such as methylmalonic aciduria, 3-methylglutaric aciduria-Bartter syndrome, glutaric aciduria, 2-hydroxyglutaric aciduria-D and L types; fatty acid β-oxidation disorders, such as medium chain acyl-CoA dehydrogenase deficiency (MCAD), long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LC3D), and hydroxyacyl-CoA dehydrogenase deficiency (LC3D). and peroxisomal disorders, such as Zellweger syndrome (CHS), X-linked adrenoleukodystrophy, and Refsum disease.

[0229] In some embodiments, the metabolic disorder is associated with body fat distribution and includes, but is not limited to, metabolic syndrome, type 2 diabetes, hyperlipidemia or dyslipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C)), hyperlipidemia or dyslipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B, or other lipid components), obesity (particularly abdominal obesity), lipodystrophy (e.g., inability to store fat locally in body fat (regional lipodystrophy) or systemically (lipoatrophy)), insulin resistance or elevated or altered insulin levels during fasting or metabolic challenge, hepatic fat deposition or fatty liver body fat distribution and its complications (e.g., cirrhosis, fibrosis, or inflammation of the liver), non-alcoholic steatohepatitis, other types of liver inflammation, elevated or elevated or altered liver enzyme levels or other markers of liver damage, inflammation or fat deposition in the liver, elevated blood pressure and / or hypertension, elevated blood sugar or glucose or hyperglycemia, metabolic syndrome, coronary artery disease and other atherosclerotic conditions and their complications. In some embodiments, the metabolic disorder is associated with body fat distribution characterized by greater fat accumulation around the waist (e.g., greater abdominal fat or larger waist circumference) and / or less fat accumulation around the hips (e.g., lower gluteofemoral fat or smaller hip circumference), resulting in a larger waist-to-hip ratio (WHR) and higher cardiometabolic risk, independent of body mass index (BMI).

[0230] In one embodiment, the metabolic disorder is metabolic syndrome. As used herein, the term "metabolic syndrome" refers to a collection of symptoms that include overnutrition, sedentary lifestyle, genetic factors, aging, and the resulting obesity. Metabolic syndrome includes abdominal obesity, insulin resistance, dyslipidemia, and elevated blood pressure, and is associated with other comorbidities, including prothrombotic state, proinflammatory state, nonalcoholic fatty liver disease, and reproductive disorders. Metabolic syndrome approximately doubles the risk of cardiovascular disease and 5 times the risk of type 2 diabetes. Abdominal obesity (e.g., excessive waist circumference (high waist-to-hip ratio)), hypertension, insulin resistance, and dyslipidemia are core components of metabolic syndrome and its various components (e.g., central obesity, fasting blood glucose (FBG) / prediabetes / diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension).

[0231] In one embodiment, the metabolic disorder is a carbohydrate disorder. In one embodiment, the carbohydrate disorder is diabetes. As used herein, the term "diabetes" refers to a collection of metabolic disorders characterized by high blood sugar (glucose) levels, which are caused by defects in insulin secretion or action or both. The two most common types of diabetes, type 1 diabetes (also referred to as "type I diabetes") and type 2 diabetes (also referred to as "type II diabetes"), are both caused by the body's inability to regulate insulin. Insulin is a hormone released by the pancreas in response to elevated blood sugar (glucose) levels.

[0232] As used herein, the term "type 1 diabetes" refers to a chronic disease that occurs when the pancreas produces too little insulin to properly regulate blood sugar levels. Type 1 diabetes is also known as insulin-dependent diabetes mellitus, IDDM, and juvenile-onset diabetes. People with type 1 diabetes (insulin-dependent diabetes) typically produce little or no insulin. Type 1 diabetes may be due to progressive autoimmune destruction of pancreatic beta cells and subsequent insulin deficiency. People with type 1 diabetes must take regular insulin injections.

[0233] In type 2 diabetes (also known as non-insulin-dependent diabetes mellitus, NDDM), the pancreas continues to produce insulin, sometimes even at higher than normal levels, yet the body becomes resistant to its effects, leading to relative insulin deficiency. Obesity is a risk factor for type 2 diabetes, and most people with the condition are obese.

[0234] In some embodiments, diabetes includes prediabetes. "Prediabetes" refers to one or more early diabetic symptoms, including impaired glucose utilization, abnormal or impaired fasting blood glucose levels, impaired glucose tolerance, impaired insulin sensitivity, and insulin resistance. Prediabetes is a major risk factor for type 2 diabetes, cardiovascular disease, and mortality. Developing therapeutic interventions to effectively treat prediabetes to prevent the development of type 2 diabetes has received widespread attention.

[0235] Diabetes can be diagnosed by performing a glucose tolerance test. Clinically, diabetes is often divided into several basic categories. The main examples of these categories include autoimmune diabetes, non-insulin dependent diabetes mellitus (type 2 NDDM or NIDDM), insulin dependent diabetes mellitus (type 1 IDDM), non-autoimmune diabetes and maturity-onset diabetes of the juvenile onset (MODY). Another classification is commonly referred to as secondary diabetes, refers to the diabetes caused by some identifiable conditions, and these conditions cause or promote the development of diabetic syndrome. The example of the second category includes but is not limited to, the diabetes caused by pancreatic disease, hormone abnormalities, the diabetes induced by drugs or chemicals, the diabetes caused by insulin receptor abnormalities, the diabetes relevant to genetic syndromes and the diabetes of other reasons.

[0236] In one embodiment, metabolic disorder is lipid metabolism disorder. " lipid metabolism disorder " or " lipid metabolism disorder " as used herein refer to any disease relevant to lipid metabolism disorder or caused by lipid metabolism disorder. The term also includes any disease, disease or symptom that can cause hyperlipidemia or be characterized as any or all lipids and / or lipoprotein levels in blood that are abnormally elevated. The term can refer to hereditary diseases, such as familial hypertriglyceridemia, type 1 familial partial lipodystrophy (FPLDI), or secondary or acquired diseases, such as due to disease, disease or symptom (such as, renal failure), diet or intake of some medicines (such as, due to treatment, such as, AIDS or HIV, and using highly effective antiretroviral therapy (HAART)) to induce or obtain disease) and induce or obtain disease. The term also refers to the disorder of fat distribution and / or storage, such as lipodystrophy.

[0237] Additional examples of lipid metabolism disorders include, but are not limited to, atherosclerosis, dyslipidemia, hypertriglyceridemia (including drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcohol-induced hypertriglyceridemia, beta-adrenergic blocking agent-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia, vitamin A-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, and familial hypertriglyceridemia. hyperlipidemia, hyperlipidemia (including familial combined hyperlipidemia), hyperlipidemia (including familial combined hyperlipidemia), hyperlipidemia (including familial combined hyperlipidemia), lipodystrophy, gout associated with hypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposits), heterogeneous LPL deficiency hyperlipidemia, high LDL and heterogeneous LPL deficiency hyperlipidemia, fatty liver disease, or nonalcoholic steatohepatitis (NASH).

[0238] In one embodiment, the metabolic disorder is cardiovascular disease. The cardiovascular disease may include, but is not limited to, coronary artery disease (also known as ischemic heart disease), hypertension, inflammation associated with coronary artery disease, restenosis, peripheral vascular disease, or stroke.

[0239] In one embodiment, the metabolic disorder is a kidney disease. The kidney disease may include, but is not limited to, chronic kidney disease, diabetic nephropathy, or gout.

[0240] In one embodiment, the metabolic disorder is a disorder related to weight. Weight disorders may include, but are not limited to, obesity, hypometabolic states, hypothyroidism, uremia, and risk of weight gain (including rapid weight gain), weight loss, sustained weight loss, or weight regain after weight loss.

[0241] In one embodiment, the metabolic disorder is a glycemic disorder. Glycemic disorders may include, but are not limited to, diabetes, hypertension, and polycystic ovary syndrome (PCOS) associated with insulin resistance.

[0242] Other exemplary metabolic disorders include, but are not limited to, renal transplantation, nephrotic syndrome, Cushing's syndrome, acromegaly, systemic lupus erythematosus, dysglobulinemia, lipodystrophy, glycogenotype 1, and Addison's disease.

[0243] In one embodiment, metabolic disorder is essential hypertension." Essential hypertension " may be the result of environmental or genetic causes (e.g., the result without obvious underlying medical causes). In another embodiment, metabolic disorder is secondary hypertension." Secondary hypertension " has an identifiable underlying disease, which may be of multiple etiologies, including kidney, blood vessel and endocrine causes, such as renal parenchymal disease (e.g., polycystic kidney disease, glomerular or interstitial disease), renal vascular disease (e.g., renal artery stenosis, fibromuscular dysplasia), endocrine disease (e.g., adrenocortical steroids or mineralocorticoids are excessive, pheochromocytoma, hyperthyroidism or hypothyroidism, growth hormone is excessive, hyperparathyroidism), coarctation of the aorta or use of oral contraceptives.

[0244] In one embodiment, the metabolic disorder is refractory hypertension. "Refractory hypertension" refers to blood pressure that remains above target (e.g., systolic blood pressure above 130 mmHg or diastolic blood pressure above 90 mmHg) despite simultaneous use of three different classes of antihypertensive drugs (one of which is a thiazide diuretic). Subjects who use four or more medications to control their blood pressure are also considered to have refractory hypertension.

[0245] In some embodiments, the expression of the target gene in the cell is inhibited, and the protein level of the target gene expression in the serum of the subject is reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In some embodiments of the present disclosure, the expression of the LPA gene or INHBE gene in the subject or cell reduces the corresponding LPA protein level or INHBE protein level in the serum of the subject by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0246] In some embodiments of the present disclosure, the dsRNAi agent is administered to a subject at a dose of about 0.01 mg / kg to about 50 mg / kg, or at a dose of about 0.10 mg / kg to about 50 mg / kg, for example, but not limited to, a dose of about 0.01 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 50 mg / kg, about 10 mg / kg to about 30 mg / kg, about 10 mg / kg to about 20 mg / kg, about 15 mg / kg to about 20 mg / kg, about 15 mg / kg to about 25 mg / kg, about 15 mg / kg to about 30 mg / kg, or about 20 mg / kg to about 30 mg / kg.

[0247] In some embodiments of the present disclosure, the method further comprises determining the target gene level (e.g., LPA or INHBE level) in a sample from the subject, for example, in a blood, serum, liver tissue, or adipose tissue sample. The target gene level in the sample from the subject can be determined before, during, and / or after administration of the dsRNAi agent to the subject (e.g., to monitor efficacy or treatment efficiency, monitor target gene mRNA and / or protein levels before, during, or after treatment, etc.).

[0248] In some embodiments of the present disclosure, the method further comprises administering to the subject an additional therapeutic agent to treat the metabolic disorder. The therapeutic agent includes but is not limited to insulin, glucagon-like peptide 1 agonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists, glucagon receptor agonists, sulfonylureas, seglininides, biguanides, thiazolidinediones, α-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, statins, and a combination of any of the foregoing drugs.

[0249] In some embodiments, the dsRNAi agent of the present disclosure is administered by injection or by infusion. In one embodiment, the double-stranded RNAi agent is administered subcutaneously. In one embodiment, the double-stranded RNAi agent is administered intramuscularly. In one embodiment, the double-stranded RNAi agent is administered intravenously. In one embodiment, the double-stranded RNAi agent is administered by systemic administration to the lungs, such as intranasal administration or oral inhalation administration.

[0250] In some embodiments, the pharmaceutical composition comprises a dsRNAi agent of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention can be used in practice for the prevention and / or treatment of various corresponding diseases or conditions. An acceptable carrier (or excipient) is a substance that is intentionally included in a drug delivery system in addition to an active pharmaceutical ingredient (API, therapeutic product, such as a dsRNA agent of the present invention). The carrier or excipient does not or is not intended to exert a therapeutic effect at the intended dose. The carrier or excipient may play the following roles: a) aids in the handling of the drug delivery system during preparation, b) protects, supports or enhances the stability, bioavailability or patient acceptability of the API; c) aids in product identification; and / or d) enhances any other properties of the overall safety, efficacy or delivery of the API during storage or use. Carriers or excipients include, but are not limited to, the following components: absorption enhancers, anti-adherents, anti-foaming agents, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, expanders, fillers, flavorings, glidants, wetting agents, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained-release matrices, sweeteners, thickeners, tonicity agents, vehicles, waterproofing agents, and wetting agents.

[0251] In some embodiments, the carrier of the pharmaceutical composition is a non-buffered solution or a buffered solution. Typical non-buffered solutions are saline or water, and buffered solutions include one or more of acetate, citrate, prolamin, carbonate, and phosphate. In some embodiments, the buffered solution is phosphate buffered saline (PBS).

[0252] The present disclosure includes all combinations of the specific embodiments described. Further embodiments of the present disclosure and the full scope of applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present disclosure, these descriptions and examples are provided by way of illustration only, as various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including citations, are incorporated herein by reference in their entirety for all purposes.

[0253] The compounds disclosed herein can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining the specific embodiments with other methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples disclosed herein.

[0254] Example

[0255] The present disclosure will be more readily understood by reference to the following examples, which are provided to illustrate the invention and are not to be construed as limiting the scope of the invention in any way.

[0256] Unless otherwise defined or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. Unless otherwise specified, the materials and instruments used in the present invention were all commercially available.

[0257] Example 1: dsRNA synthesis

[0258] 1.1 Target sequence screening

[0259] dsRNA was designed based on the full LPA mRNA sequence, all sequences of which were obtained from the NCBI gene database (https: / / www.ncbi.nlm.nih.gov / gene / ). All dsRNA designs were guaranteed to be completely identical to the human (Gene ID: 4018, SEQ ID NO: 2361) and cynomolgus macaque (Gene ID: 101865897, SEQ ID NOs: 2362-2367) sequences.

[0260] dsRNA was designed based on the full INHBE mRNA sequence, all sequences of which were obtained from the NCBI gene database (https: / / www.ncbi.nlm.nih.gov / gene / ). All dsRNA designs were guaranteed to be fully identical to the human (Gene ID: 83729, SEQ ID NO: 2368) and cynomolgus macaque (Gene ID: 102127493, SEQ ID NO: 2369) sequences.

[0261] After scanning the entire sequence, all potential 19-nucleotide siRNA sequences were obtained and simultaneously compared to the cynomolgus macaque sequence to ensure a match. All human / cynomolgus macaque sequences were then BLAST-aligned against the human full transcriptome mRNA sequence, and any dsRNAs with potential off-target effects were removed. The activity of all dsRNAs was evaluated using rational dsRNA design principles, and molecules with low theoretical activity were removed.

[0262] 1.2 dsRNA synthesis

[0263] Hypoxanthine-modified dsRNAs were designed for LPA and INHBE at various sites (as shown in Tables 1 and 2-A to 2-B). These were synthesized and annealed by Suzhou Beixin Biotechnology Co., Ltd. Blunt-ended siRNAs (e.g., LPAI-1 to LPAI-238) were synthesized with two complementary nucleotides or two dTs (i.e., dTdT) added to the 3' end of the antisense strand for in vitro activity assays. Table 3 shows the sequences of dsRNAs not modified with hypoxanthine.

[0264] Table 1: Sense and antisense strand sequences of hypoxanthine-modified dsRNA reagents

[0265] Table 2-A: Sense and antisense strand sequences of hypoxanthine-modified LPA dsRNA and INHBE dsRNA

[0266] Table 2-B: Sense and antisense strand sequences of hypoxanthine-modified LPA dsRNA and INHBE dsRNA

[0267] Table 3. dsRNA sequences not modified with hypoxanthine

[0268] Example 2: dsRNA in vitro activity detection method

[0269] 2.1 Fluorescence quantitative PCR

[0270] 2.1.1 Cell culture and transfection

[0271] (1)LPA

[0272] Huh7 cells (ATCC) were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and double antibodies (Gibco) at 5% CO2 and 37°C. After the cells grew to almost completely cover the culture flask, they were digested with trypsin and resuspended. The resuspended cells were adjusted in density and seeded into 96-well plates at 3e4 / well, and dsRNA transfection complexes were added to the suspension. The dsRNA transfection complex was obtained by mixing Opti-MEM (Gibco) containing 0.3ul / well liposome Lipofectamine 3000 (Thermo) with the dsRNA mixture in a 1:1 ratio. After 48 hours of cell culture, Dynabeads were used according to the instructions. TM Total mRNA was extracted using an mRNA isolation kit (Thermo). Heating was performed using a BIO-RAD T100 Thermal Cycler PCR instrument.

[0273] (2)INHBE

[0274] Hep3B cells were cultured in MEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and double-antibody (Gibco) in a 5% CO2, 37°C atmosphere. After the cells grew to nearly completely cover the culture flask, they were trypsinized and resuspended. The resuspended cells were adjusted to a density of 1.5×10 4 Cells were seeded into 96-well plates and suspended in dsRNA transfection complexes. The dsRNA transfection complexes were prepared by mixing Opti-MEM (Gibco) containing 0.3 μL / well Lipofectamine RNAi Max (Thermo) with dsRNA in a 1:1 ratio. After the cells were cultured for a certain period of time, Dynabeads were used according to the instructions. TM Total mRNA was extracted using an mRNA isolation kit (Thermo). Finally, mRNA was eluted with 20 μL of RNase-free H₂O at 80°C for 5 minutes, and 15 μL of the supernatant was quickly transferred to a magnetic rack. Heating was performed using a BIO-RAD T100 Thermal Cycler PCR instrument.

[0275] 2.1.2 Reverse transcription and fluorescent quantitative PCR

[0276] (1)LPA

[0277] cDNA synthesis was performed using the Vazyme Reverse Transcription Kit. mRNA from Example 2.1.1 was added and the volume was adjusted to 20 μl with RNase-free H₂O. cDNA synthesis was performed using a BIO-RAD T100 Thermal Cycler PCR instrument and the cells were stored at 4°C.

[0278] The relative mRNA levels of LPA and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were detected using the SYBR green method (TIANGEN) using the ΔΔCt method. The instrument used was a LightCycler 480 II (Roche) fluorescence quantitative system. The reaction conditions were: (1) 50°C, 15 minutes; (2) pre-denaturation at 95°C, 15 minutes; (3) denaturation at 95°C, 10 seconds, annealing and extension at 60°C, 30 seconds. Step (3) was repeated for 40 cycles. The results were normalized with the negative control to obtain the relative mRNA levels and knockdown efficiency. If IC50 was required, it was obtained by four-parameter fitting using Graphpad Prism.

[0279] (2)INHBE

[0280] cDNA synthesis and quantitative PCR were performed using a one-step method. Relative mRNA levels of INHBE and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were measured using the ΔΔCt method. To the supernatant transferred from Example 2.1.1 above, the One-Step mix buffer and One-Step Enzyme mix, along with the forward and reverse primers for the target gene, were added according to the manufacturer's instructions (HiScript II One Step RT-PCR Kit, Vazyme).

[0281] The instrument used was a LightCycler 480 II (Roche). The reaction conditions were: (1) reverse transcription at 50°C for 3 minutes; (2) pre-denaturation at 95°C for 30 seconds; (3) denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 30 seconds. Step (3) was repeated for 40 cycles. The results were normalized with the negative control (NC) to obtain the relative mRNA level and knockdown efficiency. If IC 50 The four-parameter fitting was performed using Graphpad Prism.

[0282] Among them, NC, LPA-Ref1 to Ref2 and their modified sequences, and IN-Ref1 to IN-Ref9 and their modified sequences are shown in Table 4 below. LPA-Ref1mg or LPA-Ref2mg used in subsequent experiments represent the structures of the control compounds LPA-Ref1m and LPA-Ref2m, respectively, connected to the ligand. The specific structure of LPA-Ref1m-g is referenced in WO2019092283A1; the specific structure of LPA-Ref2m-g is referenced in WO2017059223A2; IN-Ref1m-g to IN-Ref9m-g are all IN-Ref1m to IN-Ref9m connected to the ligand L96.

[0283] Table 4: Sequences of NC and Ref1-Ref8

[0284] 2.2 Reporter gene method

[0285] 2.2.1 Transient cell line reporter gene method

[0286] HEK293 cells (ATCC) were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and a double-antibody (Gibco) in a 5% CO2, 37°C atmosphere. Once the cells had grown to nearly completely cover the culture flask, they were trypsinized and resuspended. The resuspended cells were adjusted in density and seeded at 3e4 / well in a 96-well plate. The dsRNA and psiCheck-LPA plasmid transfection complex was then added to the suspension. The dsRNA and psiCheck-2-LPA plasmid transfection complex was prepared by mixing the dsRNA and psiCheck-LPA plasmid mixture in a 1:1 ratio with Opti-MEM (Gibco) containing 0.3 μl / well of Lipofectamine 3000 (Thermo). After a certain period of cell culture, an equal volume of Duo-Lite Luciferase Detection Reagent (Vazyme) was added to the cell culture to detect firefly luciferase luminescence; then, an equal volume of Duo-Lite Stop&Lite Detection Reagent was added to the original cell culture to detect Renilla luciferase luminescence.

[0287] 2.2.2 Stable cell line reporter gene method

[0288] (1)LPA

[0289] HEK293-psiCheck-LPA cells contain a tandem expression system for the LPA gene sequence and the luciferase gene. They were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco), 1 μg / ml puromycin, and a double-stranded antibody (Gibco) at 5% CO₂ and 37°C. Once the cells had grown to nearly completely cover the culture flask, they were trypsinized and resuspended. The resuspended cells were adjusted in density and seeded at 3×4 cells / well in a 96-well plate. A dsRNA transfection complex was then added. The dsRNA transfection complex consisted of a 1:1 mixture of Opti-MEM (Gibco) containing 0.3 μl / well of Lipofectamine RNAiMAX (Thermo) and dsRNA. After incubation for a specified period, the cells were lysed and Luciferase Substrate (Vazyme) was added. Then, Renilla substrate working solution was added, mixed rapidly, and fluorescence activity was measured. LPA assay results are shown in Figures 1A-2D and Tables 5-6.

[0290] Table 5 shows the IC values ​​of LPA-siRNA 50 Assay results, where the decimal numbers in the duplex numbers of the siRNA ID represent different assay batches.

[0291] Table 5

[0292] Table 6 shows the IC of LPA-siRNA-ligand 50 The results of the assay are as follows: the LPA-siRNA ligand selected in this application is the conjugate connection method of formula 5 in Table A, where the decimal point in the duplex number of the siRNA ID represents different assay batches.

[0293] Table 6

[0294] (2)INHBE

[0295] SK-Hep-1-psiCheck-INHBE cells contain a tandem expression system for the full-length INHBE mRNA sequence and the luciferase gene. They were cultured in MEM medium (Gibco) supplemented with 10% FBS (Gibco), 1 μg / ml puromycin, and a double-antibody (Gibco) in a 5% CO2, 37°C atmosphere. Once the cells had grown to nearly completely cover the flask, they were trypsinized and resuspended. The resuspended cells were adjusted to a density of 1.5×10 4Cells were seeded into 96-well plates at 1 μL / well and dsRNA transfection complexes were added. The dsRNA transfection complexes consisted of a 1:1 mixture of Opti-MEM (Gibco) containing 0.3 μL / well Lipofectamine RNAi Max (Thermo) and dsRNA. After incubation for a specified period, cells were lysed and Renilla substrate (Vazyme) was added for fluorescence activity detection.

[0296] The test results are shown in Table 7, which shows the IC of IN-siRNA. 50 Assay results. The decimal places in the duplex numbers of the siRNA IDs represent different assay batches, all of which involved human INHBE.

[0297] Table 7

[0298] Example 3: In vivo efficacy of LPA dsRNA in mice

[0299] In order to evaluate the effect of dsRNA on the in vivo activity of LPA, male LPA humanized mice were used for in vivo activity detection. Pre-dose serum samples were obtained after a 4-hour fast on day -1. The above-mentioned dsRNA conjugate was diluted with normal saline and injected subcutaneously on day 1 according to the experimental design. Blank normal saline was used as a negative control. Plasma was collected after a 4-hour fast on days 1, 3, 7, 14, 21, 28, 35, 42, and 49. The LPA protein level in plasma was detected by ELISA (Abcam) according to the experimental protocol provided by the supplier. The LPA level and blood lipid index in serum were detected using a fully automatic biochemical analyzer and handed over to Shanghai Biyuntian Biotechnology Co., Ltd. for testing.

[0300] LPA protein levels and lipid profiles were normalized for each animal. Normalization was performed by dividing each animal's LPA protein, insulin, blood glucose, and lipid profiles at a given time point by their pre-treatment levels (in this case, day -1) to determine a "normalized to pre-treatment" ratio. Expression at a specific time point was then normalized to the saline group by dividing the "normalized to pre-treatment" ratio for each individual animal by the average "normalized to pre-treatment" ratio for all mice in the saline group.

[0301] Example 4: In vivo efficacy of LPA dsRNA in healthy cynomolgus monkeys

[0302] To evaluate the in vivo activity of dsRNA against LPA, in vivo activity assays were performed using healthy cynomolgus monkeys. Pre-dose serum samples were obtained on days -7 and -14 after a 4-hour fast. The dsRNA conjugate was diluted with saline and subcutaneously injected on day 1 according to the experimental design. Blank saline was used as a negative control. Plasma was collected after a 4-hour fast on days 3, 7, 14, 28, 42, 56, 70, and 84. The LPA protein level in plasma was detected by ELISA (Abcam) according to the experimental procedures provided by the supplier. Lp(a), triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), Apo-A1, and Apo-B in serum were detected using an automatic biochemical analyzer.

[0303] LPA protein levels, Lp(a) levels, triglyceride levels, total cholesterol levels, HDL-c levels, and LDL-c levels were normalized for each animal. For normalization, the LPA protein level for each animal at a time point was divided by the animal's pre-treatment expression level (the average of the pre-experimental samples from that group) to determine a "normalized to pre-treatment" expression ratio. Expression at a specific time point was then normalized to the saline group by dividing the "normalized to pre-treatment" ratio for the individual animal by the average "normalized to pre-treatment" ratio for all mice in the saline group.

[0304] Although the present invention has been described in detail with reference to the embodiments of the present invention, these embodiments are provided to illustrate rather than limit the present invention. Other embodiments that can be obtained according to the principles of the present invention all fall within the scope defined by the claims of the present invention.

Claims

1. A modified dsRNA, characterized in that It comprises a sense strand and an antisense strand, wherein at least one of the nucleotides at positions 2 to 11 in the direction from the 5' end to the 3' end of the antisense strand contains hypoxanthine, and the nucleotide containing the hypoxanthine forms a non-canonical base pair (I:C pairing) with the cytosine at the corresponding position in the sense strand, and the modified dsRNA meets at least one of the following characteristics: (1) In the continuous sequence N1N2I0N3N4 at positions 2 to 11 of the antisense strand from the 5' end to the 3' end, at least three bases among N1, N2, N3 and N4 are adenine and / or uracil; wherein N1, N2, N3 and N4 are each independently a nucleotide containing adenine, cytosine, guanine, thymine or uracil as a base, and I0 is a nucleotide containing hypoxanthine as a base; (2) the number of adenine and uracil in positions 2 to 8 of the antisense strand is 4 or more in the direction from the 5' end to the 3' end; and / or; (3) There is a difference of at least 2°C in dissociation temperature (Tm) between the modified dsRNA and the dsRNA with canonical base pairing (G:C pairing); wherein the G:C pairing dsRNA differs from the modified dsRNA only in that guanine is used in place of hypoxanthine (G:C pairing).

2. The modified dsRNA according to claim 1, characterized in that The number of the non-canonical base pairs is 1, 2 or 3 pairs.

3. The modified dsRNA according to claim 1 or 2, characterized in that The base in at least one of the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th or 11th nucleotides from the 5' end to the 3' end of the antisense strand is hypoxanthine.

4. The modified dsRNA according to any one of claims 1 to 3, characterized in that The base of at least one nucleotide from the 2nd to the 8th position in the antisense strand from the 5' end to the 3' end is hypoxanthine.

5. The modified dsRNA according to any one of claims 1 to 4, characterized in that The base of at least one nucleotide from the 6th to the 8th position in the antisense strand from the 5' end to the 3' end is hypoxanthine, for example, the 6th, 7th and / or 8th position.

6. The modified dsRNA according to any one of claims 1 to 5, characterized in that In the continuous sequence N1N2I0N3N4 at positions 2 to 11 of the antisense strand, there is no nucleotide whose base is guanine.

7. The modified dsRNA according to any one of claims 1 to 6, characterized in that In the continuous sequence N1N2I0N3N4 in positions 2-11 of the antisense strand, the bases of N1, N2, N3 and N4 are each independently adenine and / or uracil, and optionally at least one nucleotide among N1, N2, N3, N4 and I0 also has a modified sugar group and / or a modified internucleotide bond.

8. The modified dsRNA according to any one of claims 1 to 7, characterized in that The length of the sense strand and the antisense strand is independently 17-25 nucleotides; preferably, the length of the sense strand and the antisense strand is independently 19-23 nucleotides; more preferably, the length of the sense strand and the antisense strand is independently 19-21 nucleotides.

9. The modified dsRNA according to claim 1, wherein The other nucleotides in the sense strand and the antisense strand are unmodified nucleotides; alternatively, substantially all the nucleotides in the sense strand and the antisense strand are modified nucleotides; alternatively, all the nucleotides in the sense strand and the antisense strand are modified nucleotides.

10. The modified dsRNA according to claim 1, wherein It further comprises at least one modified nucleotide selected from the group consisting of 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-F-arabino nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides and inverted nucleotides; the inverted nucleotides are preferably selected from the group consisting of inverted A nucleotides, inverted dA nucleotides, inverted T nucleotides, inverted dT nucleotides, inverted C nucleotides and inverted U nucleotides.

11. The modified dsRNA according to claim 1, wherein It further meets at least one of the following characteristics: 1) the antisense strand comprises 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8) 2'-fluoro modified nucleotides; 2) the antisense strand comprises 1-4 (e.g., 1, 2, 3, 4) phosphorothioate internucleotide bonds; 3) the antisense strand comprises 12-19 (e.g., 12, 13, 14, 15, 16, 17, 18, 19) 2'-O-methyl modified nucleotides; 4) the sense strand comprises 2-4 (e.g., 2, 3, 4) 2'-fluoro modified nucleotides; 5) the sense strand comprises 1-4 (e.g., 1, 2, 3, 4) phosphorothioate internucleotide bonds; 6) The sense strand comprises 14-17 (eg, 14, 15, 16, 17) 2'-O-methyl modified nucleotides.

12. The modified dsRNA according to any one of claims 1 to 11, characterized in that The antisense strand comprises at least 15 consecutive nucleotides that differ from any sequence shown in Table 1 or Table 2 by 0, 1, 2 or 3 nucleotides, and the sense strand has at least 15, 16, 17, 18, 19, 20 or 21 nucleotides complementary to the antisense strand; Alternatively, the antisense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ from any of the sequences shown in Table 1 or Table 2 by 0, 1, 2 or 3 nucleotides.

13. The modified dsRNA according to any one of claims 1 to 12, characterized in that The modified dsRNA comprises any one of the antisense strand or sense strand sequences in Table 1 or Table 2.

14. The modified dsRNA according to any one of claims 1 to 13, characterized in that The modified dsRNA comprises an antisense strand sequence and a sense strand sequence shown in the duplex sequence in Table 1 or Table 2.

15. The modified dsRNA according to any one of claims 1 to 14, characterized in that The antisense strand comprises a member selected from the group consisting of SEQ ID NOs: 1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1178, 1179, 1180, 1810, 1820, 1830, 1840, 1850, 1860, 1871 9, 1181, 1183, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261 or 1263 of completely consecutive nucleotides.

16. The modified dsRNA according to any one of claims 1 to 15, characterized in that The sense strand comprises a member selected from the group consisting of SEQ ID NOs: 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149, 1151, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1179, 1180 0, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, or 1264 of completely consecutive nucleotides.

17. The modified dsRNA according to any one of claims 1 to 16, characterized in that The modified dsRNA comprises a sense strand sequence and an antisense strand sequence selected from the group consisting of SEQ ID NOs: 1093 and 1094; SEQ ID NOs: 1095 and 1096; SEQ ID NOs: 1097 and 1098; SEQ ID NOs: 1099 and 1100; SEQ ID NOs: 1101 and 1102; SEQ ID NOs: 1103 and 1104; SEQ ID NOs: 1105 and 1106; SEQ ID NOs: 1107 and 1108; SEQ ID NOs: 1109 and 1110; SEQ ID NOs: 1111 and 1112; SEQ ID NOs: 1113 and 1114; SEQ ID NOs: 1115 and 1116; SEQ ID NOs: 1117 and 1118; SEQ ID NOs: 1119 and 1120; SEQ ID NOs: 1121 and 1122; SEQ ID NOs: 1123 and 1124; SEQ ID NOs: 1125 and 1126; SEQ ID NOs: 1127 and 1128; SEQ ID NOs: 1129 and 1130; SEQ ID NOs: 1131 and 1132; SEQ ID NOs: 1133 and 1134; SEQ ID NOs: 1135 and 1136; SEQ ID NOs: 1137 and 1138; SEQ ID NOs: 1139 and 1140; SEQ ID NOs: 1141 and 1142; SEQ ID NOs: 1143 and 1144; SEQ ID NOs: 1145 and 1146; SEQ ID NOs: 1125 and 1126; SEQ ID NOs: 1127 and 1128; SEQ ID NOs: 1129 and 1130; SEQ ID NOs: 1131 and 1132; SEQ ID NOs: 1133 and 1134; SEQ ID NOs: 1135 and 1136; or, SEQ ID NOs: 1137 and 1138; SEQ ID NOs: 1139 and 1140; SEQ ID NOs: 1141 and 1142; SEQ ID NOs: 1143 and 1144; SEQ ID NOs: 1145 and 1146; SEQ ID NOs: 1147 and 1148; SEQ ID NOs: 1149 and 1150; SEQ ID NOs: 1151 and 1152, SEQ ID NOs: 1153 and 1154, SEQ ID NOs: 1155 and 1156, SEQ ID NOs: 1157 and 1158, SEQ ID NOs: 1159 and 1160; SEQ ID NOs: 1159 and 1160, SEQ ID NOs: 1161 and 1162, SEQ ID NOs: 1163 and 1164, SEQ ID NOs: 1165 and 1166, SEQ ID NOs: 1167 and 1168, SEQ ID NOs: 1169 and 1170, SEQ ID NOs: 1171 and 1172, SEQ ID NOs: 1173 and 1174, SEQ ID NOs: 1174 and 1176, SEQ ID NOs: 1177 and 1178, SEQ ID NOs: 1179 and 1180, SEQ ID NOs: 1181 and 1182, SEQ ID NOs: 1183 and 1184, SEQ ID NOs: 1185 and 1186, SEQ ID NOs: 1187 and 1188, SEQ ID NOs: 1189 and 1190, SEQ ID NOs: 1191 and 1192, SEQ ID NOs: 1193 and 1194, SEQ ID NOs: 1195 and 1196, SEQ ID NOs: 1197 and 1198SEQ ID NO: 1214 and 1215, SEQ ID NO: 1216, SEQ ID NO: 1217 and 1218, SEQ ID NO: 1219 and 1220, SEQ ID NO: 1221 and 1222, SEQ ID NO: 1223 and 1224, SEQ ID NO: 1225 and 1226, SEQ ID NO: 1227 and 1228, SEQ ID NO: 1229 and 1230, SEQ ID NO: 1231 and 1232, SEQ ID NO: 1233 and 1234, SEQ ID NO: 1235 and 1236, SEQ ID NO: 1237 and 1238, SEQ ID NO: 1239 and 1240, SEQ ID NO: 1241 and 1242, SEQ ID NO: 1243 SEQ ID NO: 1249 and 1250, SEQ ID NO: 1251 and 1252, SEQ ID NO: 1253 and 1254, SEQ ID NO: 1255 and 1256, SEQ ID NO: 1257 and 1258, SEQ ID NO: 1259 and 1260, SEQ ID NO: 1261 and 1262, SEQ ID NO: 1263 and 1264, SEQ ID NO: 1265 and 1266, SEQ ID NO: 1267 and 1268, SEQ ID NO: 1269 and 1270, SEQ ID NO: 1271 and 1272, SEQ ID NO: 1273 and 1274, SEQ ID NO: 1275 and 1276, SEQ ID NO: 1277 and 1278, NO: 1257 and 1258, SEQ ID NO: 1259 and 1260, SEQ ID NO: 1261 and 1262, SEQ ID NO: 1263 and 1264.

18. The modified dsRNA according to any one of claims 1 to 17, characterized in that The modified dsRNA comprises a duplex selected from LPA-318, LPA-319, LPA-324, LPA-370, LPA-558, LPA-560, LPA-407, LPA-449, LPA-564, LPA-566, LPA-487, LPA-518, LPA-562, LPA-568, LPA-816, LPA-817, LPA-818, LPA-819, LPA-820, LPA-821, LPA-822, LPA-823, LPA-824, LPA-825, LPA-826, LPA-827, LPA-828, LPA-829, LPA-830, LPA-831, LPA-832, LPA-833, LPA-834, LPA-835, LPA-836, LPA-837, LPA-838, LPA-880, LPA-867, LPA-868, LPA-869, LPA-870, LPA-871, LPA-873, LPA-874, LPA-875, LPA-876, LPA-877, LPA-878, LPA-879, LPA-872, LPA-864, LPA-865, LPA-866, IN-267, IN-268, IN-301, IN-360, IN-361, IN-389, IN-417, IN-418, IN-439, IN-440, IN-441, IN-442, IN-443, IN-464, IN-465, IN-488, IN-489, IN-503, IN-504, IN-518, IN-519, IN-520, IN-521, IN-522, IN-523, IN-524, IN-525, IN-526, IN-527, IN-528, IN-538 or IN-539.

19. The modified dsRNA according to any one of claims 1 to 18, characterized in that The antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, and the target sequence is a sequence selected from the following targets: PCSK9, ANGPTL3, LPA, INHBE, ACVR1C, PLIN1, PDE3B, INHBC, GDF-8 (MSTN), SOD1, APP, C3, C5, HTT, DMPK, HSD17B13, PNPLA3, XDH, AGT, AKT, Kras, SHP2, TGF-β, IFN-a, IL-13, IL-6, Myc, IL-4, IL-17, TERT, KHK, Factor VII, Factor X, Factor XI, Thrombin, TPX2, apoB, SAA, TTR, RSV, PDGFβ, Erb-B, Src, CRK, GRB2, MEKK, JNK, RAF, Erk1 / 2, MYB, JUN, FOS, BCL- 2. Hepciden, PC, CCND, VEGF, EGFR, CCNA, CCNE, WNT-1, β-catenin, c-MET, PKC, NFKB, STAT3, survivin, TOP1 and TOP2A.

20. A double-stranded RNAi agent comprising the modified dsRNA of any one of claims 1-19, and optionally comprising a targeting ligand.

21. The double-stranded RNAi agent according to claim 20, characterized in that The double-stranded RNAi agent comprises the dsRNA of any one of claims 1 to 19, and at least one targeting ligand, wherein the 3' end of the sense strand of the dsRNA is conjugated to the targeting ligand.

22. The double-stranded RNAi agent according to claim 20 or 21, characterized in that The targeting ligand comprises N-acetyl-galactosamine (GalNAc), or the targeting ligand is a GalNAc derivative.

23. The double-stranded RNAi agent according to claim 20, characterized in that The structure of the double-stranded RNAi agent is selected from Table A, wherein R2 is the modified dsRNA of any one of claims 1 to 19.

24. A pharmaceutical composition, characterized in that It comprises the double-stranded RNAi agent or a pharmaceutically acceptable salt thereof according to any one of claims 20 to 23, and a pharmaceutically acceptable carrier.

25. The pharmaceutical composition according to claim 24, characterized in that The pharmaceutical composition is formulated for administration by injection or infusion.

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

27. The pharmaceutical composition according to any one of claims 24 to 26, characterized in that The carrier is a non-buffered solution or a buffered solution.

28. A method for inhibiting the expression of a target gene in a cell, characterized in that: The cell is contacted with the double-stranded RNAi agent according to any one of claims 20 to 23, so that the mRNA transcript of the target gene is degraded, thereby inhibiting the expression of the target gene in the cell.

29. The method of claim 28, wherein: The target gene is selected from any one of the following groups: PCSK9, ANGPTL3, LPA, INHBE, ACVR1C, PLIN1, PDE3B, INHBC, GDF-8 (MSTN), SOD1, APP, C3, C5, HTT, DMPK, HSD17B13, PNPLA3, XDH, AGT, AKT, Kras, SHP2, TGF-β, IFN-a, IL-13, IL-6, Myc, IL-4, IL-17, TERT, KHK, Factor VII, Factor X, Factor XI, Thrombin, TPX2, apoB, SAA, TTR, RSV, PDGFβ, Erb-B, Src, CRK, GRB2, MEKK, JNK, RAF, Erk1 / 2, MYB, JUN, FOS, BCL- 2. Hepciden, PC, CCND, VEGF, EGFR, CCNA, CCNE, WNT-1, β-catenin, c-MET, PKC, NFKB, STAT3, survivin, TOP1 and TOP2A.

30. The method of claim 28, wherein: The cell is a hepatocyte or an adipocyte.

31. The method of claim 28, wherein: Target gene expression is inhibited by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

32. Use of a double-stranded RNAi agent as claimed in any one of claims 20 to 23 for the preparation of a medicament for treating a subject having a disorder mediated by target gene expression.

33. The use according to claim 32, characterized in that The subject suffers from an LPA-related disease or disorder or suffers from an INHBE-related disease or disorder.

34. The use according to claim 32, characterized in that The LPA-associated disease or condition is one or more of Bergey's disease, metabolic syndrome, aortic regurgitation, aortic dissection, retinal artery occlusion, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, heterozygous or homozygous familial hypercholesterolemia, hyperlipobetalipoproteinemia, cerebrovascular atherosclerosis, venous thrombosis, congestive heart failure, ischemic heart disease, carotid artery disease, myocardial infarction, stroke, atrial fibrillation, heart failure, hyperlipidemia, type 2 diabetes, and non-alcoholic fatty liver disease; The INHBE-related disease or condition is one or more of metabolic disorders, metabolic syndrome, type 2 diabetes, obesity, prediabetes, hypertriglyceridemia, dyslipidemia, liver inflammation, fatty liver, hypercholesterolemia, diseases associated with elevated liver enzymes, non-alcoholic steatohepatitis, cardiovascular disease, kidney disease, abdominal obesity, insulin resistance, hypertension, dyslipidemia, cardiometabolic disorders, and cancer associated with INHBE expression.

Images