Compositions and methods for inhibiting synuclein alpha (SNCA) gene expression

Abstract

Compositions and methods useful for reducing synuclein alpha (SNCA) gene expression and for treating SNCA-related diseases and disorders are provided. SNCA dsRNA agents, SNCA antisense polynucleotide agents, compositions comprising SNCA dsRNA agents, and compositions comprising SNCA antisense polynucleotide agents useful for reducing SNCA expression in cells and subjects are provided.

Description

Technical Field

[0001] This invention relates in part to compositions and methods for inhibiting the expression of the synuclein alpha (SNCA) gene. Background Technology

[0002] Expression of the SNCA gene produces the protein α-synuclein. Mutations in the SNCA gene and SNCA gene duplication are associated with familial Parkinson's disease (PD), the second most prevalent neurodegenerative disease in the Western world. Clinically, the disease manifests as tremor, bradykinesia, and muscle rigidity, as well as impaired posture and gait. These symptoms are associated with the loss of dopaminergic neurons in the substantia nigra of the brain, leading to striatal dopamine deficiency. Neuronal cell death in most PD patients is caused by the inclusion and accumulation of aggregates of the protein α-synuclein. α-synuclein aggregates are frequently observed in the brains of PD patients. Similar aggregates have also been observed in patients diagnosed with sporadic PD, Alzheimer's disease, multiple system atrophy, and Lewy body dementia. Currently available treatments cannot prevent or cure Parkinson's disease.

[0003] Therefore, there is an urgent need to develop new treatments for all synucleinosis, and siRNA therapy that silences SNCA represents a new approach for treating SNCA-related diseases. Summary of the Invention

[0004] Generally, this disclosure provides novel SNCA gene-specific RNAi agents, compositions comprising SNCA RNAi agents, and methods for inhibiting SNCA gene expression in vitro and / or in vivo using the SNCA RNAi agents and compositions comprising SNCA RNAi agents described herein. The SNCA RNAi agents described herein can selectively and effectively reduce, inhibit, or silence SNCA gene expression in subjects (e.g., human or animal subjects).

[0005] According to one aspect of the invention, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of synuclein α (SNCA) is provided, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides differing from the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 by no more than 1, 2 or 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides differing from the nucleotide sequence of SEQ ID NO: 2, 4, 6 or 8 by no more than 1, 2 or 3 nucleotides, wherein the sense strand and the antisense strand may be partially, substantially or completely complementary to each other.

[0006] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region partially complementary to the mRNA encoding SNCA, comprising at least 15, 16, 17, 18, or 19 consecutive nucleotides having 0, 1, 2, 3, 4, or 5 mismatches. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region complementary to a target region of the SNCA mRNA transcript, comprising at least 15, 16, 17, 18, or 19 consecutive nucleotides having 0, 1, 2, 3, 4, or 5 mismatches.

[0007] In some implementations, the SNCA mRNA transcript is SEQ ID NO: 1.

[0008] In some implementations, the target region of the SNCA mRNA transcript is SEQ ID NO: 1. Any of the following nucleotide sequences: 776-796, 699-719, 628-648, 614-634, 913-933, 828-848, 238-258, 325-345, 326-346, 337-357, 397-417, 587-607, 591-611, 201-221, 202-222, 203-223, 204-224, 206-226, 207-227, 209-229, 214-234, 215-235, 216-236, 217-237, 220-240, 223-243, 224-244, 225-245. 226-246, 227-247, 228-248, 230-250, 232-252, 235-255, 236-256, 238-258, 239-259, 241-261, 268-288, 275-295, 277-297, 278-298, 306-326, 307-327, 308-328, 322-342, 324-344, 326-346, 327-347, 328-348, 338-358, 378-398, 386-406, 392-412, 394-414, 396-416, 398-418, 399-419, 400-420, 401-421, 402-422, 403-423, 405-425, 406-426, 409-429, 506-526, 513-533, 580-600, 583-603, 584-604, 585-605, 587-607, 594-614, 595-615, 598-618, 616-636, 617-637, 620-640, 623-643, 635-655, 638-658, 640-660, 642-662, 644-664, 654-674, 657-677, 664-684, 667-687, 697-717, 700-720, 704-724, 705-725, 719-739, 722-742, 726-746, 728-748, 729-749, 731-751, 735-755, 737-757, 771-791, 773-793, 775-795, 778-798, 781-801, 782-802, 783-803, 822-842, 825-845, 826-846, 831-851, 832-852, 835-855, 837-857, 839-859, 856-876, 864-884, 867-887,869-889、872-892、873-893、875-895、876-896、906-926、911-931、912-932、915-935、919-939、920-940、921-941、923-943、925-945、926-946、929-949、938-958、943-963、945-965、948-968、949-969、957-977、962-982、963-983、967-987、968-988、970-990、971-991、976-996、979-999、980-1000、1003-1023、1005-1025、1006-1026、1010-1030、1011-1031、1012-1032、1014-1034、1016-1036、1017-1037、1023-1043、1026-1046、1028-1048、1049-1069、1066-1086、1081-1101、1083-1103、1085-1105、1086-1106、1088-1108、1089-1109、1090-1110、1091-1111、1092-1112、1097-1117、1099-1119、1105-1125、1106-1126、1107-1127、1166-1186、1168-1188、778-796、701-719、630-648、616-634、915-933、830-848、240-258、327-345、328-346、339-357、399-417、589-607、593-611、203-221、204-222、205-223、206-224、208-226、209-227、211-229、216-234、217-235、218-236、219-237、222-240、225-243、226-244、227-245、228-246、229-247、230-248、232-250、234-252、237-255、238-256、240-258、241-259、243-261、270-288、277-295、279-297、280-298、308-326、309-327、310-328、324-342、326-344、328-346、329-347、330-348、340-358、380-398、388-406、394-412、396-414、398-416、400-418、401-419、402-420、403-421、404-422、405-423、407-425、408-426、411-429、508-526、515-533、582-600、585-603、586-604、587-605、589-607、596-614、597-615、600-618、618-636、619-637、622-640、625-643、637-655、640-658、642-660、644-662、646-664、656-674、659-677、666-684、669-687、699-717、702-720、706-724、707-725、721-739、724-742、728-746、730-748、731-749、733-751、737-755、739-757、773-791、775-793、777-795、778-796、780-798、783-801、784-802、785-803、824-842、827-845、828-846、833-851、834-852、837-855、839-857、841-859、858-876、866-884、869-887、871-889、874-892、875-893、877-895、878-896、908-926、913-931、914-932、917-935、921-939、922-940、923-941、925-943、927-945、928-946、931-949、940-958、945-963、947-965、950-968、951-969、959-977、964-982、965-983、969-987、970-988、972-990、973-991、978-996、981-999、982-1000、1005-1023、1007-1025、1008-1026、1012-1030、1013-1031、1014-1032、1016-1034、1018-1036、1019-1037、1025-1043、1028-1046、1030-1048、1051-1069、1068-1086、1083-1101、1085-1103、1087-1105、1088-1106、1090-1108、1091-1109、1092-1110、1093-1111、1094-1112、1099-1117、1101-1119、1107-1125、1108-1126、1109-1127、1168-1186、1170-1188、777-795、700-718、629-647、615-633、914-932、829-847、239-257、326-344、327-345、338-356、398-416、588-606、592-610、202-220、203-221、204-222、205-223、207-225、208-226、210-228、215-233、216-234、217-235、218-236、221-239、224-242、225-243、226-244、227-245、228-246、229-247、231-249、233-251、236-254、237-255、239-257、240-258、242-260、269-287、276-294、278-296、279-297、307-325、308-326、309-327、323-341、325-343、327-345、328-346、329-347、339-357、379-397、387-405、393-411、395-413、397-415、399-417、400-418、401-419、402-420、403-421、404-422、406-424、407-425、410-428、507-525、514-532、581-599、584-602、585-603、586-604、588-606、595-613、596-614、599-617、617-635、618-636、621-639、624-642、636-654、639-657、641-659、643-661、645-663、655-673、658-676、665-683、668-686、698-716、701-719、705-723、706-724、720-738、723-741、727-745、729-747、730-748、732-750、736-754、738-756、772-790、774-792、776-794、777-795、779-797、782-800、783-801、784-802、823-841、826-844、827-845、832-850、833-851、836-854、838-856、840-858、857-875、865-883、868-886、870-888、873-891、874-892、876-894、877-895、907-925、912-930、913-931、916-934、920-938、921-939、922-940、924-942、926-944、927-945、930-948、939-957、944-962、946-964、949-967、950-968、958-976、963-981、964-982、968-986、969-987、971-989、972-990、977-995、980-998、981-999、1004-1022、1006-1024、1007-1025、1011-1029、1012-1030、1013-1031、1015-1033、1017-1035、1018-1036、1024-1042、1027-1045、1029-1047、1050-1068、1067-1085、1082-1100、1084-1102、1086-1104、1087-1105、1089-1107、1090-1108、1091-1109、1092-1110、1093-1111、1098-1116、1100-1118、1106-1124、1107-1125、1108-1126、1167-1185、1169-1187、777-793、700-716、629-645、615-631、914-930、829-845、239-255、326-342、327-343、338-354、398-414、588-604、592-608、202-218、203-219、204-220、205-221、207-223、208-224、210-226、215-231、216-232、217-233、218-234、221-237、224-240、225-241、226-242、227-243、228-244、229-245、231-247、233-249、236-252、237-253、239-255、240-256、242-258、269-285、276-292、278-294、279-295、307-323、308-324、309-325、323-339、325-341、327-343、328-344、329-345、339-355、379-395、387-403、393-409、395-411、397-413、399-415、400-416、401-417、402-418、403-419、404-420、406-422、407-423、410-426、507-523、514-530、581-597、584-600、585-601、586-602、588-604、595-611、596-612、599-615、617-633、618-634、621-637、624-640、636-652、639-655、641-657、643-659、645-661、655-671、658-674、665-681、668-684、698-714、701-717、705-721、706-722、720-736、723-739、727-743、729-745、730-746、732-748、736-752、738-754、772-788、774-790、776-792、777-793、779-795、782-798、783-799、784-800、823-839、826-842、827-843、832-848、833-849、836-852、838-854、840-856、857-873、865-881、868-884、870-886、873-889、874-890、876-892、877-893、907-923、912-928、913-929、916-932、920-936、921-937、922-938、924-940、926-942、927-943、930-946、939-955、944-960、946-962、949-965、950-966、958-974、963-979、964-980、968-984、969-985、971-987、972-988、977-993、980-996、981-997、1004-1020、1006-1022、1007-1023、1011-1027、1012-1028、1013-1029、1015-1031、1017-1033、1018-1034、1024-1040、1027-1043、1029-1045、1050-1066、1067-1083、1082-1098、1084-1100、1086-1102、1087-1103、1089-1105、1090-1106、1091-1107、1092-1108、1093-1109、1098-1114, 1100-1116, 1106-1122, 1107-1123, 1108-1124, 1167-1183, or 1169-1185.

[0009] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a region similar to SEQ ID NO: 1. The nucleotide sequence of any of the following nucleotides differs from the nucleotide sequence of any one of them by 0, 1, 2 or 3 nucleotides by at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides: 776-796, 699-719, 628-648, 614-634, 913-933, 828-848, 238-258, 325-345, 326-346, 337-357, 397-417, 587-607, 591-611, 201-221, 202-222, 203-223, 204-224, 206-226, 207-227, 209-229, 214-234, 215-235, 2 16-236, 217-237, 220-240, 223-243, 224-244, 225-245, 226-246, 227-247, 228-248, 230-250, 232-252, 235-255, 236-256, 238-258, 239-259, 241-261, 268-288, 275-295, 277-297, 278-298, 306-326, 307-327, 308-328, 322-342, 324-344, 326-346, 327-347, 328-348, 338-358, 378-39 8, 386-406, 392-412, 394-414, 396-416, 398-418, 399-419, 400-420, 401-421, 402-422, 403-423, 405-425, 406-426, 409-429, 506-526, 513-533, 580-600, 583-603, 584-604, 585-605, 587-607, 594-614, 595-615, 598-618, 616-636, 617-637, 620-640, 623-643, 635-655, 638-658, 640 -660, 642-662, 644-664, 654-674, 657-677, 664-684, 667-687, 697-717, 700-720, 704-724, 705-725, 719-739, 722-742, 726-746, 728-748, 729-749, 731-751, 735-755, 737-757, 771-791, 773-793, 775-795, 776-796, 778-798, 781-801, 782-802, 783-803, 822-842, 825-845, 826-846,831-851、832-852、835-855、837-857、839-859、856-876、864-884、867-887、869-889、872-892、873-893、875-895、876-896、906-926、911-931、912-932、915-935、919-939、920-940、921-941、923-943、925-945、926-946、929-949、938-958、943-963、945-965、948-968、949-969、957-977、962-982、963-983、967-987、968-988、970-990、971-991、976-996、979-999、980-1000、1003-1023、1005-1025、1006-1026、1010-1030、1011-1031、1012-1032、1014-1034、1016-1036、1017-1037、1023-1043、1026-1046、1028-1048、1049-1069、1066-1086、1081-1101、1083-1103、1085-1105、1086-1106、1088-1108、1089-1109、1090-1110、1091-1111、1092-1112、1097-1117、1099-1119、1105-1125、1106-1126、1107-1127、1166-1186、1168-1188、778-796、701-719、630-648、616-634、915-933、830-848、240-258、327-345、328-346、339-357、399-417、589-607、593-611、203-221、204-222、205-223、206-224、208-226、209-227、211-229、216-234、217-235、218-236、219-237、222-240、225-243、226-244、227-245、228-246、229-247、230-248、232-250、234-252、237-255、238-256、240-258、241-259、243-261、270-288、277-295、279-297、280-298、308-326、309-327、310-328、324-342、326-344、328-346、329-347、330-348、340-358、380-398、388-406、394-412、396-414、398-416、400-418、401-419、402-420、403-421、404-422、405-423、407-425、408-426、411-429、508-526、515-533、582-600、585-603、586-604、587-605、589-607、596-614、597-615、600-618、618-636、619-637、622-640、625-643、637-655、640-658、642-660、644-662、646-664、656-674、659-677、666-684、669-687、699-717、702-720、706-724、707-725、721-739、724-742、728-746、730-748、731-749、733-751、737-755、739-757、773-791、775-793、777-795、778-796、780-798、783-801、784-802、785-803、824-842、827-845、828-846、833-851、834-852、837-855、839-857、841-859、858-876、866-884、869-887、871-889、874-892、875-893、877-895、878-896、908-926、913-931、914-932、917-935、921-939、922-940、923-941、925-943、927-945、928-946、931-949、940-958、945-963、947-965、950-968、951-969、959-977、964-982、965-983、969-987、970-988、972-990、973-991、978-996、981-999、982-1000、1005-1023、1007-1025、1008-1026、1012-1030、1013-1031、1014-1032、1016-1034、1018-1036、1019-1037、1025-1043、1028-1046、1030-1048、1051-1069、1068-1086、1083-1101、1085-1103、1087-1105、1088-1106、1090-1108、1091-1109、1092-1110、1093-1111、1094-1112、1099-1117、1101-1119、1107-1125、1108-1126、1109-1127、1168-1186、1170-1188、777-795、700-718、629-647、615-633、914-932、829-847、239-257、326-344、327-345、338-356、398-416、588-606、592-610、202-220、203-221、204-222、205-223、207-225、208-226、210-228、215-233、216-234、217-235、218-236、221-239、224-242、225-243、226-244、227-245、228-246、229-247、231-249、233-251、236-254、237-255、239-257、240-258、242-260、269-287、276-294、278-296、279-297、307-325、308-326、309-327、323-341、325-343、327-345、328-346、329-347、339-357、379-397、387-405、393-411、395-413、397-415、399-417、400-418、401-419、402-420、403-421、404-422、406-424、407-425、410-428、507-525、514-532、581-599、584-602、585-603、586-604、588-606、595-613、596-614、599-617、617-635、618-636、621-639、624-642、636-654、639-657、641-659、643-661、645-663、655-673、658-676、665-683、668-686、698-716、701-719、705-723、706-724、720-738、723-741、727-745、729-747、730-748、732-750、736-754、738-756、772-790、774-792、776-794、777-795、779-797、782-800、783-801、784-802、823-841、826-844、827-845、832-850、833-851、836-854、838-856、840-858、857-875、865-883、868-886、870-888、873-891、874-892、876-894、877-895、907-925、912-930、913-931、916-934、920-938、921-939、922-940、924-942、926-944、927-945、930-948、939-957、944-962、946-964、949-967、950-968、958-976、963-981、964-982、968-986、969-987、971-989、972-990、977-995、980-998、981-999、1004-1022、1006-1024、1007-1025、1011-1029、1012-1030、1013-1031、1015-1033、1017-1035、1018-1036、1024-1042、1027-1045、1029-1047、1050-1068、1067-1085、1082-1100、1084-1102、1086-1104、1087-1105、1089-1107、1090-1108、1091-1109、1092-1110、1093-1111、1098-1116、1100-1118、1106-1124、1107-1125、1108-1126、1167-1185、1169-1187、777-793、700-716、629-645、615-631、914-930、829-845、239-255、326-342、327-343、338-354、398-414、588-604、592-608、202-218、203-219、204-220、205-221、207-223、208-224、210-226、215-231、216-232、217-233、218-234、221-237、224-240、225-241、226-242、227-243、228-244、229-245、231-247、233-249、236-252、237-253、239-255、240-256、242-258、269-285、276-292、278-294、279-295、307-323、308-324、309-325、323-339、325-341、327-343、328-344、329-345、339-355、379-395、387-403、393-409、395-411、397-413、399-415、400-416、401-417、402-418、403-419、404-420、406-422、407-423、410-426、507-523、514-530、581-597、584-600、585-601、586-602、588-604、595-611、596-612、599-615、617-633、618-634、621-637、624-640、636-652、639-655、641-657、643-659、645-661、655-671、658-674、665-681、668-684、698-714、701-717、705-721、706-722、720-736、723-739、727-743、729-745、730-746、732-748、736-752、738-754、772-788、774-790、776-792、777-793、779-795、782-798、783-799、784-800、823-839、826-842、827-843、832-848、833-849、836-852、838-854、840-856、857-873、865-881、868-884、870-886、873-889、874-890、876-892、877-893、907-923、912-928、913-929、916-932、920-936、921-937、922-938、924-940、926-942、927-943、930-946、939-955、944-960、946-962、949-965、950-966、958-974、963-979、964-980、968-984、969-985、971-987、972-988、977-993、980-996、981-997、1004-1020、1006-1022、1007-1023、1011-1027、1012-1028、1013-1029、1015-1031、1017-1033、1018-1034、1024-1040、1027-1043、1029-1045、1050-1066、1067-1083、1082-1098、1084-1100、1086-1102、The sequence numbers are 1087-1103, 1089-1105, 1090-1106, 1091-1107, 1092-1108, 1093-1109, 1098-1114, 1100-1116, 1106-1122, 1107-1123, 1108-1124, 1167-1183, or 1169-1185, and the antisense strand contains at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides differing from the corresponding nucleotide sequence of SEQ ID NO: 2 by 0, 1, 2, or 3 nucleotides.

[0010] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a region similar to SEQ ID NO: 1. The nucleotide sequence of any of the following nucleotides differs from the nucleotide sequence of any one of them by 0, 1, 2 or 3 nucleotides by at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides: 776-796, 699-719, 628-648, 614-634, 913-933, 828-848, 238-258, 325-345, 326-346, 337-357, 397-417, 587-607, 591-611, 201-221, 202-222, 203-223, 204-224, 206-226, 207-227, 209-229, 214-234, 215-235, 2 16-236, 217-237, 220-240, 223-243, 224-244, 225-245, 226-246, 227-247, 228-248, 230-250, 232-252, 235-255, 236-256, 238-258, 239-259, 241-261, 268-288, 275-295, 277-297, 278-298, 306-326, 307-327, 308-328, 322-342, 324-344, 326-346, 327-347, 328-348, 338-358, 378-39 8, 386-406, 392-412, 394-414, 396-416, 398-418, 399-419, 400-420, 401-421, 402-422, 403-423, 405-425, 406-426, 409-429, 506-526, 513-533, 580-600, 583-603, 584-604, 585-605, 587-607, 594-614, 595-615, 598-618, 616-636, 617-637, 620-640, 623-643, 635-655, 638-658, 640 -660, 642-662, 644-664, 654-674, 657-677, 664-684, 667-687, 697-717, 700-720, 704-724, 705-725, 719-739, 722-742, 726-746, 728-748, 729-749, 731-751, 735-755, 737-757, 771-791, 773-793, 775-795, 776-796, 778-798, 781-801, 782-802, 783-803, 822-842, 825-845, 826-846,831-851、832-852、835-855、837-857、839-859、856-876、864-884、867-887、869-889、872-892、873-893、875-895、876-896、906-926、911-931、912-932、915-935、919-939、920-940、921-941、923-943、925-945、926-946、929-949、938-958、943-963、945-965、948-968、949-969、957-977、962-982、963-983、967-987、968-988、970-990、971-991、976-996、979-999、980-1000、1003-1023、1005-1025、1006-1026、1010-1030、1011-1031、1012-1032、1014-1034、1016-1036、1017-1037、1023-1043、1026-1046、1028-1048、1049-1069、1066-1086、1081-1101、1083-1103、1085-1105、1086-1106、1088-1108、1089-1109、1090-1110、1091-1111、1092-1112、1097-1117、1099-1119、1105-1125、1106-1126、1107-1127、1166-1186、1168-1188、778-796、701-719、630-648、616-634、915-933、830-848、240-258、327-345、328-346、339-357、399-417、589-607、593-611、203-221、204-222、205-223、206-224、208-226、209-227、211-229、216-234、217-235、218-236、219-237、222-240、225-243、226-244、227-245、228-246、229-247、230-248、232-250、234-252、237-255、238-256、240-258、241-259、243-261、270-288、277-295、279-297、280-298、308-326、309-327、310-328、324-342、326-344、328-346、329-347、330-348、340-358、380-398、388-406、394-412、396-414、398-416、400-418、401-419、402-420、403-421、404-422、405-423、407-425、408-426、411-429、508-526、515-533、582-600、585-603、586-604、587-605、589-607、596-614、597-615、600-618、618-636、619-637、622-640、625-643、637-655、640-658、642-660、644-662、646-664、656-674、659-677、666-684、669-687、699-717、702-720、706-724、707-725、721-739、724-742、728-746、730-748、731-749、733-751、737-755、739-757、773-791、775-793、777-795、778-796、780-798、783-801、784-802、785-803、824-842、827-845、828-846、833-851、834-852、837-855、839-857、841-859、858-876、866-884、869-887、871-889、874-892、875-893、877-895、878-896、908-926、913-931、914-932、917-935、921-939、922-940、923-941、925-943、927-945、928-946、931-949、940-958、945-963、947-965、950-968、951-969、959-977、964-982、965-983、969-987、970-988、972-990、973-991、978-996、981-999、982-1000、1005-1023、1007-1025、1008-1026、1012-1030、1013-1031、1014-1032、1016-1034、1018-1036、1019-1037、1025-1043、1028-1046、1030-1048、1051-1069、1068-1086、1083-1101、1085-1103、1087-1105、1088-1106、1090-1108、1091-1109、1092-1110、1093-1111、1094-1112、1099-1117、1101-1119、1107-1125、1108-1126、1109-1127、1168-1186、1170-1188、777-795、700-718、629-647、615-633、914-932、829-847、239-257、326-344、327-345、338-356、398-416、588-606、592-610、202-220、203-221、204-222、205-223、207-225、208-226、210-228、215-233、216-234、217-235、218-236、221-239、224-242、225-243、226-244、227-245、228-246、229-247、231-249、233-251、236-254、237-255、239-257、240-258、242-260、269-287、276-294、278-296、279-297、307-325、308-326、309-327、323-341、325-343、327-345、328-346、329-347、339-357、379-397、387-405、393-411、395-413、397-415、399-417、400-418、401-419、402-420、403-421、404-422、406-424、407-425、410-428、507-525、514-532、581-599、584-602、585-603、586-604、588-606、595-613、596-614、599-617、617-635、618-636、621-639、624-642、636-654、639-657、641-659、643-661、645-663、655-673、658-676、665-683、668-686、698-716、701-719、705-723、706-724、720-738、723-741、727-745、729-747、730-748、732-750、736-754、738-756、772-790、774-792、776-794、777-795、779-797、782-800、783-801、784-802、823-841、826-844、827-845、832-850、833-851、836-854、838-856、840-858、857-875、865-883、868-886、870-888、873-891、874-892、876-894、877-895、907-925、912-930、913-931、916-934、920-938、921-939、922-940、924-942、926-944、927-945、930-948、939-957、944-962、946-964、949-967、950-968、958-976、963-981、964-982、968-986、969-987、971-989、972-990、977-995、980-998、981-999、1004-1022、1006-1024、1007-1025、1011-1029、1012-1030、1013-1031、1015-1033、1017-1035、1018-1036、1024-1042、1027-1045、1029-1047、1050-1068、1067-1085、1082-1100、1084-1102、1086-1104、1087-1105、1089-1107、1090-1108、1091-1109、1092-1110、1093-1111、1098-1116、1100-1118、1106-1124、1107-1125、1108-1126、1167-1185、1169-1187、777-793、700-716、629-645、615-631、914-930、829-845、239-255、326-342、327-343、338-354、398-414、588-604、592-608、202-218、203-219、204-220、205-221、207-223、208-224、210-226、215-231、216-232、217-233、218-234、221-237、224-240、225-241、226-242、227-243、228-244、229-245、231-247、233-249、236-252、237-253、239-255、240-256、242-258、269-285、276-292、278-294、279-295、307-323、308-324、309-325、323-339、325-341、327-343、328-344、329-345、339-355、379-395、387-403、393-409、395-411、397-413、399-415、400-416、401-417、402-418、403-419、404-420、406-422、407-423、410-426、507-523、514-530、581-597、584-600、585-601、586-602、588-604、595-611、596-612、599-615、617-633、618-634、621-637、624-640、636-652、639-655、641-657、643-659、645-661、655-671、658-674、665-681、668-684、698-714、701-717、705-721、706-722、720-736、723-739、727-743、729-745、730-746、732-748、736-752、738-754、772-788、774-790、776-792、777-793、779-795、782-798、783-799、784-800、823-839、826-842、827-843、832-848、833-849、836-852、838-854、840-856、857-873、865-881、868-884、870-886、873-889、874-890、876-892、877-893、907-923、912-928、913-929、916-932、920-936、921-937、922-938、924-940、926-942、927-943、930-946、939-955、944-960、946-962、949-965、950-966、958-974、963-979、964-980、968-984、969-985、971-987、972-988、977-993、980-996、981-997、1004-1020、1006-1022、1007-1023、1011-1027、1012-1028、1013-1029、1015-1031、1017-1033、1018-1034、1024-1040、1027-1043、1029-1045、1050-1066、1067-1083、1082-1098、1084-1100、1086-1102、The sequence numbers are 1087-1103, 1089-1105, 1090-1106, 1091-1107, 1092-1108, 1093-1109, 1098-1114, 1100-1116, 1106-1122, 1107-1123, 1108-1124, 1167-1183, or 1169-1185, and the antisense strand contains at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides differing from the corresponding nucleotide sequence of SEQ ID NO: 2 by 0, 1, 2, or 3 nucleotides.

[0011] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a region similar to SEQ ID NO: 1. The nucleotide sequence of any of the following nucleotides differs from the nucleotide sequence of any one of them by 0, 1, 2 or 3 nucleotides by at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides: 776-796, 699-719, 628-648, 614-634, 913-933, 828-848, 238-258, 325-345, 326-346, 337-357, 397-417, 587-607, 591-611, 201-221, 202-222, 203-223, 204-224, 206-226, 207-227, 209-229, 214-234, 215-235, 2 16-236, 217-237, 220-240, 223-243, 224-244, 225-245, 226-246, 227-247, 228-248, 230-250, 232-252, 235-255, 236-256, 238-258, 239-259, 241-261, 268-288, 275-295, 277-297, 278-298, 306-326, 307-327, 308-328, 322-342, 324-344, 326-346, 327-347, 328-348, 338-358, 378-39 8, 386-406, 392-412, 394-414, 396-416, 398-418, 399-419, 400-420, 401-421, 402-422, 403-423, 405-425, 406-426, 409-429, 506-526, 513-533, 580-600, 583-603, 584-604, 585-605, 587-607, 594-614, 595-615, 598-618, 616-636, 617-637, 620-640, 623-643, 635-655, 638-658, 640 -660, 642-662, 644-664, 654-674, 657-677, 664-684, 667-687, 697-717, 700-720, 704-724, 705-725, 719-739, 722-742, 726-746, 728-748, 729-749, 731-751, 735-755, 737-757, 771-791, 773-793, 775-795, 776-796, 778-798, 781-801, 782-802, 783-803, 822-842, 825-845, 826-846,831-851、832-852、835-855、837-857、839-859、856-876、864-884、867-887、869-889、872-892、873-893、875-895、876-896、906-926、911-931、912-932、915-935、919-939、920-940、921-941、923-943、925-945、926-946、929-949、938-958、943-963、945-965、948-968、949-969、957-977、962-982、963-983、967-987、968-988、970-990、971-991、976-996、979-999、980-1000、1003-1023、1005-1025、1006-1026、1010-1030、1011-1031、1012-1032、1014-1034、1016-1036、1017-1037、1023-1043、1026-1046、1028-1048、1049-1069、1066-1086、1081-1101、1083-1103、1085-1105、1086-1106、1088-1108、1089-1109、1090-1110、1091-1111、1092-1112、1097-1117、1099-1119、1105-1125、1106-1126、1107-1127、1166-1186、1168-1188、778-796、701-719、630-648、616-634、915-933、830-848、240-258、327-345、328-346、339-357、399-417、589-607、593-611、203-221、204-222、205-223、206-224、208-226、209-227、211-229、216-234、217-235、218-236、219-237、222-240、225-243、226-244、227-245、228-246、229-247、230-248、232-250、234-252、237-255、238-256、240-258、241-259、243-261、270-288、277-295、279-297、280-298、308-326、309-327、310-328、324-342、326-344、328-346、329-347、330-348、340-358、380-398、388-406、394-412、396-414、398-416、400-418、401-419、402-420、403-421、404-422、405-423、407-425、408-426、411-429、508-526、515-533、582-600、585-603、586-604、587-605、589-607、596-614、597-615、600-618、618-636、619-637、622-640、625-643、637-655、640-658、642-660、644-662、646-664、656-674、659-677、666-684、669-687、699-717、702-720、706-724、707-725、721-739、724-742、728-746、730-748、731-749、733-751、737-755、739-757、773-791、775-793、777-795、778-796、780-798、783-801、784-802、785-803、824-842、827-845、828-846、833-851、834-852、837-855、839-857、841-859、858-876、866-884、869-887、871-889、874-892、875-893、877-895、878-896、908-926、913-931、914-932、917-935、921-939、922-940、923-941、925-943、927-945、928-946、931-949、940-958、945-963、947-965、950-968、951-969、959-977、964-982、965-983、969-987、970-988、972-990、973-991、978-996、981-999、982-1000、1005-1023、1007-1025、1008-1026、1012-1030、1013-1031、1014-1032、1016-1034、1018-1036、1019-1037、1025-1043、1028-1046、1030-1048、1051-1069、1068-1086、1083-1101、1085-1103、1087-1105、1088-1106、1090-1108、1091-1109、1092-1110、1093-1111、1094-1112、1099-1117、1101-1119、1107-1125、1108-1126、1109-1127、1168-1186、1170-1188、777-795、700-718、629-647、615-633、914-932、829-847、239-257、326-344、327-345、338-356、398-416、588-606、592-610、202-220、203-221、204-222、205-223、207-225、208-226、210-228、215-233、216-234、217-235、218-236、221-239、224-242、225-243、226-244、227-245、228-246、229-247、231-249、233-251、236-254、237-255、239-257、240-258、242-260、269-287、276-294、278-296、279-297、307-325、308-326、309-327、323-341、325-343、327-345、328-346、329-347、339-357、379-397、387-405、393-411、395-413、397-415、399-417、400-418、401-419、402-420、403-421、404-422、406-424、407-425、410-428、507-525、514-532、581-599、584-602、585-603、586-604、588-606、595-613、596-614、599-617、617-635、618-636、621-639、624-642、636-654、639-657、641-659、643-661、645-663、655-673、658-676、665-683、668-686、698-716、701-719、705-723、706-724、720-738、723-741、727-745、729-747、730-748、732-750、736-754、738-756、772-790、774-792、776-794、777-795、779-797、782-800、783-801、784-802、823-841、826-844、827-845、832-850、833-851、836-854、838-856、840-858、857-875、865-883、868-886、870-888、873-891、874-892、876-894、877-895、907-925、912-930、913-931、916-934、920-938、921-939、922-940、924-942、926-944、927-945、930-948、939-957、944-962、946-964、949-967、950-968、958-976、963-981、964-982、968-986、969-987、971-989、972-990、977-995、980-998、981-999、1004-1022、1006-1024、1007-1025、1011-1029、1012-1030、1013-1031、1015-1033、1017-1035、1018-1036、1024-1042、1027-1045、1029-1047、1050-1068、1067-1085、1082-1100、1084-1102、1086-1104、1087-1105、1089-1107、1090-1108、1091-1109、1092-1110、1093-1111、1098-1116、1100-1118、1106-1124、1107-1125、1108-1126、1167-1185、1169-1187、777-793、700-716、629-645、615-631、914-930、829-845、239-255、326-342、327-343、338-354、398-414、588-604、592-608、202-218、203-219、204-220、205-221、207-223、208-224、210-226、215-231、216-232、217-233、218-234、221-237、224-240、225-241、226-242、227-243、228-244、229-245、231-247、233-249、236-252、237-253、239-255、240-256、242-258、269-285、276-292、278-294、279-295、307-323、308-324、309-325、323-339、325-341、327-343、328-344、329-345、339-355、379-395、387-403、393-409、395-411、397-413、399-415、400-416、401-417、402-418、403-419、404-420、406-422、407-423、410-426、507-523、514-530、581-597、584-600、585-601、586-602、588-604、595-611、596-612、599-615、617-633、618-634、621-637、624-640、636-652、639-655、641-657、643-659、645-661、655-671、658-674、665-681、668-684、698-714、701-717、705-721、706-722、720-736、723-739、727-743、729-745、730-746、732-748、736-752、738-754、772-788、774-790、776-792、777-793、779-795、782-798、783-799、784-800、823-839、826-842、827-843、832-848、833-849、836-852、838-854、840-856、857-873、865-881、868-884、870-886、873-889、874-890、876-892、877-893、907-923、912-928、913-929、916-932、920-936、921-937、922-938、924-940、926-942、927-943、930-946、939-955、944-960、946-962、949-965、950-966、958-974、963-979、964-980、968-984、969-985、971-987、972-988、977-993、980-996、981-997、1004-1020、1006-1022、1007-1023、1011-1027、1012-1028、1013-1029、1015-1031、1017-1033、1018-1034、1024-1040、1027-1043、1029-1045、1050-1066、1067-1083、1082-1098、1084-1100、1086-1102、The sequence numbers are 1087-1103, 1089-1105, 1090-1106, 1091-1107, 1092-1108, 1093-1109, 1098-1114, 1100-1116, 1106-1122, 1107-1123, 1108-1124, 1167-1183, or 1169-1185, and the antisense strand contains at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides differing from the corresponding nucleotide sequence of SEQ ID NO: 2 by 0, 1, 2, or 3 nucleotides.

[0012] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a region similar to SEQ ID NO: 1. The nucleotide sequence of any of the following nucleotides differs from the nucleotide sequence of any one of them by 0, 1, 2 or 3 nucleotides by at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides: 776-796, 699-719, 628-648, 614-634, 913-933, 828-848, 238-258, 325-345, 326-346, 337-357, 397-417, 587-607, 591-611, 201-221, 202-222, 203-223, 204-224, 206-226, 207-227, 209-229, 214-234, 215-235, 2 16-236, 217-237, 220-240, 223-243, 224-244, 225-245, 226-246, 227-247, 228-248, 230-250, 232-252, 235-255, 236-256, 238-258, 239-259, 241-261, 268-288, 275-295, 277-297, 278-298, 306-326, 307-327, 308-328, 322-342, 324-344, 326-346, 327-347, 328-348, 338-358, 378-39 8, 386-406, 392-412, 394-414, 396-416, 398-418, 399-419, 400-420, 401-421, 402-422, 403-423, 405-425, 406-426, 409-429, 506-526, 513-533, 580-600, 583-603, 584-604, 585-605, 587-607, 594-614, 595-615, 598-618, 616-636, 617-637, 620-640, 623-643, 635-655, 638-658, 640 -660, 642-662, 644-664, 654-674, 657-677, 664-684, 667-687, 697-717, 700-720, 704-724, 705-725, 719-739, 722-742, 726-746, 728-748, 729-749, 731-751, 735-755, 737-757, 771-791, 773-793, 775-795, 776-796, 778-798, 781-801, 782-802, 783-803, 822-842, 825-845, 826-846,831-851、832-852、835-855、837-857、839-859、856-876、864-884、867-887、869-889、872-892、873-893、875-895、876-896、906-926、911-931、912-932、915-935、919-939、920-940、921-941、923-943、925-945、926-946、929-949、938-958、943-963、945-965、948-968、949-969、957-977、962-982、963-983、967-987、968-988、970-990、971-991、976-996、979-999、980-1000、1003-1023、1005-1025、1006-1026、1010-1030、1011-1031、1012-1032、1014-1034、1016-1036、1017-1037、1023-1043、1026-1046、1028-1048、1049-1069、1066-1086、1081-1101、1083-1103、1085-1105、1086-1106、1088-1108、1089-1109、1090-1110、1091-1111、1092-1112、1097-1117、1099-1119、1105-1125、1106-1126、1107-1127、1166-1186、1168-1188、778-796、701-719、630-648、616-634、915-933、830-848、240-258、327-345、328-346、339-357、399-417、589-607、593-611、203-221、204-222、205-223、206-224、208-226、209-227、211-229、216-234、217-235、218-236、219-237、222-240、225-243、226-244、227-245、228-246、229-247、230-248、232-250、234-252、237-255、238-256、240-258、241-259、243-261、270-288、277-295、279-297、280-298、308-326、309-327、310-328、324-342、326-344、328-346、329-347、330-348、340-358、380-398、388-406、394-412、396-414、398-416、400-418、401-419、402-420、403-421、404-422、405-423、407-425、408-426、411-429、508-526、515-533、582-600、585-603、586-604、587-605、589-607、596-614、597-615、600-618、618-636、619-637、622-640、625-643、637-655、640-658、642-660、644-662、646-664、656-674、659-677、666-684、669-687、699-717、702-720、706-724、707-725、721-739、724-742、728-746、730-748、731-749、733-751、737-755、739-757、773-791、775-793、777-795、778-796、780-798、783-801、784-802、785-803、824-842、827-845、828-846、833-851、834-852、837-855、839-857、841-859、858-876、866-884、869-887、871-889、874-892、875-893、877-895、878-896、908-926、913-931、914-932、917-935、921-939、922-940、923-941、925-943、927-945、928-946、931-949、940-958、945-963、947-965、950-968、951-969、959-977、964-982、965-983、969-987、970-988、972-990、973-991、978-996、981-999、982-1000、1005-1023、1007-1025、1008-1026、1012-1030、1013-1031、1014-1032、1016-1034、1018-1036、1019-1037、1025-1043、1028-1046、1030-1048、1051-1069、1068-1086、1083-1101、1085-1103、1087-1105、1088-1106、1090-1108、1091-1109、1092-1110、1093-1111、1094-1112、1099-1117、1101-1119、1107-1125、1108-1126、1109-1127、1168-1186、1170-1188、777-795、700-718、629-647、615-633、914-932、829-847、239-257、326-344、327-345、338-356、398-416、588-606、592-610、202-220、203-221、204-222、205-223、207-225、208-226、210-228、215-233、216-234、217-235、218-236、221-239、224-242、225-243、226-244、227-245、228-246、229-247、231-249、233-251、236-254、237-255、239-257、240-258、242-260、269-287、276-294、278-296、279-297、307-325、308-326、309-327、323-341、325-343、327-345、328-346、329-347、339-357、379-397、387-405、393-411、395-413、397-415、399-417、400-418、401-419、402-420、403-421、404-422、406-424、407-425、410-428、507-525、514-532、581-599、584-602、585-603、586-604、588-606、595-613、596-614、599-617、617-635、618-636、621-639、624-642、636-654、639-657、641-659、643-661、645-663、655-673、658-676、665-683、668-686、698-716、701-719、705-723、706-724、720-738、723-741、727-745、729-747、730-748、732-750、736-754、738-756、772-790、774-792、776-794、777-795、779-797、782-800、783-801、784-802、823-841、826-844、827-845、832-850、833-851、836-854、838-856、840-858、857-875、865-883、868-886、870-888、873-891、874-892、876-894、877-895、907-925、912-930、913-931、916-934、920-938、921-939、922-940、924-942、926-944、927-945、930-948、939-957、944-962、946-964、949-967、950-968、958-976、963-981、964-982、968-986、969-987、971-989、972-990、977-995、980-998、981-999、1004-1022、1006-1024、1007-1025、1011-1029、1012-1030、1013-1031、1015-1033、1017-1035、1018-1036、1024-1042、1027-1045、1029-1047、1050-1068、1067-1085、1082-1100、1084-1102、1086-1104、1087-1105、1089-1107、1090-1108、1091-1109、1092-1110、1093-1111、1098-1116、1100-1118、1106-1124、1107-1125、1108-1126、1167-1185、1169-1187、777-793、700-716、629-645、615-631、914-930、829-845、239-255、326-342、327-343、338-354、398-414、588-604、592-608、202-218、203-219、204-220、205-221、207-223、208-224、210-226、215-231、216-232、217-233、218-234、221-237、224-240、225-241、226-242、227-243、228-244、229-245、231-247、233-249、236-252、237-253、239-255、240-256、242-258、269-285、276-292、278-294、279-295、307-323、308-324、309-325、323-339、325-341、327-343、328-344、329-345、339-355、379-395、387-403、393-409、395-411、397-413、399-415、400-416、401-417、402-418、403-419、404-420、406-422、407-423、410-426、507-523、514-530、581-597、584-600、585-601、586-602、588-604、595-611、596-612、599-615、617-633、618-634、621-637、624-640、636-652、639-655、641-657、643-659、645-661、655-671、658-674、665-681、668-684、698-714、701-717、705-721、706-722、720-736、723-739、727-743、729-745、730-746、732-748、736-752、738-754、772-788、774-790、776-792、777-793、779-795、782-798、783-799、784-800、823-839、826-842、827-843、832-848、833-849、836-852、838-854、840-856、857-873、865-881、868-884、870-886、873-889、874-890、876-892、877-893、907-923、912-928、913-929、916-932、920-936、921-937、922-938、924-940、926-942、927-943、930-946、939-955、944-960、946-962、949-965、950-966、958-974、963-979、964-980、968-984、969-985、971-987、972-988、977-993、980-996、981-997、1004-1020、1006-1022、1007-1023、1011-1027、1012-1028、1013-1029、1015-1031、1017-1033、1018-1034、1024-1040、1027-1043、1029-1045、1050-1066、1067-1083、1082-1098、1084-1100、1086-1102、The sequence numbers are 1087-1103, 1089-1105, 1090-1106, 1091-1107, 1092-1108, 1093-1109, 1098-1114, 1100-1116, 1106-1122, 1107-1123, 1108-1124, 1167-1183, or 1169-1185, and the antisense strand contains at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides differing from the corresponding nucleotide sequence of SEQ ID NO: 2 by 0, 1, 2, or 3 nucleotides.

[0013] In some embodiments, the antisense strand comprises at least 14, 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides differing by 0, 1, 2, or 3 nucleotides from the complementary sequence of the following nucleotide sequence of SEQ ID NO: 1: 776-796, 699-719, 628-648, 614-634, 913-933, 828-848, 238-258, 325-345, 326-346, 337-357, 397-417, 587-607, 591-611, 201-221, 202-222, 203-223, 204-224, 206-226, 207-227, 209-229, 214-234, 215-235, 216-236. 217-237, 220-240, 223-243, 224-244, 225-245, 226-246, 227-247, 228-248, 230-250, 232-252, 235-255, 236-256, 238-258, 239-259, 241-261, 268-288, 275-295, 277-297, 278-298, 306-326, 307-327, 308-328, 322-342, 324-344, 326-346, 327-347, 328-348, 338-358, 378-398, 386-406, 39 2-412, 394-414, 396-416, 398-418, 399-419, 400-420, 401-421, 402-422, 403-423, 405-425, 406-426, 409-429, 506-526, 513-533, 580-600, 583-603, 584-604, 585-605, 587-607, 594-614, 595-615, 598-618, 616-636, 617-637, 620-640, 623-643, 635-655, 638-658, 640-660, 642-662, 644-6 64, 654-674, 657-677, 664-684, 667-687, 697-717, 700-720, 704-724, 705-725, 719-739, 722-742, 726-746, 728-748, 729-749, 731-751, 735-755, 737-757, 771-791, 773-793, 775-795, 776-796, 778-798, 781-801, 782-802, 783-803, 822-842, 825-845, 826-846, 831-851, 832-852, 835-855,837-857、839-859、856-876、864-884、867-887、869-889、872-892、873-893、875-895、876-896、906-926、911-931、912-932、915-935、919-939、920-940、921-941、923-943、925-945、926-946、929-949、938-958、943-963、945-965、948-968、949-969、957-977、962-982、963-983、967-987、968-988、970-990、971-991、976-996、979-999、980-1000、1003-1023、1005-1025、1006-1026、1010-1030、1011-1031、1012-1032、1014-1034、1016-1036、1017-1037、1023-1043、1026-1046、1028-1048、1049-1069、1066-1086、1081-1101、1083-1103、1085-1105、1086-1106、1088-1108、1089-1109、1090-1110、1091-1111、1092-1112、1097-1117、1099-1119、1105-1125、1106-1126、1107-1127、1166-1186、1168-1188、778-796、701-719、630-648、616-634、915-933、830-848、240-258、327-345、328-346、339-357、399-417、589-607、593-611、203-221、204-222、205-223、206-224、208-226、209-227、211-229、216-234、217-235、218-236、219-237、222-240、225-243、226-244、227-245、228-246、229-247、230-248、232-250、234-252、237-255、238-256、240-258、241-259、243-261、270-288、277-295、279-297、280-298、308-326、309-327、310-328、324-342、326-344、328-346、329-347、330-348、340-358、380-398、388-406、394-412、396-414、398-416、400-418、401-419、402-420、403-421、404-422、405-423、407-425、408-426、411-429、508-526、515-533、582-600、585-603、586-604、587-605、589-607、596-614、597-615、600-618、618-636、619-637、622-640、625-643、637-655、640-658、642-660、644-662、646-664、656-674、659-677、666-684、669-687、699-717、702-720、706-724、707-725、721-739、724-742、728-746、730-748、731-749、733-751、737-755、739-757、773-791、775-793、777-795、778-796、780-798、783-801、784-802、785-803、824-842、827-845、828-846、833-851、834-852、837-855、839-857、841-859、858-876、866-884、869-887、871-889、874-892、875-893、877-895、878-896、908-926、913-931、914-932、917-935、921-939、922-940、923-941、925-943、927-945、928-946、931-949、940-958、945-963、947-965、950-968、951-969、959-977、964-982、965-983、969-987、970-988、972-990、973-991、978-996、981-999、982-1000、1005-1023、1007-1025、1008-1026、1012-1030、1013-1031、1014-1032、1016-1034、1018-1036、1019-1037、1025-1043、1028-1046、1030-1048、1051-1069、1068-1086、1083-1101、1085-1103、1087-1105、1088-1106、1090-1108、1091-1109、1092-1110、1093-1111、1094-1112、1099-1117、1101-1119、1107-1125、1108-1126、1109-1127、1168-1186、1170-1188、777-795、700-718、629-647、615-633、914-932、829-847、239-257、326-344、327-345、338-356、398-416、588-606、592-610、202-220、203-221、204-222、205-223、207-225、208-226、210-228、215-233、216-234、217-235、218-236、221-239、224-242、225-243、226-244、227-245、228-246、229-247、231-249、233-251、236-254、237-255、239-257、240-258、242-260、269-287、276-294、278-296、279-297、307-325、308-326、309-327、323-341、325-343、327-345、328-346、329-347、339-357、379-397、387-405、393-411、395-413、397-415、399-417、400-418、401-419、402-420、403-421、404-422、406-424、407-425、410-428、507-525、514-532、581-599、584-602、585-603、586-604、588-606、595-613、596-614、599-617、617-635、618-636、621-639、624-642、636-654、639-657、641-659、643-661、645-663、655-673、658-676、665-683、668-686、698-716、701-719、705-723、706-724、720-738、723-741、727-745、729-747、730-748、732-750、736-754、738-756、772-790、774-792、776-794、777-795、779-797、782-800、783-801、784-802、823-841、826-844、827-845、832-850、833-851、836-854、838-856、840-858、857-875、865-883、868-886、870-888、873-891、874-892、876-894、877-895、907-925、912-930、913-931、916-934、920-938、921-939、922-940、924-942、926-944、927-945、930-948、939-957、944-962、946-964、949-967、950-968、958-976、963-981、964-982、968-986、969-987、971-989、972-990、977-995、980-998、981-999、1004-1022、1006-1024、1007-1025、1011-1029、1012-1030、1013-1031、1015-1033、1017-1035、1018-1036、1024-1042、1027-1045、1029-1047、1050-1068、1067-1085、1082-1100、1084-1102、1086-1104、1087-1105、1089-1107、1090-1108、1091-1109、1092-1110、1093-1111、1098-1116、1100-1118、1106-1124、1107-1125、1108-1126、1167-1185、1169-1187、777-793、700-716、629-645、615-631、914-930、829-845、239-255、326-342、327-343、338-354、398-414、588-604、592-608、202-218、203-219、204-220、205-221、207-223、208-224、210-226、215-231、216-232、217-233、218-234、221-237、224-240、225-241、226-242、227-243、228-244、229-245、231-247、233-249、236-252、237-253、239-255、240-256、242-258、269-285、276-292、278-294、279-295、307-323、308-324、309-325、323-339、325-341、327-343、328-344、329-345、339-355、379-395、387-403、393-409、395-411、397-413、399-415、400-416、401-417、402-418、403-419、404-420、406-422、407-423、410-426、507-523、514-530、581-597、584-600、585-601、586-602、588-604、595-611、596-612、599-615、617-633、618-634、621-637、624-640、636-652、639-655、641-657、643-659、645-661、655-671、658-674、665-681、668-684、698-714、701-717、705-721、706-722、720-736、723-739、727-743、729-745、730-746、732-748、736-752、738-754、772-788、774-790、776-792、777-793、779-795、782-798、783-799、784-800、823-839、826-842、827-843、832-848、833-849、836-852、838-854、840-856、857-873、865-881、868-884、870-886、873-889、874-890、876-892、877-893、907-923、912-928、913-929、916-932、920-936、921-937、922-938、924-940、926-942、927-943、930-946、939-955、944-960、946-962、949-965、950-966、958-974、963-979、964-980、968-984、969-985、971-987、972-988、977-993、980-996、981-997、1004-1020、1006-1022、1007-1023、1011-1027、1012-1028、1013-1029、1015-1031、1017-1033、1018-1034、1024-1040、1027-1043、1029-1045、1050-1066、1067-1083、1082-1098、1084-1100、1086-1102、1087-1103、1089-1105、1090-1106, 1091-1107, 1092-1108, 1093-1109, 1098-1114, 1100-1116, 1106-1122, 1107-1123, 1108-1124, 1167-1183 or 1169-1185.

[0014] In some embodiments, the dsRNA agent comprises at least one modified nucleotide. In some embodiments, all or substantially all nucleotides of the antisense strand are modified nucleotides. In some embodiments, all or substantially all nucleotides of both the sense and antisense strands are modified nucleotides. In some embodiments, at least one modified nucleotide comprises: 2'-O-methyl nucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2'3'-seco nucleotide mimic, locked nucleotide, unlocked nucleic acid nucleotide (UNA), glycol nucleic acid nucleotide (GNA), 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, baseless nucleotide, ribitol, reverse nucleotide, reverse baseless nucleotide, reverse 2'-OMe nucleotide, reverse 2'-deoxynucleotide, isomannitol nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholinonucleotide, and 3'-OMe nucleotide, nucleotide containing a 5'-thiophosphate group, nucleotide modified with 5'-phosphonate, nucleotide modified with 5'-phosphate or 5'-phosphate mimic, or a terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid bis(decyl)amide group, 2'-amino-modified nucleotide, phosphoramide, or nucleotide containing a non-natural base.

[0015] In some embodiments, the antisense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methylnucleotides, 2'-fluoronucleotides, and UNA-modified nucleotides, wherein fewer than 6 of the modified nucleotides are 2'-fluoronucleotides. In some embodiments, the antisense strand comprises 3 or 5 2'-fluoronucleotides, preferably 5 2'-fluoronucleotides. In some embodiments, the sense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methylnucleotides and 2'-fluoronucleotides, wherein fewer than 4 of the modified nucleotides are 2'-fluoronucleotides. In some embodiments, the sense strand comprises 3 2'-fluoronucleotides. In some embodiments, the antisense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methylnucleotides and 2'-fluoronucleotides, wherein at least 14 of the modified nucleotides are 2'-O-methylnucleotides, and the nucleotides at positions 2, 5, 7, 12, 14, 16, and / or 18, counting from the first matching position from the 5' end of the antisense strand, are independently 2'-fluoronucleotides. In some embodiments, the antisense strand comprises at least one UNA-modified nucleotide and five 2'-fluoronucleotides. In some embodiments, the antisense strand comprises one UNA-modified nucleotide counting from the first matching position at the 7th position starting from the 5' end, and five 2'-fluoronucleotides at positions 2, 5, 12, 14, and 16, with the remainder being a 2'-O-methylnucleotide. In some embodiments, the antisense strand comprises five 2'-fluoronucleotides counting from the first matching position at the 2nd, 5th, 12th, 14th, and 18th positions starting from the 5' end, with the remainder being a 2'-O-methylnucleotide. In some embodiments, the antisense strand comprises five 2'-fluoronucleotides counting from the first matching position at the 2nd, 7th, 12th, 14th, and 16th positions starting from the 5' end, with the remainder being a 2'-O-methylnucleotide. In some embodiments, the sense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methylnucleotides and 2'-fluoronucleotides. In some embodiments, at least 18 modified nucleotides are 2'-O-methyl nucleotides, and the nucleotides at positions 9, 11, and / or 13, counted starting from the first matching position from the 3' end of the sense strand, are 2'-fluoronucleotides. In some embodiments, at least 18 modified nucleotides are 2'-O-methyl nucleotides, and the nucleotides at positions 8, 11, and / or 13, counted starting from the first matching position from the 3' end of the sense strand, are 2'-fluoronucleotides.

[0016] In some embodiments, the dsRNA agent further comprises a phosphate ester or a phosphate ester mimic. In some embodiments, the dsRNA agent comprises one or more nucleotides modified with a 5'-phosphate ester or a 5'-phosphate ester mimic. In some embodiments, the phosphate ester mimic is 5'-vinyl phosphonate (VP). In some embodiments, the nucleotide modified with a 5'-phosphate ester or a 5'-phosphate ester mimic is introduced at the 5' end of the antisense strand.

[0017] In some implementations, the phosphate mimicry of the 5'-terminal nucleotide has a fragment represented by the following formula:

[0018]

[0019] Where: Q8 is O, S, SO, SO2, PR 16 R 17 or NR 11 ;R 16 and R 17 Independently selected from (=O), (=S), OH, SH, C1-C6 alkyl and NR 18 R 19 ;

[0020] Ra and Rc are each independently selected from hydroxyl or protected hydroxyl, mercapto or protected mercapto, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, protected or optionally substituted amino, native or modified nucleoside; and R b For O, S, or NR 12 R 12 Protected by hydrogen, C1-C6 alkyl and amino groups;

[0021] The substituents in the substituted amino group are selected from: optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, sulfinyl, sulfonyl and acetyl groups;

[0022] R 11 R 18 and R 19 Independently selected from H, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, methanesulfonyl, and sulfonic acid groups;

[0023] Each substituent comprises one or more substituents optionally and independently selected from the following: halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl mercapto, and CN;

[0024] This indicates a bond that links to a segment of the 5'-terminal nucleotide. In some embodiments, Q8 is bonded to the 4'-carbon or 5'-carbon of the sugar or sugar-substitute portion of the 5'-terminal nucleotide.

[0025] In some implementations, the dsRNA agent contains an E-vinylphosphonate nucleotide at the 5' end of the guide strand.

[0026] In some embodiments, the dsRNA agent contains a 5'-phosphate mimic nucleotide, or its stereoisomer or racemate, represented by formula (VIII) at the 5' end of the guide strand:

[0027]

[0028] Where: Q8 is O, S, SO, SO2, PR 16 R 17 or NR 11 ;R 16 and R 17 Independently, it is (=O), (=S), OH, SH, C1-C6 alkyl, NR 18 R 19 Ra and Rc are each independently selected from hydroxyl or protected hydroxyl, mercapto or protected mercapto, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, protected or optionally substituted amino, native or modified nucleoside; and R b For O, S, or NR 12 R 12 Protected by hydrogen, C1-C6 alkyl, or amino groups;

[0029] Q1 and Q2 are each independently H, halogen, -CN, or optionally substituted C1-C6 alkyl;

[0030] The substituents in the substituted amino group are selected from: optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, sulfinyl, sulfonyl, acetyl;

[0031] R 11 R 18 and R 19 Independently, it is H, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, methanesulfonyl, and sulfonic acid groups;

[0032] Z is a nucleoside containing a sugar or a sugar-substituted portion;

[0033] T3 is an internucleotide linker that links the 5' terminal nucleotide of formula (VIII) or its stereoisomer to an existing guide strand;

[0034] Each substituent contains one or more substituents optionally selected independently from the following: halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl mercapto, CN.

[0035] In one specific embodiment, Q8 in formula (VIII) is S, SO, or SO2. In one specific embodiment, Q1 and Q2 in formula (VIII) are each independently H. In one specific embodiment, the sugar or sugar substitution moiety in formula (VIII) comprises a 5-membered furanyl ring, a non-furanyl ring, or a 5- to 6-membered carbon ring system or an open ring system. In one specific embodiment, the sugar substitution moiety in formula (VIII) is selected from morpholinyl, cyclohexenyl, cyclohexyl, cyclopentyl, pyranyl, or cyclohexanol. In one specific embodiment, the sugar moiety in formula (VIII) is a furanyl sugar. In one specific embodiment, the sugar or sugar substitution moiety in formula (VIII) comprises an unlocked nucleobase analog (UNA) or a glycerol nucleobase analog (GNA). In one specific embodiment, the sugar or sugar substitution moiety in formula (VIII) comprises a locked nucleic acid (LNA) or a bridged nucleic acid (BNA). In one embodiment, Q8 in formula (VIII) is bonded to the 4'-carbon or 5'-carbon of the sugar or sugar-substituted portion. In one embodiment, Rb in formula (VIII) is oxygen. In one embodiment, Ra and Rc in formula (VIII) are each independently selected from OH, SH, NH2, or NHSO2CH3.

[0036] In some embodiments, the dsRNA agent comprises a 5'-phosphate mimic modified nucleotide at the 5' end of the guide strand, wherein the 5'-phosphate mimic modified nucleotide is any of the following structures or their stereoisomers or racemates:

[0037] Or its stereoisomers or racemates,

[0038] This indicates the key that connects to the end of the existing boot chain 5'.

[0039] In some embodiments, the internucleotide linker is independently selected from phosphodiester linkers, phosphotriester linkers, thiophosphate linkers, dithiophosphate linkers, alkylphosphonates, aminophosphonates, phosphonates, hypophosphonates, phosphorothioamidates, or aminophosphates. To illustrate a 5'-phosphate mimic modified nucleotide having a phosphodiester linker, a non-limiting example is Phos-15-1* having the following structure:

[0040] .

[0041] In some embodiments, the dsRNA agent comprises at least one modified nucleotide and also comprises one or more targeting groups or linker groups. In some embodiments, one or more targeting ligands are independently selected from the group consisting of small molecules, lipids and lipid derivatives, peptides, aptamers, polysaccharides, protein ligands, nanobodies, and antibodies. In some embodiments, one or more targeting groups or linker groups target receptors mediating delivery to CNS tissues or liver tissue, such as hydrophilic ligands. In some embodiments, one or more targeting groups or linker groups target brain tissue or spinal cord tissue, such as the striatum or dorsal root ganglia. In some embodiments, one or more targeting groups or linker groups are conjugated to a sense strand. In some embodiments, the dsRNA agent comprises a targeting group conjugated to the 5' end of a sense strand. In some embodiments, the dsRNA agent comprises a targeting group conjugated to the 3' end of a sense strand. In some embodiments, the targeting group or linker group comprises N-acetylgalactosamine (GalNAc).

[0042] In some implementations, the targeting group has a structure as shown in formula (X):

[0043]

[0044] Each n” is independently selected from 1 or 2.

[0045] In some implementations, the targeting group has the following structure:

[0046]

[0047]

[0048]

[0049] .

[0051] In some embodiments, the dsRNA agent contains a targeting group conjugated to the 5' end of the sense strand. Preferably, the targeting group is selected from any one of GLO-1 to GLO-16 and GLS-1* to GLS-16*. More preferably, the targeting group is GLS-15*.

[0052] In some embodiments, one or more lipophilic portions are attached to one or more end or internal positions on at least one chain. In some embodiments, one or more lipophilic portions are attached to one or more internal positions on at least one chain via a connector or carrier. In some embodiments, internal positions include all positions except those two positions from each end of at least one chain. In some embodiments, internal positions include all positions except those three positions from each end of at least one chain. In some embodiments, internal positions do not include the cleavage site region of the sense chain. In some embodiments, internal positions include all positions except positions 9 through 11, counting from the first matching position starting from the 3' end of the sense chain. In some embodiments, internal positions include all positions except positions 11 through 13, counting from the first matching position starting from the 3' end of the sense chain. In some embodiments, internal positions do not include the cleavage site region of the sense chain. In some embodiments, internal positions do not include the cleavage site region of the antisense chain. In some embodiments, internal positions include all positions except positions 12 through 14, counting from the 5' end of the antisense chain. In some implementations, the internal positions include all positions except for positions 11 through 13 on the sense chain, counting from the 3' end, and positions 12 through 14 on the antisense chain, counting from the 5' end (both starting from the first matching position).

[0053] In some embodiments, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound. In some embodiments, the lipophilic moiety is selected from lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-hexadecylglycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytriphenylmethyl, or phenethyl. Azine. In some embodiments, the lipophilic moiety comprises saturated or unsaturated C4-C... 30 The hydrocarbon chain, and optionally a functional group selected from hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. In some embodiments, the lipophilic moiety comprises a saturated or unsaturated C6-C group. 18Hydrocarbon chain. In some embodiments, the lipophilic moiety comprises a saturated or unsaturated C16 hydrocarbon chain.

[0054] In some embodiments, the lipophilic moiety is conjugated by a carrier that replaces one or more nucleotides in the internal position or double-stranded region. In some embodiments, the carrier is selected from pyrrolidinyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazineyl, [1,3]dioxolaneyl, etc. azolealkyl, isopropyl Cyclic groups of azole alkyl, morpholinyl, thiazolyl, isothiazolyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or non-cyclic portions based on a serine alcohol skeleton or a diethanolamine skeleton.

[0055] In some embodiments, the lipophilic moiety is conjugated to a double-stranded RNAi agent via a linker comprising an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, click reaction product, or carbamate. In some embodiments, the lipophilic moiety is conjugated to a nucleotide, sugar moiety, or nucleoside. In some embodiments, the lipophilic moiety or targeting ligand is conjugated via a biocleavable linker selected from DNA, RNA, disulfides, amides, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0056] In some embodiments, the antisense strand includes an invab at its 3' end. In some embodiments, the sense strand includes one or two invab residues and / or one or two imann residues at its 3' and / or 5' ends. In some embodiments, each end of the sense strand includes an invab residue. In some embodiments, each end of the sense strand includes an imann residue. In some embodiments, the dsRNA agent has two blunt ends. In some embodiments, at least one strand includes a 3' overhang having at least one nucleotide. In some embodiments, at least one strand includes a 3' overhang having at least two nucleotides.

[0057] In some embodiments, the antisense strand includes an inverted abase residue at its 3' end. In some embodiments, the sense strand includes one or two inverted abase residues and / or one or two Imann residues at its 3' and / or 5' ends. In some embodiments, the 3' and 5' ends of the sense strand each independently include an inverted abase residue. In some embodiments, the 3' and 5' ends of the sense strand each independently include an Imann residue. In some embodiments, the sense strand includes two inverted abase residues at its 3' and 5' ends, and either residue at the 3' or 5' end is further conjugated to a targeting group, preferably the aforementioned GLS-15*. In some embodiments, the sense strand includes an inverted abase residue at its 3' end, and the 5' end is further conjugated to a targeting group, preferably the aforementioned GLS-15*. In some embodiments, the sense strand includes two Imann residues at its 3' and 5' ends, and either residue at the 3' or 5' end is further conjugated to a targeting group, preferably the aforementioned GLS-15*.

[0058] In some embodiments, at least one link of the sense strand and / or antisense strand is a phosphodiester (PO) link. In some embodiments, at least one link of the sense strand and / or antisense strand is a modified link. In some embodiments, at least one link of the sense strand and / or antisense strand is a phosphate thioester (PS) link. In some embodiments, the dsRNA agent comprises at least one phosphate thioester nucleoside link. In some embodiments, the sense strand comprises at least one phosphate thioester nucleoside link. In some embodiments, the antisense strand comprises at least one phosphate thioester nucleoside link. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphate thioester nucleoside links. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphate thioester nucleoside links. In some embodiments, at least one phosphate thioester (PS) link is introduced at the 5' end, 3' end, or both ends of the sense strand and / or antisense strand. In some embodiments, one, two, three, four, five, or six phosphate-thioester (PS) links are independently introduced at the 5' end, 3' end, or both ends of the sense and / or antisense strand. In some embodiments, the 5' end of the antisense strand comprises two phosphate-thioester nucleoside links. In some embodiments, the 3' end of the antisense strand comprises two phosphate-thioester nucleoside links. In some embodiments, the 5' end and the 3' end of the antisense strand independently comprise two phosphate-thioester nucleoside links. In some embodiments, at least two modified or unmodified nucleotides at one or both ends of the antisense strand are linked by phosphate-thioester links. In some embodiments, three modified or unmodified nucleotides at one or both ends of the antisense strand are linked by phosphate-thioester links. In some embodiments, at least two modified or unmodified nucleotides at one or both ends of the sense strand are linked by phosphate-thioester links. In some embodiments, the terminal three modified or unmodified nucleotides at one or both ends of the sense strand are linked by phosphate thioester bonds. In some embodiments, the terminal three modified or unmodified nucleotides at the 5' end of the sense strand are linked by phosphate thioester bonds, and the terminal two modified or unmodified nucleotides at the 3' end of the sense strand are linked by phosphate thioester bonds. In some embodiments, one or more inverted abase residues or one or more Imann residues are conjugated to either or both ends of the sense strand by phosphate thioester bonds. In some embodiments, the targeting group is also conjugated to either end of the sense strand by phosphate thioester bonds. In some embodiments, the targeting group is also conjugated to the 5' end of the sense strand by phosphate thioester bonds.

[0059] In some embodiments, the sense strand sequence of the SNCA dsRNA agent of the present invention can be represented by formula (I):

[0060]

[0061] in:

[0062] Each N' F This indicates a nucleotide modified with 2'-fluorine; N' N1 、N' N2 、N' N3 、N' N4 、N' N5 and N' N6 Each N' represents a modified or unmodified nucleotide independently; L Each nucleotide can be independently represented as a modified or unmodified nucleotide, but not a nucleotide with 2'-fluorine modification, and m' and n' are each an independent integer from 0 to 7.

[0063] In some implementations, the modified nucleotide is the modified nucleotide defined above.

[0064] In some implementations, the modified nucleotide is a 2'-OMe modified nucleotide or a 2'-F modified nucleotide.

[0065] In some implementation schemes, N' N1 and N' N3 Each can be used independently to represent a nucleotide modified with 2'-fluorine.

[0066] In some implementation schemes, N' N4 and N' N5 Each can be used independently to represent a nucleotide modified with 2'-fluorine.

[0067] In some implementation schemes, N' N2 and N' N4 Each can be used independently to represent a nucleotide modified with 2'-fluorine.

[0068] In some implementations, m' is 1 and n' is 3, or m' is 1 and n' is 4, or m' is 1 and n' is 5.

[0069] In some implementations, m' is 4 and n' is 3.

[0070] In some implementations, m' is 2 and n' is 3, or m' is 2 and n' is 4, or m' is 2 and n' is 5.

[0071] In some implementation schemes, N' N4 and N' N5 Each of these independently represents a 2′-fluorine-modified nucleotide, where m' is 2 and N' is... L 、N' N1 、N'N2 、N' N3 and N' N6 Each can be used independently to represent a 2′-O-methyl nucleotide.

[0072] In some implementation schemes, N' N2 and N' N4 Each of these represents a 2'-fluorinated nucleotide independently, m' is 4, and N' L 、N' N1 、N' N3 、N' N5 and N' N6 Each can be used independently to represent a 2'-O-methyl nucleotide.

[0073] In some embodiments, the antisense strand sequence of the SNCA dsRNA agent of the present invention can be independently represented by formula (II):

[0074]

[0075] in:

[0076] Each N F This indicates a nucleotide modified with 2'-fluorine; N M1 N M2 N M3 N M4 N M5 N M6 N M7 N M8 and N M9 Each can be used independently to represent a modified or unmodified nucleotide; N L and N Z Each of these terms independently represents a modified or unmodified nucleotide, but not a nucleotide modified with 2'-fluorine; and n is an integer from 0 to 7.

[0077] In some implementations, the modified nucleotide is the modified nucleotide defined above.

[0078] In some embodiments, the modified nucleotide is a 2'-OMe modified nucleotide, a 2'-F modified nucleotide, an UNA modified nucleotide, or a nucleotide containing a phosphate ester mimic.

[0079] In some implementation schemes, N M1 N M3 and N M7 Each can be used independently to represent a nucleotide modified with 2'-fluorine.

[0080] In some implementation schemes, N M2 N M3 and N M6Each can be used independently to represent a nucleotide modified with 2'-fluorine.

[0081] In some implementation schemes, N M2 N M3 and N M7 Each independently represents a 2'-fluorinated nucleotide, and N M6 This indicates a nucleotide modified with UNA;

[0082] In some implementation schemes, N M2 N M3 and N M6 Each independently represents a 2'-fluorinated nucleotide, and N M1 N M4 N M5 N M7 N M8 and N L Each can be used independently to represent a 2'-O-methyl nucleotide.

[0083] In some implementation schemes, N M2 N M3 and N M7 Each independently represents a 2'-fluorinated nucleotide, and N M6 This indicates a nucleotide modified with UNA, and N M1 N M4 N M5 N M8 and N L Each can be used independently to represent a 2'-O-methyl nucleotide.

[0084] In some implementation schemes, N Z This indicates a nucleotide modified with a 5'-phosphonate.

[0085] In some implementation schemes, N Z It is a vinylphosphonate-modified nucleotide.

[0086] In some implementation schemes, N Z It is Vpu*, which has a structure

[0087] .

[0088] In some implementation schemes, N Z Selected from the following or their stereoisomers or racemates:

[0089] .

[0090] In some implementations, n is 1, or n is 2, or n is 3.

[0091] In some embodiments, the SNCA dsRNA duplex of the present invention may be represented by formula (III), and the complementary region comprises at least 15 consecutive nucleotides, wherein,

[0092]

[0093] in:

[0094] Each chain is approximately 17 to 30 nucleotides in length;

[0095] N F and N' F Each independently represents a nucleotide modified with 2'-fluorine; N M1 N M2 N M3 N M4 N M5 N M6 N M7 N M8 N M9 、N' N1 、N' N2 、N' N3 、N' N4 、N' N5 and N' N6 Each can be used independently to represent a modified or unmodified nucleotide; N Z N L and N' L Each of these characters independently represents a modified or unmodified nucleotide, but not a nucleotide modified with 2'-fluorine, and m', n', and n are each independent integers from 0 to 7.

[0096] In some implementations, the modified nucleotide is the modified nucleotide defined above.

[0097] In some embodiments, the modified nucleotide is a 2'-OMe modified nucleotide, a 2'-F modified nucleotide, an UNA modified nucleotide, or a nucleotide containing a phosphate ester mimic.

[0098] In some implementation schemes, N' N1 、N' N2 、N' N3 、N' N4 、N' N5 and N' N6 Each can be used independently to represent a 2'-fluorine modified nucleotide or a 2'-O-methyl nucleotide.

[0099] In some implementation schemes, N' L and N L Each can be used independently to represent a 2'-O-methyl nucleotide.

[0100] In some implementation schemes, N M1 N M2 N M3 N M4 N M5 N M6 N M7 N M8 N M9 and N Z Each of these can be used independently to represent a 2'-fluorine modified nucleotide, a 2'-O-methyl nucleotide, an UNA modified nucleotide, or a nucleotide containing a phosphate ester mimic.

[0101] In some implementation schemes, N' N1 and N' N3 Each can be used independently to represent a nucleotide modified with 2'-fluorine.

[0102] In some implementation schemes, N' N2 and N' N4 Each can be used independently to represent a nucleotide modified with 2'-fluorine.

[0103] In some implementation schemes, N' N4 and N' N5 Each can be used independently to represent a nucleotide modified with 2'-fluorine.

[0104] In some implementation schemes, N M1 N M3 and N M7 Each can be used independently to represent a nucleotide modified with 2'-fluorine.

[0105] In some implementation schemes, N M2 N M3 and N M6 Each independently represents a 2'-fluorinated nucleotide; in some embodiments, N M2 N M3 and N M6 All of them are nucleotides modified with 2'-fluorine.

[0106] In some implementation schemes, N M2 N M3 and N M7 Each independently represents a 2'-fluorinated nucleotide, and N M6 This indicates a nucleotide modified with UNA.

[0107] In some implementation schemes, N M2 N M6 and N M9 Each can be used independently to represent a nucleotide modified with 2'-fluorine.

[0108] In some implementation schemes, NM2 N M7 and N M9 Each independently represents a 2'-fluorinated nucleotide, and N M6 This indicates a nucleotide modified with UNA.

[0109] In some implementation schemes, N Z This indicates a nucleotide modified with a 5'-phosphonate.

[0110] In some implementation schemes, N Z It is a vinylphosphonate-modified nucleotide.

[0111] In some implementation schemes, N Z It is Vpu*, which has a structure

[0112] .

[0113] In some implementation schemes, N Z Selected from the following or their stereoisomers or racemates

[0114] .

[0115] In some implementations, n' is 1 and m' is 2, n' is 2 and m' is 2, or n' is 1 and m' is 4, or n' is 3 and m' is 2, or n' is 3 and m' is 4, or n' is 4 and m' is 2, or n' is 5 and m' is 2, or m' is 1 and n' is 3, or m' is 1 and n' is 4, or m' is 1 and n' is 5.

[0116] In some implementations, n is 1, or n is 2, or n is 3.

[0117] In some embodiments of formula (II) or (III), the antisense strand includes an inverted abase residue at its 3' end. In some embodiments of formula (I) or (III), the sense strand includes one or two inverted abase residues and / or one or two imann residues at its 3' and / or 5' ends. In some embodiments, the 3' and 5' ends of the sense strand each independently include an inverted abase residue. In some embodiments of formula (I) or (III), the 3' end of the sense strand independently includes an inverted abase residue. In some embodiments of formula (I) or (III), the 3' and 5' ends of the sense strand each independently include an imann residue. In some embodiments of formula (I) or (III), the sense strand includes an inverted abase residue at both its 3' and 5' ends, which is further conjugated to a targeting group (e.g., a hydrophilic ligand) mediating delivery to CNS tissue or liver tissue, said targeting group optionally being the aforementioned GLS-15*. In some embodiments of formula (I) or (III), the sense strand comprises an inverted abase-free residue at both the 3' and 5' ends, and either residue at the 3' or 5' end is further conjugated to a targeting group (e.g., a hydrophilic ligand) mediating delivery to CNS or liver tissue, wherein the targeting group is optionally the aforementioned GLS-15*. In some embodiments of formula (I) or (III), the sense strand comprises an imann residue at both the 3' and 5' ends, and either residue at the 3' or 5' end is further conjugated to a targeting group (e.g., a hydrophilic ligand) mediating delivery to CNS or liver tissue, wherein the targeting group is optionally the aforementioned GLS-15*. In some embodiments, the aforementioned dsRNA agent has two blunt ends. In some embodiments of formula (I), (II), or (III), at least one strand comprises a 3' overhang having at least one nucleotide. In some embodiments of formula (I), (II), or (III), at least one strand comprises a 3' overhang having at least two nucleotides.

[0118] In some embodiments of formula (I), (II) or (III), N M1 N M2 N M3 N M4 N M5 N M6 N M7 N M8 N M9 、N' N1 、N' N2 、N' N3 、N' N4 、N' N5 、N' N6 、N' L N L and NZ Each nucleotide is independently linked to an adjacent nucleotide via a phosphodiester (PO) bond. In some embodiments of formula (I), (II), or (III), N M1 N M2 N M3 N M4 N M5 N M6 N M7 N M8 N M9 、N' N1 、N' N2 、N' N3 、N' N4 、N' N5 、N' N6 、N' L N L and N Z At least one of the nucleotides is linked to an adjacent nucleotide via a phosphate thioester (PS) bond. In some embodiments of formulas (I), (II), or (III) above, a reverse abase residue, an imann residue, and / or a targeting group are also included, and the bonds at positions 1 to 10 of the terminal position of each end of the chain independently include 1, 2, 3, 4, 5, or 6 phosphate thioester (PS) bonds. In some embodiments of formulas (I), (II), or (III) above that include a reverse abase residue, an imann residue, and / or a targeting group, the bonds at positions 1 to 5 of the terminal position of each end of the chain independently include 1, 2, or 3 phosphate thioester (PS) bonds. In some embodiments of formulas (I), (II), or (III) above that include a reverse abase residue, an imann residue, and / or a targeting group, the bonds at positions 1 to 3 of the terminal position of each end of the chain independently include 1 or 2 phosphate thioester (PS) bonds.

[0119] In some implementations, any of the semantic chains in Table 1 may also be modified in the manner shown in formula (I) or (III) above.

[0120] In some implementations, any of the antisense chains in Table 1 may also be modified in the manner shown in formula (II) or (III) above.

[0121] In some implementations, any of the double strands in Table 1 may also be modified in the manner shown in the aforementioned formula (III).

[0122] In some embodiments, the antisense strand of the dsRNA agent is independently 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some embodiments, the sense strand of the dsRNA agent is independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides long. In some embodiments, both the sense and antisense strands are 23 nucleotides long. In some embodiments, both the sense and antisense strands are 21 nucleotides long.

[0123] In some embodiments of the aforementioned dsRNA agent, the SNCA mRNA transcript is SEQ ID NO: 1.

[0124] In some implementations, the dsRNA agent targets the corresponding portions of the SNCA mRNA transcripts disclosed in Table 1 and / or the target regions of the aforementioned SNCA mRNA transcripts.

[0125] In some embodiments of the aforementioned dsRNA agent, the region complementary to a portion of the mRNA encoding SNCA comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides that differ from the complementary sequence of any of the aforementioned target regions of the SNCA mRNA transcript by no more than 0, 1, 2, or 3 nucleotides.

[0126] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein nucleotides 2 to 18 of the antisense strand contain a region complementary to the SNCA mRNA transcript, the complementary region comprising at least 15, 16, or 17 consecutive nucleotides differing by 0, 1, 2, or 3 nucleotides from one of the antisense sequences listed in one of Tables 1 to 3, and optionally contains one or more targeting ligands.

[0127] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region complementary to a portion of the mRNA encoding SNCA, and comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides differing from any of the antisense sequences listed in any of Tables 1 to 3 by no more than 1, 2, or 3 nucleotides. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region complementary to a portion of the mRNA encoding SNCA, and comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides from any of the antisense sequences listed in any of Tables 1 to 3.

[0128] In some embodiments, the antisense strand of the dsRNA agent is at least substantially complementary to any of the target regions of SEQ ID NO: 1, and is preferably provided in any of Tables 1 to 3. In some embodiments, the antisense strand of the dsRNA agent is completely complementary to any of the target regions of SEQ ID NO: 1, and is preferably provided in any of Tables 1 to 3. In some embodiments, the dsRNA agent comprises a sense strand sequence shown in any of Tables 1 to 3, wherein the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent. In some embodiments, the dsRNA agent comprises a sense strand sequence shown in any of Tables 1 to 3, wherein the sense strand sequence is completely complementary to the antisense strand sequence in the dsRNA agent. In some embodiments, the dsRNA agent comprises an antisense strand sequence shown in any of Tables 1 to 3. In some embodiments, the dsRNA agent comprises a sequence shown as a double-stranded sequence in any of Tables 1 to 3.

[0129] In some embodiments, the modified sense strand has a modification pattern shown in any of Tables 2 to 3. In some embodiments, the modified antisense strand has a modification pattern shown in any of Tables 2 to 3. In some embodiments, the modified sense strand is a modified sense strand sequence shown in one of Tables 2 to 3. In some embodiments, the modified antisense strand is a modified antisense strand sequence shown in one of Tables 2 to 3.

[0130] According to one aspect of the invention, a composition is provided comprising any embodiment of the foregoing dsRNA pharmaceutical aspect of the invention. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises one or more additional therapeutic agents. In some embodiments, the composition is packaged in a kit, container, packaging, dispenser, pre-filled syringe, or vial. In some embodiments, the composition is formulated for subcutaneous administration, for intrathecal administration, for intracranial administration, for intraventricular administration, for intracerebral administration, or for intravenous (IV) administration.

[0131] According to another aspect of the invention, a cell is provided that comprises any embodiment of the foregoing dsRNA pharmaceutical aspect of the invention. In some embodiments, the cell is a mammalian cell, optionally a human cell. In some embodiments, the cell is a neuron (e.g., a primary sensory neuron).

[0132] According to another aspect of the invention, a method for inhibiting SNCA gene expression in cells is provided, the method comprising: (i) preparing cells containing an effective amount of any embodiment of the aforementioned dsRNA agent of the invention or any embodiment of the aforementioned compositions of the invention. In some embodiments, the method further comprises: (ii) maintaining the prepared cells for a time sufficient to allow degradation of the mRNA transcript of the SNCA gene, thereby inhibiting SNCA gene expression in the cells. In some embodiments, the cells are administered subcutaneously to the subject and the dsRNA agent is administered subcutaneously to the subject. In some embodiments, the cells are administered intravenously to the subject and the dsRNA agent is administered via IV. In some embodiments, the cells are administered intracranially or intrathecally to the subject and the dsRNA agent is administered intrathecally, intraventricularly, or intracerebrally to the subject. In some embodiments, the method further includes assessing inhibition of the SNCA gene after administration of the dsRNA agent to the subject, wherein the means of assessment include: (i) identifying one or more physiological characteristics of an SNCA-related disease or condition in the subject, and (ii) comparing the identified physiological characteristics with pre-treatment baseline physiological characteristics of the SNCA-related disease or condition and / or control physiological characteristics of the SNCA-related disease or condition, wherein the comparison indicates the presence or absence of one or more of inhibition of SNCA gene expression in the subject. In some embodiments, the physiological characteristic is one or more of the following: the level of SNCA mRNA, α-synuclein, or other parameters functionally related to SNCA expression levels. A decrease in SNCA expression can also be indirectly assessed by measuring a decrease in SNCA biological activity, such as a decrease in the level of one or more of SNCA mRNA, α-synuclein, or other parameters functionally related to SNCA expression levels.

[0133] Another aspect of this disclosure provides a method for identifying a subject as suffering from or at risk of developing a disease or disorder characterized by enlarged endosomes in neuronal cells, and for selecting a treatment for the subject, the method comprising: a) obtaining a nucleic acid sample from the subject; b) identifying the subject as having a mutation in synuclein α (SNCA), which is associated with enlarged endosomes in neuronal cells with SNCA mutations; and c) selecting a double-stranded RNA inhibitor (dsRNAi) agent targeting synuclein α (SNCA) for administration to the subject in an amount sufficient to reduce SNCA levels in the subject's neuronal cells, thereby identifying the subject as suffering from or at risk of developing a disease or disorder characterized by enlarged endosomes in neuronal cells, and for selecting a treatment for the subject.

[0134] According to another aspect of the invention, a method for inhibiting SNCA gene expression in a subject is provided, the method comprising administering to the subject an effective amount of an embodiment of the aforementioned dsRNA agent of the invention or an embodiment of the aforementioned composition of the invention. In some embodiments, the dsRNA agent is administered subcutaneously to the subject. In some embodiments, the dsRNA agent is administered to the subject via intravenous (IV) administration. In some embodiments, the dsRNA agent is administered to the subject intracranially, intrathecally, intraventricularly, or intracerebrally. In some embodiments, the method further comprises: after administration of the dsRNA agent, evaluating the inhibition of the SNCA gene, wherein the means for evaluation include: (i) identifying one or more physiological characteristics of an SNCA-related disease or condition in the subject, and (ii) comparing the identified physiological characteristics with pre-treatment baseline physiological characteristics of the SNCA-related disease or condition and / or control physiological characteristics of the SNCA-related disease or condition, wherein the comparison indicates the presence or absence of one or more of inhibition of SNCA gene expression in the subject. In some implementations, SNCA gene expression can be assessed based on the level or changes in the level of any variable associated with SNCA gene expression, such as the level of SNCA mRNA, α-synuclein, or other parameters functionally related to SNCA expression levels. Decreased SNCA expression can also be indirectly assessed by characteristics of clinical synucleinopathy, such as chronic and progressive declines in motor, cognitive, behavioral, and autonomic functions, depending on the distribution of lesions in the brain.

[0135] According to another aspect of the invention, a method for treating a disease or condition associated with the presence of SNCA protein is provided, the method comprising: administering to a subject an effective amount of any of the aforementioned embodiments of the dsRNA agent of the invention or any of the aforementioned embodiments of the composition of the invention to inhibit SNCA gene expression. In some embodiments, the disease or condition is an SNCA-related disease. In some embodiments, the disease or condition is a synucleinopathy. In some implementation schemes, the diseases, disorders, or conditions associated with SNCA are selected from: Parkinson's disease (PD), multiple system atrophy (MSA), Lewy body dementia (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, dementia pugilistica, chromosome 17-related Parkinson's syndrome, Lytico-Bodig disease, tangle predominant dementia, argyrophilic grain disease, silver-philic granulosis, gangliocytoma, meningioma, subacute sclerotic encephalitis, lead-poisoning encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease. Diseases, lipofuscin deposition, cortical basal ganglia degeneration, frontotemporal dementia, frontotemporal lobe degeneration, Alzheimer's disease, Huntington's disease, Down's syndrome, psychosis, schizophrenia and / or Crotzfeldt-Jab disease, or other diseases associated with SNCA expression.

[0136] In some embodiments, the method further includes administering an additional treatment to the subject. In some embodiments, the additional treatment includes treatment for SNCA-related diseases or conditions. In some embodiments, the additional treatment includes administering one or more of the present invention's SNCA antisense polynucleotides to the subject, administering a non-SNCA dsRNA therapeutic agent to the subject, and behavioral alterations in the subject. In some embodiments, the additional treatment is selected from the following categories: antipruritics, astringents, local anesthetics, anti-inflammatory agents, cholinesterase inhibitors, muscarinic agonists, antioxidants, or anti-inflammatory drugs.In some implementation schemes, additional treatment options include one or more of the following: carbidopa-levodopa, levodopa, entacapone, tolcapone, opicapone, pramipexole, ropinirole, apomorphine, rotigotine, selegiline, rasagiline, safinamide, amantadine, istradefylline, trihexyphenidyl, benztropine, rivastigmine, donepezil, galantamine, acetyl-L-carnitine, vinpocetine, and huperzine. A), alpha-lipoic acid, vitamin E, rhodiola, biotin, reminyl, tacrine (Cognex), selegiline, physostigmine, revistigmin, donepezil, rivastigmine (Exelon), metrifonate, milameline, xanomeline, saeluzole, idebenone, ENA-713, mermic, quetiapine, neurostrol, idebenone, propentofylline, neuromidal, and memantine, as well as physical, occupational, and speech therapies, including movement programs for cardiopulmonary, resistance, flexibility, and gait and balance exercises, and deep brain stimulation (DBS) involving the implantation of electrodes into target brain regions. In some embodiments, dsRNA agents are administered subcutaneously to the subject. In some embodiments, the dsRNA agent is administered to the subject via intravenous (IV) administration. In some embodiments, the dsRNA agent is administered to the subject intracranially, intrathecally, intraventricularly, or intracerebrally. Intrathecal administration of bifilar RNAi agents can reduce the expression of SNCA target genes in brain (e.g., striatum) or spinal cord tissue (e.g., cortex, cerebellum, cervical, lumbar, and thoracic vertebrae).In some embodiments, the dsRNA agent and non-SNCA dsRNA therapeutic agent may be administered simultaneously and / or in the same combination, or the non-SNCA dsRNA therapeutic agent may be administered as part of a separate composition or at separate times and / or by other methods known in the art or described herein. In some embodiments, the method further includes determining the potency of the administered double-stranded RNA (dsRNA) agent in the subject. In some embodiments, means of determining the potency of the treatment in the subject include: (i) determining one or more physiological characteristics of an SNCA-related disease or condition in the subject, and (ii) comparing the determined physiological characteristics to pre-treatment baseline physiological characteristics of the SNCA-related disease or condition, wherein the comparison indicates one or more of the presence, absence, and level of potency of the double-stranded RNA (dsRNA) agent administered to the subject. In some embodiments, SNCA gene expression may be assessed based on the level or change in the level of any variable associated with SNCA gene expression, such as the level of SNCA mRNA, α-synuclein, or other parameters functionally related to SNCA expression levels.

[0137] According to another aspect of the invention, a method is provided to reduce the level of SNCA protein in a subject compared to a baseline pre-treatment level, the method comprising administering to the subject an effective amount of any of the aforementioned dsRNA agents of the invention or any of the aforementioned compositions of the invention to reduce the level of SNCA gene expression. In some embodiments, the dsRNA agent is administered subcutaneously to the subject, or intracranially, intrathecally, intraventricularly, or intracerebrally, or via intravenous administration. By administering the dsRNA agent intrathecally, this method can reduce the expression of SNCA target genes in the brain (e.g., striatum) or spinal cord tissue (e.g., cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae).

[0138] According to another aspect of the invention, a method is provided to alter the physiological characteristics of an SNCA-related disease or condition in a subject compared to a pre-treatment baseline physiological characteristic. The method comprises administering to the subject an effective amount of any of the aforementioned dsRNA agents of the invention or any of the aforementioned compositions of the invention to alter the physiological characteristics of the SNCA-related disease or condition in the subject. In some embodiments, the dsRNA agent is administered subcutaneously, intracranially, intrathecally, intraventricularly, or intracerebrally, or via intravenous administration. In some embodiments, the physiological characteristic is one or more of the following: the level of SNCA mRNA, α-synuclein, or other parameters functionally related to SNCA expression levels in the subject.

[0139] According to another aspect of the invention, the aforementioned dsRNA agent is provided for use in a method of treating a disease or condition associated with the presence of SNCA protein. In some embodiments, the disease or condition is an SNCA-related disease. In some embodiments, the disease or condition is a synucleinopathy. In some implementations, the disease or condition is one or more of the following: Parkinson's disease (PD), multiple system atrophy (MSA), Lewy body dementia (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, boxing dementia, chromosomal-associated Parkinson's syndrome, Lytico-Bodig disease, tangled dominant dementia, aurophilic granuloma, aurophilic granulozoanosis, gangliocytoma, meningioma, subacute sclerosing encephalitis, lead poisoning encephalopathy, tuberous sclerosis, Hallewarden-Schpattz disease, lipofuscin deposition, corticobasal degeneration, frontotemporal dementia, frontotemporal lobe degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia and / or Crotzfeldt-Jab disease, or other diseases associated with SNCA expression.

[0140] According to another aspect of the invention, an antisense polynucleotide agent for inhibiting SNCA protein expression is provided, the agent comprising 10 to 30 consecutive nucleotides, wherein at least one of the consecutive nucleotides is a modified nucleotide, and wherein the nucleotide sequence of the agent has approximately 80% complementarity over its full length with an equivalent region of the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the equivalent region is any of the target regions of SEQ ID NO: 1, and the complementary sequence is a sequence provided in one of Tables 1 to 3. In some embodiments, the antisense polynucleotide agent comprises one of the antisense sequences provided in one of Tables 1 to 3.

[0141] According to another aspect of the invention, compositions are provided comprising any of the aforementioned antisense polynucleotide pharmaceutical agents. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises one or more additional therapeutic agents for treating SNCA-related diseases or conditions. In some embodiments, the composition is packaged in a kit, container, packaging, dispenser, pre-filled syringe, or vial. In some embodiments, the composition is formulated for subcutaneous, intrathecal, intracranial, intraventricular, or intracerebral administration, or intravenously.

[0142] According to another aspect of the invention, embodiments of cells comprising any of the aforementioned antisense polynucleotide agents are provided. In some embodiments, the cells are mammalian cells, optionally human cells.

[0143] According to another aspect of the invention, a method for inhibiting SNCA gene expression in cells is provided, the method comprising: (i) preparing cells containing an effective amount of any of the aforementioned antisense polynucleotide agents according to an embodiment. In some embodiments, the method further comprises (ii) maintaining the cells prepared in (i) for a time sufficient to allow degradation of the mRNA transcript of the SNCA gene, thereby inhibiting the expression of the SNCA gene in the cells.

[0144] According to another aspect of the invention, a method for inhibiting SNCA gene expression in a subject is provided, the method comprising administering an effective amount of any of the aforementioned antisense polynucleotide agents to the subject.

[0145] According to another aspect of the invention, a method for treating a disease or condition associated with the presence of SNCA protein, the method comprising administering to a subject an effective amount of any of the aforementioned antisense polynucleotide agents of the invention or any of the aforementioned compositions to inhibit SNCA gene expression. In some embodiments, the disease or condition is an SNCA-related disease. In some embodiments, the disease or condition is a synucleinopathy. In some implementations, the disease or condition is one or more of the following: Parkinson's disease (PD), multiple system atrophy (MSA), Lewy body dementia (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, boxing dementia, chromosomal-associated Parkinson's syndrome, Lytico-Bodig disease, tangled dominant dementia, aurophilic granuloma, aurophilic granulozoanosis, gangliocytoma, meningioma, subacute sclerosing encephalitis, lead poisoning encephalopathy, tuberous sclerosis, Hallewarden-Schpattz disease, lipofuscin deposition, corticobasal degeneration, frontotemporal dementia, frontotemporal lobe degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia and / or Crotzfeldt-Jab disease, or other diseases associated with SNCA expression.

[0146] According to another aspect of the invention, a method is provided for reducing SNCA protein levels in a subject compared to baseline pre-treatment levels, the method comprising administering to the subject an effective amount of any of the aforementioned antisense polynucleotide agents or any of the aforementioned compositions of the invention to reduce SNCA gene expression levels. In some embodiments, the antisense polynucleotide agent is administered to the subject subcutaneously, intracranially, intrathecally, intracardiacly, or intracerebrally, or via intravenous administration.

[0147] According to another aspect of the invention, an antisense polynucleotide agent for inhibiting SNCA gene expression is provided, the agent comprising 10 to 30 consecutive nucleotides, wherein at least one of the consecutive nucleotides is a modified nucleotide, and wherein the nucleotide sequence of the agent has about 80% or about 85% complementarity over its full length with the equivalent region of the nucleotide sequence of SEQ ID NO: 1.

[0148] According to another aspect of the invention, a method is provided to alter the physiological characteristics of an SNCA-related disease or condition in a subject compared to a pre-treatment baseline physiological characteristic, the method comprising administering to the subject an effective amount of any of the aforementioned antisense polynucleotide agents or any of the aforementioned compositions of the invention to alter the physiological characteristics of the SNCA disease or condition in the subject. In some embodiments, the antisense polynucleotide agent is administered to the subject subcutaneously, intrathecally, or via IV. In some embodiments, the physiological characteristic is one or more of the following: the level of SNCA mRNA, α-synuclein, or other parameters functionally related to SNCA expression levels, etc.

[0149] Sequence Summary

[0150] SEQ ID NO: 1 and SEQ ID NO: 2 (reverse complements) are Homo sapiens synuclein α (SNCA) mRNA [NCBI reference sequence: NM_000345.4].

[0151] SEQ ID NO: 3 and SEQ ID NO: 4 (reverse complement) are predicted synuclein α (SNCA) mRNAs from cynomolgus macaques [NCBI reference sequence: XM_005555422.3].

[0152] SEQ ID NO: 5 and SEQ ID NO: 6 (reverse complement) are predicted rhesus monkey (Macaca mulatta) synuclein α (SNCA) mRNA [NCBI reference sequence: XM_015138783.2].

[0153] SEQ ID NO: 7 and SEQ ID NO: 8 (reverse complements) are synuclein α (SNCA) mRNA from brown rat (Rattus norvegicus) [NCBI reference sequence: NM_019169.3].

[0154] SEQ ID NO: 9 to 368, 1861 to 1862 are shown in Table 1 and are sense chain sequences.

[0155] SEQ ID NO: 369 to 728, 1863 to 1864 are shown in Table 1 and are antisense sequences.

[0156] Chemically modified SEQ ID NOs: 729 to 1088, 1355 to 1860 are shown in Table 2.

[0157] SEQ ID NOs: 1089 to 1202, 1203 to 1354 are shown in Table 3. The delivery molecule is indicated as “GLX-_” at the 3' or 5' end of each sense strand. Detailed Implementation

[0158] This invention partially includes RNAi agents, such as, but not limited to, double-stranded (ds) RNAi agents, which are capable of inhibiting the expression of the synuclein α (SNCA) gene. This invention also partially includes compositions comprising SNCA RNAi agents and methods of using the compositions. The SNCA RNAi agents disclosed herein can be linked to a delivery compound for delivery to cells, including CNS (e.g., brain) cells and hepatocytes. Pharmaceutical compositions of this invention may comprise at least one dsRNAi SNCA agent and a delivery compound. In some embodiments of the compositions and methods of this invention, the delivery compound is a delivery compound comprising GalNAc. SNCA RNAi agents delivered to cells can inhibit SNCA gene expression, thereby reducing the activity of the SNCA protein product of this gene in cells. The dsRNAi agents of this invention can be used to treat SNCA-related diseases and conditions.

[0159] In some embodiments of the invention, reducing SNCA expression in cells or objects treats diseases or conditions associated with SNCA expression in cells or objects. In some embodiments, dsRNA leads to a reduction in SNCA gene mRNA in brain (e.g., striatum) or spinal cord tissue (e.g., hippocampus, striatum, cortex, cerebellum, thalamus, hypothalamus, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae, as well as the spinal cord). Some non-limiting examples of diseases and conditions that can be treated by reducing SNCA activity include: Parkinson's disease (PD), multiple system atrophy (MSA), Lewy body dementia (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, boxing dementia, chromosomal-related Parkinson's syndrome, Lytico-Bodig disease, tangled dominant dementia, aurophilic granuloma, aurophilic granulozoanosis, gangliocytoma, meningioma, subacute sclerosing encephalitis, lead poisoning encephalopathy, tuberous sclerosis, Hallewarden-Schpattz disease, lipofuscin deposition, corticobasal degeneration, frontotemporal dementia, frontotemporal lobe degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia and / or Crotzfeldt-Jab disease, or other diseases where reducing SNCA protein levels and activity is medically beneficial.

[0160] As used herein, “G,” “C,” “A,” and “U” typically represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. However, it should be understood that the terms “ribonucleotide” or “nucleotide” can also refer to modified nucleotides, or alternative substitutions, as further detailed below. Those skilled in the art will understand that guanine, cytosine, adenine, and uracil can be substituted with other portions without substantially altering the base-pairing characteristics of the oligonucleotide containing such a substitution. For example, but not limited to, a nucleotide containing inosine as its base can base-pair with a nucleotide containing adenine, cytosine, or uracil. Therefore, in the nucleotide sequences of this invention, nucleotides containing uracil, guanine, or adenine can be substituted with nucleotides containing, for example, inosine. Sequences containing such substitutions are some embodiments of this invention.

[0161] Unless otherwise stated, the term "synuclein α," used herein and interchangeably with "SNCA," refers to the naturally occurring gene encoding synuclein α from any vertebrate or mammalian source, including but not limited to humans, cattle, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys, and guinea pigs. The term also refers to fragments and variants of natural SNCA that maintain at least one in vivo or in vitro activity of natural SNCA. The amino acid sequence and complete coding sequence of the human SNCA gene reference sequence can be found, for example, in GenBank Ref Seq accession No. NM_000345.4 (SEQ ID NO: 1 and SEQ ID NO: 2). Mammal orthologs of the human SNCA gene can be found, for example, in the following: GenBank Ref Seq Accession No. XM_005555422.3, cynomolgus macaque (SEQ ID NO: 3 and SEQ ID NO: 4); GenBank Ref Seq Accession No. XM_015138783.2, rhesus monkey (SEQ ID NO: 5 and SEQ ID NO: 6); GenBank Ref Seq Accession No. NM_019169.3, brown rat (SEQ ID NO: 7 and SEQ ID NO: 8). Other examples of SNCAmRNA sequences are readily available from publicly available databases such as GenBank, UniProt, Ensembl, and OMIM. Those skilled in the art will understand that replacing T with U in any target sequence or mRNA transcript (e.g., SEQ ID NO: 1 to SEQ ID NO: 8) is not considered a difference.

[0162] The following describes how to prepare and use compositions containing SNCA single-stranded (ssRNA) and dsRNA agents to inhibit SNCA gene expression, as well as compositions and methods for treating diseases and conditions caused by or regulated by SNCA gene expression. The term "RNAi" is also known in the art and may be referred to as "siRNA".

[0163] As used herein, the term "RNAi" refers to an agent that contains RNA and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. As known in the art, an RNAi target region, also defined as a "target region" or "target moiety," refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during gene transcription, including messenger RNA (mRNA), a product of RNA processing of primary transcripts. The target moiety or target region of the sequence will be at least long enough to serve as a substrate for targeted RNAi cleavage at or near that portion. Target sequences can be 8 to 30 nucleotides long (including end values), 10 to 30 nucleotides long (including end values), 12 to 25 nucleotides long (including end values), 15 to 23 nucleotides long (including end values), 16 to 23 nucleotides long (including end values), or 18 to 23 nucleotides long (including end values), encompassing all shorter lengths within each specified range. In some embodiments of the invention, the target sequence is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides long. In some embodiments, the target sequence is 9 to 26 nucleotides long (including end values), including all subranges and integers therebetween. For example, although not intended to be limiting, in some embodiments of the invention, the target sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long, wherein the sequence is completely or at least substantially complementary to at least a portion of the RNA transcript of the SNCA gene. Some aspects of the invention include pharmaceutical compositions comprising one or more SNCA dsRNA agents and pharmaceutically acceptable carriers. In some embodiments of the invention, SNCA RNAi, as described herein, inhibits the expression of SNCA proteins.

[0164] As used herein, “dsRNA agent” means a composition of RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that are capable of degrading or inhibiting the translation of messenger RNA (mRNA) transcripts of target mRNA in a sequence-specific manner. While not wishing to be limited by any particular theory, the dsRNA agents of the present invention may function through RNA interference mechanisms (i.e., by inducing RNA interference through interaction with RNA interference pathways in mammalian cells, such as RNA-induced silencing complexes or RISC), or through any alternative mechanism or pathway. Methods for silencing genes in plant, invertebrate, and vertebrate cells are well known in the art [see, for example, (Sharp et al., Genes Dev. 2001, 15:485; Bernstein, et al., (2001) Nature 409:363; Nykanen, et al., (2001) Cell107:309; and Elbashir, et al., (2001) Genes Dev. 15:188)], the disclosures of which are incorporated herein by reference in their entirety. Gene silencing procedures known in the art can be used in conjunction with the disclosures provided herein to suppress SNCA expression.

[0165] The dsRNA agents disclosed herein consist of a sense strand and an antisense strand, and include, but are not limited to, short interfering RNA (siRNA), RNAi agents, microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrates. The antisense strand of the dsRNA agents described herein is at least partially complementary to the targeted mRNA. It is understood in the art that dsRNA duplex structures of different lengths can be used to repress target gene expression. For example, dsRNAs with duplex structures of 19, 20, 21, 22, and 23 base pairs are known to effectively induce RNA interference (Elbashire et al., EMBO 2001, 20:6877-6888). It is also known in the art that shorter or longer RNA duplex structures also effectively induce RNA interference. In some embodiments, the sense strand and antisense strand may be of the same or different lengths. In some embodiments, each strand is no more than 40 nucleotides in length. In some embodiments, the length of each strand does not exceed 30 nucleotides. In some embodiments, the length of each strand does not exceed 25 nucleotides. In some embodiments, the length of each strand does not exceed 23 nucleotides. In some embodiments, the length of each strand does not exceed 21 nucleotides. In some embodiments, the length of the sense and antisense strands of the RNAi agent can each be 15 to 49 nucleotides. In some embodiments, the length of the antisense strand is independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the sense strand is independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides. The terms “double-stranded region,” “double-stranded area,” and “complementary region” as used herein are used interchangeably and refer to regions where the sense strand and antisense strand are complementary or substantially complementary, as is known in the art. In some embodiments, both the sense strand and antisense strand are 21 nucleotides long. In some embodiments, the sense strand and antisense strand are complementary or substantially complementary, and the length of the complementary region is 15 to 23 nucleotides. In some embodiments, the length of the complementary region is 19 to 21 nucleotides. In some implementations, the length of the complementary region is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.In some embodiments of the invention, the SNCA dsRNA may comprise at least one strand of a minimum length of 21 nt, or, compared to the dsRNAs shown in Tables 1 to 3, may have a shorter duplex based on one of the sequences shown in any of Tables 1 to 3, but which is also effective by subtracting 1, 2, 3, or 4 nucleotides from one or both ends. In some embodiments of the invention, the SNCA dsRNA agents may have partial sequences of at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotides from one or more sequences in Tables 1 to 3, and their ability to inhibit SNCA gene expression differs from the inhibition level produced by dsRNAs containing the complete sequence by no more than 5%, 10%, 15%, 20%, 25%, or 30%. The sense sequences, antisense sequences, and duplexes disclosed in Tables 1 to 3 may be referred to herein as “parental” sequences, meaning that the sequences disclosed in Tables 1 to 3 may be modified, shortened, lengthened, included with substitutions, etc., as shown herein, wherein the resulting sequences retain all or at least part of the potency of their parental sequences in the methods and compositions of the invention. The sense and antisense strands included in the dsRNA of the present invention are independently selected. The term "independently selected" as used herein means that two or more similar elements are each selected independently of the selection of other elements. For example, although not intended to be limiting, in preparing the dsRNA of the present invention, "elements" of both strands may be selected to be included in the duplex. One selected element, the sense sequence, may be SEQ ID NO: 729 (shown in Table 2), and the other selected element, the antisense sequence, may be SEQ ID NO: 909, or may be SEQ ID NO: 909 modified, shortened, lengthened, and / or containing one, two, or three substitutions compared to its parental sequence SEQ ID NO: 909. It should be understood that the duplex of the present invention does not need to include both the sense and antisense sequences shown as duplex pairings in Tables 1 to 3. Each sense and antisense sequence in the tables is immediately followed by its SEQ ID NO.

[0166] Some embodiments of the compositions and methods of the present invention include single-stranded RNA in the composition and / or applied to a subject. For example, the antisense strand, such as those listed in any of Tables 1 to 3, may be a composition applied to a subject or in a composition applied to a subject to reduce the activity of the SNCA polypeptide and / or the expression of the SNCA gene in the subject. Table 1 shows the core extended base sequences of the antisense and sense strands of certain SNCA dsRNA agents. Single-stranded antisense molecules that may be included in certain compositions of the present invention and / or applied in certain methods of the present invention are referred to herein as “single-stranded antisense agents” or “antisense polynucleotide agents”. Single-stranded sense molecules that may be included in certain compositions of the present invention and / or applied in certain methods of the present invention are referred to herein as “single-stranded sense agents” or “sense polynucleotide agents”. The term “base sequence” as used herein refers to a polynucleotide sequence without chemical modification or delivery of a compound. For example, the sense strands shown in Table 1 (SEQ ID NO: 22) is the base sequence of SEQ ID NO: 742 in Table 2 and SEQ ID NO: 1106 in Table 3, wherein SEQ ID NO: 742 and SEQ ID NO: 1106 are shown together with their chemically modified and delivery compounds. The sequences disclosed herein may be assigned identifiers. For example, a single-stranded sense sequence may be identified as “sense strand SS#”; a single-stranded antisense sequence may be identified as “antisense strand AS#”; and a duplex comprising a sense strand and an antisense strand may be identified as “duplex AD# / AV#”.

[0167] Table 1 includes sense and antisense strands and provides identification numbers for duplexes formed by sense and antisense strands in the same row of Table 1. In some embodiments of the invention, the antisense sequence contains nucleobase u or nucleobase a at position 1 of the antisense sequence. In some embodiments of the invention, the antisense sequence contains nucleobase u at position 1 of the antisense sequence. The term "matching position" in sense and antisense strands as used herein refers to the position in each strand that is "paired" when the two strands are duplexes. For example, in a 21-nucleobase sense strand and a 21-nucleobase antisense strand, the nucleobase at position 1 of the sense strand and the nucleobase at position 21 of the antisense strand are at a "matching position". In another non-limiting example, in a 23-nucleobase sense strand and a 23-nucleobase antisense strand, the nucleobase 2 of the sense strand and the nucleobase at position 22 of the antisense strand are at a matching position. In another non-limiting example, in an 18-base sense strand and an 18-base antisense strand, the nucleus at position 1 of the sense strand and nucleus 18 of the antisense strand are in a matching position, and nucleus 4 of the sense strand and nucleus 15 of the antisense strand are in a matching position. Those skilled in the art will understand how to identify the matching positions in the sense and antisense strands of a double-stranded strand and its paired strands.

[0168] The first column of Table 1 represents a duplex AV# containing both the meaningful and antisense sequences from the same row. For example, Table 1 discloses a duplex assigned AV# AV03096.um, which contains a meaningful strand SEQ ID NO:22 and an antisense strand SEQ ID NO:382. Therefore, each row in Table 1 identifies a duplex of the present invention, each containing both the meaningful and antisense sequences shown in the same row, wherein the identifier assigned to each duplex is shown in the first column of that row.

[0169] In some embodiments of the method of the present invention, an RNAi agent comprising any of the polynucleotide sequences shown in Tables 1 to 3 is applied to a target. In some embodiments of the present invention, the RNAi agent applied to the target comprises a double strand comprising at least one of the base sequences shown in Table 1, comprising sequence modifications of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments of the method of the present invention, an RNAi agent comprising any of the polynucleotide sequences shown in Tables 1 to 3 is linked to a delivery molecule, a non-limiting example of which is a delivery compound comprising a GalNAc compound or a GLS-15* compound.

[0170] Table 1: Antisense and sense sequences of unmodified SNCA RNAi agents. All sequences are shown in the 5' to 3' orientation. The duplex AV# is the number of the duplex assigned to the two strands in the same row of the table.

[0171]

[0172]

[0173] Table 2 shows the antisense and sense sequences of certain chemically modified SNCA RNAi agents of the present invention. In some embodiments of the method of the present invention, the RNAi agent having the polynucleotide sequence shown in Table 2 is applied to cells and / or objects. In some embodiments of the method of the present invention, the RNAi agent having the polynucleotide sequence shown in Table 2 is applied to objects. In some embodiments of the present invention, the RNAi agent applied to the object comprises a duplex identified in the first column of the row in Table 2, and comprises the sequence modifications shown in the sense and antisense sequences in the third and sixth columns of the same row in Table 2, respectively. In some embodiments of the method of the present invention, the sequences shown in Table 2 may be linked (also referred to herein as “conjugated with”) to compounds capable of delivering the RNAi agent to cells and / or tissues in the object. A non-limiting example of a delivery compound that can be used in certain embodiments of the present invention is a compound containing GalNAc or a compound containing (GLS-15*). In Table 2, the first column represents the duplex AV# as shown in Table 1. Table 2 discloses the double-stranded AV# and also shows the chemical modifications contained in the sense and antisense sequences of the double-stranded AV#. For example, Table 1 shows the single-stranded base sequences SEQ ID NO: 22 (sense) and SEQ ID NO: 382 (antisense), which together are a double-stranded AV# AV03096.um, and Table 2 lists the double-stranded AV# AV03096, which indicates that the double-stranded SEQ ID NO: 742 and SEQ ID NO: 922 contain the base sequences of SEQ ID NO: 22 and SEQ ID NO: 382, ​​respectively, but have the chemical modifications shown in the sense and antisense sequences shown in columns 3 and 6, respectively. The “sense chain SS#” in column 2 of Table 2 is an identifier assigned to the sense sequence (containing modifications) shown in column 3 of the same row. The “antisense chain AS#” in column 5 of Table 2 is an identifier assigned to the antisense sequence (containing modifications) shown in column 6.

[0174] Table 2: Provides the antisense and sense sequences of chemically modified SNCA RNAi agents. All sequences are shown from 5' to 3'. These sequences were used in some of the in vitro assays described herein.

[0175]

[0176] Table 3 shows the antisense and sense strand sequences of certain chemically modified SNCA RNAi agents of the present invention. In some embodiments of the method of the present invention, the RNAi agents shown in Table 3 are applied to cells and / or subjects. In some embodiments of the method of the present invention, RNAi agents having the polynucleotide sequences shown in Table 3 are applied to subjects. In some embodiments of the present invention, the RNAi agent applied to the subject comprises a duplex identified in the first column of the row in Table 3, and comprises sequence-modifying and / or delivery compounds shown in the sense and antisense strand sequences in the third and sixth columns of the same row in Table 3, respectively. These sequences are used in certain in vivo assay studies described elsewhere herein. In some embodiments of the method of the present invention, the sequences shown in Table 3 may be linked (also referred to herein as “conjugated with”) to a compound for delivery, a non-limiting example of which is a compound containing GalNAc, wherein the delivery compound is identified as “GLX-n” on the sense strand in the third column of Table 3. As used in this article, “GLX-n” is used to denote “GLS-n*” or “GLO-n” delivery compounds (“X” can be “S” or “O”) that can be linked to the 3' end of an oligonucleotide during synthesis. As used herein and as shown in Table 3, “GLX-n” is used to indicate that the linked GalNAc-containing compound is any one of the following compounds: GLS-1*, GLS-2*, GLS-3*, GLS-4*, GLS-5*, GLS-6*, GLS-7*, GLS-8*, GLS-9*, GLS-10*, GLS-11*, GLS-12*, GLS-13*, GLS-14*, GLS-15*, GLS-16*, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16, the structures of which are provided elsewhere herein. Those skilled in the art will be able to prepare and use the dsRNA compounds of the present invention, wherein the attached delivery compound is one of the following: GLS-1*, GLS-2*, GLS-3*, GLS-4*, GLS-5*, GLS-6*, GLS-7*, GLS-8*, GLS-9*, GLS-10*, GLS-11*, GLS-12*, GLS-13*, GLS-14*, GLS-15*, GLS-16*, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16.

[0177] The first column of Table 3 provides the duplex AD# of the duplexes of the sense and antisense sequences assigned to the rows of this table. For example, duplex AD# AD01746 is a duplex of the sense strand SEQ ID NO: 1106 and the antisense strand SEQ ID NO: 1163. Each row in Table 3 provides the sense and antisense strands and discloses the duplexes of the sense and antisense strands shown. The “Sense Strand SS#” in the second column of Table 3 is an identifier assigned to the sense sequence (including modifications) shown in the third column of the same row. The “Antisense Strand AS#” in the fifth column of Table 3 is an identifier assigned to the antisense sequence (including modifications) shown in the sixth column. The identifiers for certain linked “GLO-n” or “GLS-n*” compounds containing GalNAc are shown as GLS-5* or GLS-15*, wherein the resulting compounds are included in embodiments of the methods and / or compositions of the present invention.

[0178] Table 3 provides the antisense and sense strand sequences of the chemically modified SNCA RNAi agents. All sequences are shown from 5' to 3'. These sequences were used in certain in vivo assays described elsewhere in this document. Delivery molecules used in in vivo studies are indicated as "GLO-n" or "GLS-n*" at the 3' or 5' end of each sense strand.

[0179]

[0180] In some embodiments of the invention, dsRNA (also referred to herein as a “double strand”) is one disclosed in one of Tables 1 to 3. Each row in Tables 1 to 3 discloses a double strand containing a sense strand sequence and an antisense strand sequence contained in that row. In addition to the double strands disclosed in Tables 1 to 3, it should be understood that in some embodiments, the double strand of the invention may comprise the sense and antisense sequences shown in Tables 1 to 3, differing from the sequences shown in Tables 1 to 3 by 0, 1, 2, or 3 nucleotides. Therefore, as a non-limiting example, in some embodiments, the antisense strand in the duplex of the present invention may be one of the following SEQ ID NO: 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, or 1170, having 0, 1, 2, or 3 different nucleotides compared to the nucleotides in SEQ ID NO: 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, or 1170.

[0181] It should be understood that the sense and antisense sequences in the duplex of the present invention can be selected independently. Therefore, the dsRNA of the present invention may comprise the sense and antisense strands of the duplexes disclosed in the rows of Tables 1 to 3. Alternatively, in the dsRNA of the present invention, one or both of the selected sense and antisense strands may comprise the sequences shown in Tables 1 to 3, but wherein one or both of the sense and antisense sequences contain one, two, three, or more nucleobase substitutions relative to the parental sequence. In some embodiments, the selected sequence may be longer or shorter than its parental sequence. Therefore, the dsRNA agents included in the present invention may, but do not necessarily, comprise the exact sequences of the sense and antisense pairs disclosed as duplexes in Tables 1 to 3.

[0182] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein nucleotides 2 to 18 of the antisense strand comprise a complementary region to the SNCA RNA transcript, wherein said complementary region comprises at least 15 consecutive nucleotides differing from one of the antisense sequences listed in one of Tables 1 to 3 by 0, 1, 2, or 3 nucleotides, and optionally comprises a targeting ligand. In some cases, the complementary region to the SNCA RNA transcript comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides differing from one of the antisense sequences listed in one of Tables 1 to 3 by no more than 3 nucleotides. In some embodiments of the dsRNA agent of the present invention, the antisense strand of the dsRNA is at least substantially complementary to any of the target regions of SEQ ID NO: 1, and is provided in any of Tables 1 to 3. In some embodiments, the antisense strand of the dsRNA agent of the present invention is completely complementary to any of the target regions of SEQ ID NO: 1, and is provided in any of Tables 1 to 3. In some embodiments, the dsRNA agent comprises a sense sequence shown in any of Tables 1 to 3, and the sense sequence is at least substantially complementary to the antisense sequence in the dsRNA agent. In other embodiments, the dsRNA agent of the present invention comprises a sense sequence shown in any of Tables 1 to 3, and the sense sequence is completely complementary to the antisense sequence in the dsRNA agent. In some cases, the dsRNA agent of the present invention comprises an antisense sequence shown in any of Tables 1 to 3. Some embodiments of the dsRNA agent of the present invention comprise a sense sequence and an antisense sequence disclosed as a duplex in any of Tables 1 to 3. As described herein, it should be understood that the sense and antisense strands in the duplex of the present invention can be selected independently.

[0183] Mismatch

[0184] Those skilled in the art know that mismatches are tolerable for efficacy in dsRNAs, especially mismatches in the terminal regions of dsRNAs. Some mismatches are more tolerable; for example, mismatches with wobble base pairs G:U and A:C are more tolerable for efficacy (Du et al., A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Res. 2005 Mar 21;33(5):1671-7. Doi: 10.1093 / nar / gki312. Nucleic Acids Res. 2005;33(11):3698). In some embodiments of the methods and compounds of the present invention, the SNCA dsRNA agent may contain one or more mismatches with the SNCA target sequence. In some embodiments, the SNCA dsRNA agent of the present invention does not contain mismatches. In some embodiments, the SNCA dsRNA agent of the present invention contains no more than one mismatch. In some embodiments, the SNCA dsRNA agent of the present invention contains no more than two mismatches. In some embodiments, the SNCA dsRNA agent of the present invention contains no more than three mismatches. In some embodiments of the present invention, the antisense strand of the SNCA dsRNA agent contains a mismatch with the SNCA target sequence that is not located at the center of the complementary region. In some embodiments, the antisense strand of the SNCA dsRNA agent contains one, two, three, four, or more mismatches that are located in the last 5, four, three, two, or one nucleotide from one or both of the 5' or 3' ends of the complementary region. The methods described herein and / or methods known in the art can be used to determine whether an SNCA dsRNA agent containing a mismatch with the SNCA target sequence effectively inhibits the expression of the SNCA gene.

[0185] Complementarity

[0186] Unless otherwise stated, the term "complementarity" as used herein, when used to describe a first nucleotide sequence (e.g., a sense strand of an SNCA dsRNA agent or targeting SNCA mRNA) associated with a second nucleotide sequence (e.g., an antisense strand of an SNCA dsRNA agent or a single-stranded antisense polynucleotide), means the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleotide sequence [forming base-pair hydrogen bonds under mammalian physiological conditions (or similar in vitro conditions)] and to form a double-stranded or double-helix structure under certain conditions. Other conditions may also apply, such as physiologically relevant conditions that can be encountered in vivo. Those skilled in the art will be able to determine the set of conditions most suitable for testing the complementarity of the two sequences based on the final application of the hybridized nucleotides. At least to the extent that the hybridization requirements described above are met, the complementary sequence contains Watson-Crick base pairs or non-Watson-Crick base pairs and contains native or modified nucleotides or nucleotide mimics. Sequence identity or complementarity is independent of modification.

[0187] Complementary sequences, such as those within SNCA dsRNA as described herein, comprise base pairings of an oligonucleotide or polynucleotide containing a first nucleotide sequence along the full length of one or two nucleotide sequences with an oligonucleotide or polynucleotide containing a second nucleotide sequence. Such sequences may be referred to herein as “perfectly complementary” with respect to each other. It should be understood that in some embodiments, when two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs are not considered mismatches with respect to defined complementarity herein. For example, an SNCA dsRNA agent comprising an oligonucleotide of 19 nucleotides in length and another oligonucleotide of 20 nucleotides in length, wherein the longer oligonucleotide contains a 19-nucleotide sequence perfectly complementary to the shorter oligonucleotide, may still be referred to as “perfectly complementary” for the purposes described herein. Therefore, “perfectly complementary” as used herein means that all (100%) bases in the sequential sequence of the first polynucleotide will hybridize with the same number of bases in the sequential sequence of the second polynucleotide. The sequential sequence may comprise all or a portion of the first or second nucleotide sequence.

[0188] The term “substantially complementary” as used herein means that, in the hybridized nucleobase sequence pairs, at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not all, of the bases in the first polynucleotide sequence will hybridize with the same number of bases in the second polynucleotide sequence. The term “substantially complementary” can be used to refer to the first sequence relative to the second sequence if the two sequences, when hybridizing into a duplex of up to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs (bp), contain one or more (e.g., at least 1, 2, 3, 4, or 5) mismatched base pairs, while simultaneously retaining the ability to hybridize under conditions most relevant to its final application (e.g., repressing SNCA gene expression via a RISC pathway).

[0189] The term "partial complementarity" may be used herein to refer to hybridized nucleobase sequence pairs in which at least 75% but not all of the bases in the sequential sequence of the first polynucleotide will hybridize with the same number of bases in the sequential sequence of the second polynucleotide. In some embodiments, "partial complementarity" means that at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bases in the sequential sequence of the first polynucleotide will hybridize with the same number of bases in the sequential sequence of the second polynucleotide.

[0190] The terms “complementary,” “fully complementary,” “substantially complementary,” and “partially complementary” are used herein to refer to base matching between the sense and antisense strands of an SNCA dsRNA agent, between the antisense strand of an SNCA dsRNA agent and the target SNCA mRNA sequence, or between a single-stranded antisense oligonucleotide and the target SNCA mRNA sequence. It should be understood that the term “antisense strand of an SNCA dsRNA agent” can refer to the same sequence as an “SNCA antisense polynucleotide agent.”

[0191] The terms "substantially identical" or "substantially identical" as used herein when referring to nucleic acid sequences mean a nucleic acid sequence that has at least about 85% sequence identity or more, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to a reference sequence. The percentage of sequence identity is determined by comparing two best-aligned sequences within a comparison window. The percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base occurs to generate a number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The invention disclosed herein covers nucleotide sequences that are substantially identical to the nucleotide sequences disclosed herein (e.g., in Tables 1 to 3). In some implementations, the sequences disclosed herein are identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the sequences disclosed herein (e.g., in Tables 1 to 3).

[0192] As used herein, the term "chain containing a sequence" refers to an oligonucleotide containing a nucleotide chain described by a sequence referenced using standard nucleotide nomenclature. The term "double-stranded RNA" or "dsRNA" as used herein refers to RNAi containing an RNA molecule or molecular complex having a hybrid double-stranded region comprising two antiparallel and substantially or completely complementary nucleic acid strands, described as having a "sense" and "antisense" orientation relative to the target SNCA RNA. The double-stranded region can be of any length allowing for specific degradation of the desired target SNCA RNA via a RISC pathway, but is typically 9 to 30 base pairs long, for example, 15 to 30 base pairs in length. Consider duplexes of 9 to 30 base pairs, which can be of any length within that range, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and any subranges thereof, including but not limited to 15 to 30 base pairs, 15 to 26 base pairs, 15 to 23 base pairs, 15 to 22 base pairs, 15 to 21 base pairs, 15 to 20 base pairs, 15 to 19 base pairs, 15 to 18 base pairs, 15 to 17 base pairs, 18 to 30 base pairs, 18 to 26 base pairs, 18... Up to 23 base pairs, 18 to 22 base pairs, 18 to 21 base pairs, 18 to 20 base pairs, 19 to 30 base pairs, 19 to 26 base pairs, 19 to 23 base pairs, 19 to 22 base pairs, 19 to 21 base pairs, 19 to 20 base pairs, 20 to 30 base pairs, 20 to 26 base pairs, 20 to 25 base pairs, 20 to 24 base pairs, 20 to 23 base pairs, 20 to 22 base pairs, 20 to 21 base pairs, 21 to 30 base pairs, 21 to 26 base pairs, 21 to 25 base pairs, 21 to 24 base pairs, 21 to 23 base pairs, or 21 to 22 base pairs. SNCA dsRNA agents produced in cells by treatment with cleatase and similar enzymes are typically 19 to 22 base pairs in length. One strand of the duplex region of the SNCA dsDNA agent contains a sequence substantially complementary to the target SNCA RNA region. The two strands forming the duplex structure can originate from a single RNA molecule having at least one self-complementary region, or can be formed from two or more separate RNA molecules. When the duplex region is formed from the two strands of a single molecule, the molecule may have a duplex region separated by a single-stranded nucleotide chain (referred to herein as a “hairpin loop”) between the 3’ end of one strand forming the duplex structure and the 5’ end of the corresponding other strand.In some embodiments of the invention, the hairpin loop comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more unpaired nucleotides. When the two substantially complementary strands of an SNCA dsRNA agent are composed of separate RNA molecules, those molecules do not need to be but can be covalently linked. When the two strands are covalently linked by means other than a hairpin loop, the linking structure is referred to as a “connector”. The term “siRNA” is also used herein to refer to the dsRNA agent as described herein.

[0193] In some embodiments of the present invention, the SNCA dsRNA agent may contain sense and antisense sequences at one or both ends of the dsRNA agent having no unpaired nucleotides or nucleotide analogs. Ends without unpaired nucleotides are referred to as "blunt ends" and have no nucleotide overhangs. If both ends of the dsRNA agent are blunt ends, the dsRNA is referred to as "blunt-terminated". In some embodiments of the present invention, the first end of the dsRNA agent is blunt-terminated; in some embodiments, the second end of the dsRNA agent is blunt-terminated; and in some embodiments of the present invention, both ends of the SNCA dsRNA agent are blunt-terminated.

[0194] In some embodiments of the dsRNA agent of the present invention, the dsRNA does not have one or two blunt ends. In such cases, there is at least one unpaired nucleotide at the end of the strand of the dsRNA agent. For example, a nucleotide overhang is present when the 3' end of one strand of the dsRNA extends beyond the 5' end of another strand, or vice versa. The dsRNA may contain overhangs having at least 1, 2, 3, 4, 5, 6 or more nucleotides. The nucleotide overhangs may contain or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. It should be understood that in some embodiments, the nucleotide overhangs are on the sense strand of the dsRNA agent, on the antisense strand of the dsRNA agent, or at both ends of the dsRNA agent, and the nucleotides of the overhangs may be present at the 5' end, 3' end, or both ends of the antisense strand or sense strand of the dsRNA. In some embodiments of the present invention, one or more nucleotides of the overhang are replaced by nucleoside phosphate thioesters.

[0195] As used herein, the terms "antisense strand" or "guide strand" refer to a strand of an SNCA dsRNA agent that contains a region substantially complementary to the SNCA target sequence. The terms "sense strand" or "follower strand" refer to a strand of an SNCA dsRNA agent that contains a region substantially complementary to the antisense strand of the SNCA dsRNA agent.

[0196] Modification

[0197] In some embodiments of the invention, the RNA of the SNCA RNAi agent is chemically modified to enhance stability and / or one or more other beneficial characteristics. The nucleic acids in some embodiments of the invention can be synthesized and / or modified by methods well established in the art, for example those described in “Current protocols in Nucleic Acid Chemistry,” Beaucage, SL et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. Modifications that may be present in certain embodiments of the SNCA dsRNA reagent of the present invention include, for example: (a) terminal modifications, such as 5' terminal modifications (phosphorylation, conjugation, reverse bonding, etc.), 3' terminal modifications (conjugation, DNA nucleotides, reverse bonding, etc.); (b) base modifications, such as substitution with a stable base, destabilizing a base, or a base paired with an extended partner library base, removal of a base (base-free nucleotide), or conjugation of a base; (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitution; and (d) backbone modifications, including modification or substitution of phosphodiester bonds. Specific examples of RNA compounds that may be used in certain embodiments of the SNCA dsRNA reagent, SNCA antisense polynucleotide, and SNCA sense polynucleotide of the present invention include, but are not limited to, RNA comprising a modified backbone or non-natural internucleotide bonds. As a non-limiting example, RNA having a modified backbone may not have a phosphorus atom in its backbone. RNA that does not have a phosphorus atom in its internucleotide backbone may be referred to as an oligonucleotide. In some embodiments of the present invention, the modified RNA has phosphorus atoms in its internucleotide backbone.

[0198] It should be understood that the terms "RNA molecule" or "RNA" or "ribonucleic acid molecule" cover not only RNA molecules expressed or found in nature, but also analogs and derivatives of RNA containing one or more ribonucleotide / ribonucleoside analogs or derivatives as described herein or known in the art. The terms "ribonucleoside" and "ribonucleotide," "nucleoside" and "nucleotide" are used interchangeably herein. RNA molecules may be modified in their nucleobase structure or ribose-phosphate backbone structure (e.g., as described below), and molecules containing ribonucleoside analogs or derivatives must retain the ability to form double strands. As some non-limiting examples, the RNA molecule may also contain at least one modified ribonucleoside, including but not limited to 2'-O-methyl modified ribonucleoside, ribonucleoside containing a 5'-thiophosphate group, terminal ribonucleoside linked to a cholesterol derivative or a dodecanoic acid bis(decanoic acid) group, locked ribonucleoside, baseless ribonucleoside, 2'-deoxy-2'-fluoro modified ribonucleoside, 2'-amino modified ribonucleoside, 2'-alkyl modified ribonucleoside, 5'-phosphonate modified ribonucleoside, morpholino ribonucleoside, aminophosphate ester, or ribonucleoside containing a non-natural base, or any combination thereof. In some embodiments of the invention, the RNA molecule contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more ribonucleosides whose full length is a modified ribonucleoside. For each of such multiple modified ribonucleosides in the RNA molecule, the modification need not be the same.

[0199] In some embodiments, the dsRNA agents, SNCA antisense polynucleotides, and / or SNCA sense polynucleotides of the present invention may comprise one or more independently selected modified nucleotides and / or one or more independently selected non-phosphodiester bonds. The terms “nucleotide-to-nucleotide bond,” “nucleoside-to-nucleotide bond,” “bond,” “backbone bond,” and “linker” as used herein are used interchangeably and refer to the linking group in the backbone of the dsRNA of the present invention, which may specifically indicate a bond between one and / or more unmodified or modified nucleosides and one or more residues in an oligonucleotide chain, and / or between an unmodified or modified nucleoside and one or more target groups. In some embodiments, the bond may be independently selected from phosphodiester (PO) bonds, phosphate thioester (PS) bonds, and / or phosphate dithioester (PS2) bonds of dinucleotides at any position in a single-stranded or double-stranded oligonucleotide. The term “independently selected” as used herein when referring to selected elements (e.g., modified nucleotides, non-phosphodiester bonds, etc.) means that two or more selected elements may, but do not necessarily, be identical to each other.

[0200] As used herein, “nucleotide base,” “nucleotide,” or “nucleobase” refers to heterocyclic pyrimidine or purine compounds, which are standard components of all nucleic acids and include bases that form the nucleotides adenine, guanine, cytosine, thymine, and uracil. Nucleobases may be further modified to include, but are not intended to limit, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. The terms “ribonucleotide” or “nucleotide” may be used herein to refer to unmodified nucleotides, modified nucleotides, nucleotide analogs, or substituted moieties. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil may be substituted with other moieties without substantially altering the base-pairing properties of the oligonucleotide containing the nucleotide with such substituted moieties.

[0201] As used herein, “optional” or “optionally” means that an event or environment described later may, but does not necessarily, occur, including the possibility that the event or environment may or may not occur. For example, “C1-6 alkyl group optionally substituted with a halogen or cyano group” means that a halogen or cyano group may, but does not necessarily, be present, including cases where the alkyl group is substituted with a halogen or cyano group and cases where the alkyl group is not substituted with a halogen or cyano group.

[0202] As used herein, in the chemical structure of the compounds disclosed herein, bonds This indicates an unspecified configuration; that is, if a chiral isomer exists in the chemical structure, then the bond... It can be or or and Both configurations. Although some of the above structural formulas are described as isomers for simplicity, this disclosure may include all isomers, such as tautomers, rotational isomers, and mixtures thereof. Suitable chiral compounds include: geometric isomers, diastereomers, racemates, and enantiomers.

[0203] As used herein, the chemical formulas used in this disclosure, within the scope of the invention as described herein, are... or It can be attached to any one or more groups.

[0204] In one embodiment, the modified RNA used in the methods and compositions described herein is considered to be a peptide nucleic acid (PNA), which has the ability to form the desired double-stranded structure and allows or mediates the specific degradation of the target RNA via the RISC pathway. In some embodiments of the invention, the SNCA RNA disruptor comprises a single-stranded RNA that interacts with the target SNCA RNA sequence to guide the cleavage of the target SNCA RNA.

[0205] The modified RNA backbone may include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methylphosphonates, and other alkylphosphonates including 3'-alkylenephosphonates, as well as chiral phosphonates, hypophosphonates, aminophosphates including 3'-aminoaminophosphates and aminoalkylaminophosphates, thioaminophosphates, thioalkylphosphonates, thioalkyl phosphate triesters, and borate phosphates having normal 3'-5' linkages, analogs of their 2'-5' linkages, and those with opposite polarities in which adjacent pairs of nucleoside units are linked in a 3'-5' to 5'-3' or 2'-5'-2' manner. Various salts, mixed salts, and free acid forms are also included. Methods for preparing phosphorus-linked RNA are conventional practices in the art, and such methods can be used to prepare certain modified SNCA dsRNA agents of the present invention, certain modified SNCA antisense polynucleotides, and / or certain modified SNCA sense polynucleotides.

[0206] The modified RNA backbones that do not contain phosphorus atoms have a backbone formed by short-chain alkyl or cycloalkyl nucleosides linked together, mixed heteroatoms linked together and alkyl or cycloalkyl nucleosides linked together, or one or more short-chain heteroatoms or heterocyclic nucleosides linked together. These include those having: morpholine links (partially formed by the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thiomethacetyl backbones; methylene formacetyl and thiomethacetyl backbones; olefin-containing backbones; aminosulfonate backbones; methylene imino and methylene hydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2 component moieties. Methods for preparing phosphorus-free modified RNA backbones are conventional practices in the art, and such methods can be used to prepare certain modified SNCA dsRNA agents, certain modified SNCA antisense polynucleotides, and / or certain modified SNCA sense polynucleotides of the present invention.

[0207] In some embodiments of the invention, the RNA mimic is contained in SNCA dsRNA, SNCA antisense polynucleotides, and / or SNCA sense polynucleotides, for example, but not limited to, replacing the sugar and nucleoside links of the nucleotide units, i.e., the backbone, with novel groups. In some such embodiments, the base units are retained for hybridization with suitable SNCA nucleic acid target compounds. One such oligomeric compound, an RNA mimic exhibiting excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by a backbone containing an amide, particularly an aminoethylglycine backbone. Nucleobases are retained and directly or indirectly bound to the aza-nitrogen atoms of the backbone amide moiety. Methods for preparing RNA mimics are conventional practices in the art, and such methods can be used to prepare certain modified SNCA dsRNA agents of the present invention.

[0208] Some embodiments of the present invention comprise RNA having a phosphate thioester backbone and oligonucleotides having a heteroatomic backbone, wherein the heteroatomic backbone is particularly -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [referred to as a methylene (methylimino) or MMI backbone], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2- [wherein the native phosphodiester backbone is represented as -OPO-CH2-]. Methods for preparing RNA having a phosphate thioester backbone and oligonucleotides having a heteroatomic backbone are conventional practices in the art, and such methods can be used to prepare certain modified SNCA dsRNA agents, certain SNCA antisense polynucleotides, and / or certain SNCA sense polynucleotides of the present invention.

[0209] Modified RNA may also contain one or more substituted sugar moieties. The SNCA dsRNA, SNCA antisense polynucleotide, and / or SNCA sense polynucleotide of the present invention may contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C2 groups. 10 Alkyl or C2 to C 10 Alkenyl and ynyl groups. Some exemplary suitable modifications include O[(CH2)] n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2)n CH3)]2, where n and m are 1 to about 10. In other embodiments, the dsRNA contains one of the following at the 2' position: C1 to C 10 Lower alkyl groups, substituted lower alkyl groups, alkylaryl groups, aryl groups, O-alkylaryl or O-aryl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkylaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleaving groups, reporter groups, intercalating agents, groups used to improve the pharmacokinetic properties of SNCA dsRNA agents, or groups used to improve the pharmacodynamic properties of SNCA dsRNA agents, SNCA antisense polynucleotides and / or SNCA sense polynucleotides, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the following examples, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2. Methods for preparing modified RNA (such as those described) are conventional practices in the art, and such methods can be used to prepare certain modified SNCA dsRNA agents of the present invention.

[0210] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluorine (2'-F). Similar modifications may also be made at other positions on the RNA of the SNCA dsRNA agents, SNCA antisense polynucleotides, and / or SNCA sense polynucleotides of the present invention, particularly at the 3' terminal nucleotide or at the 3' position of the sugar in the 2'-5' linked SNCA dsRNA, SNCA antisense polynucleotide, or SNCA sense polynucleotide, and at the 5' position of the 5' terminal nucleotide. The SNCA dsRNA agents, SNCA antisense polynucleotides, and / or SNCA sense polynucleotides may also have sugar mimics, such as replacing the cyclobutyl moiety of furanopentose. Methods for preparing modified RNA (such as those described above) are conventional practices in the art, and such methods can be used to prepare certain modified SNCA dsRNA agents, SNCA antisense polynucleotides, and / or SNCA sense polynucleotides of the present invention.

[0211] In some embodiments, the SNCA dsRNA agent, SNCA antisense polynucleotide, and / or SNCA sense polynucleotide may contain nucleobase (generally referred to in the art simply as "base") modifications or substitutions. "Unmodified" or "natural" nucleobases as used herein include purine bases adenine and guanine, and pyrimidine bases thymine, cytosine, and uracil. Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-Me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives of adenine and guanine, and other alkyl derivatives, 2-propyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, Cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deadenine and 7-deadenine, and 3-deadenine and 3-deadenine. Additional nucleosides that may be included in certain embodiments of the SNCA dsRNA agent of the present invention are known in the art, see, for example: Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. Ed. Wiley-VCH, 2008; The Concise Encyclopedia Of Polymer Science and Engineering, pp. 858-859, Kroschwitz, J. L, Ed. John Wiley & Sons, 1990, English et al., Angewandte Chemie, International Edition, 1991, 30, 613, Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B., Ed., CRC Publications, 1993.Methods for preparing dsRNAs, SNCA antisense polynucleotides, and / or SNCA sense polynucleotides containing nucleobase modifications and / or substitutions (such as those described herein) are conventional practices in the art, and such methods can be used to prepare certain modified SNCA dsRNA agents, SNCA sense polynucleotides, and / or SNCA antisense polynucleotides of the present invention.

[0212] Certain embodiments of the SNCA dsRNA agents, SNCA antisense polynucleotides, and / or SNCA sense polynucleotides of the present invention comprise RNA modified to contain one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides having a modified ribose moiety containing an additional bridging link between the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. Adding locked nucleic acids to the SNCA dsRNA agents, SNCA antisense polynucleotides, and / or SNCA sense polynucleotides of the present invention can improve serum stability and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) MolCanc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). The methods for preparing dsRNA agents, SNCA antisense polynucleotides, and / or SNCA sense polynucleotides containing locked nucleic acids are conventional practices in the art, and such methods can be used to prepare certain modified SNCA dsRNA agents of the present invention.

[0213] Certain embodiments of the SNCA dsRNA compounds, sense polynucleotides, and / or antisense polynucleotides of the present invention comprise at least one modified nucleotide, wherein the at least one modified nucleotide comprises: 2'-O-methyl nucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2'3'-seco nucleotide mimic, locked nucleotide, 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholinonucleotide, and 3'-OMe nucleotide, nucleotide comprising a 5'-phosphothioester group, nucleotide comprising a vinylphosphonate, nucleotide comprising adenosine-ethylene glycol nucleic acid (GNA), and nucleotide comprising... Nucleotides containing the S-isomer of thymidine-glycol nucleic acid (GNA), nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, nucleotides containing 2'-deoxyadenosine-3'-phosphate, nucleotides containing 2'-deoxycytidine-3'-phosphate, nucleotides containing 2'-deoxyuridine-3'-phosphate, or terminal nucleotides linked to a cholesterol derivative or a dodecanoic acid bis(decanoic acid) amide group, 2'-amino-modified nucleotides, phosphoramidates, or nucleotides containing non-natural bases. In some embodiments, the SNCA dsRNA compound contains an E-vinylphosphonate nucleotide at the 5' end of the antisense strand (also referred to herein as the guide strand).

[0214] Some embodiments of the SNCA dsRNA compounds of the present invention, the 3' and 5' ends of sense polynucleotides, and / or the 3' end of antisense polynucleotides, comprise at least one modified nucleotide, wherein the at least one modified nucleotide comprises: abase-free nucleotide, ribitol, inverted nucleotide, inverted abase-free nucleotide, inverted 2'-OMe nucleotide, or inverted 2'-deoxy nucleotide. It is known to those skilled in the art that the inclusion of abase-free or inverted abase-free nucleotide at the end of an oligonucleotide enhances stability (Czauderna et al. Structural variations and stabilizing modifications of synthetic siRNAs in mammalian cells. Nucleic Acids Res. 2003;31(11):2705-2716. doi:10.1093 / nar / gkg393). In some embodiments, the SNCA dsRNA compounds comprise one or more inverted abase-free residues at the 3' or 5' end, or both the 3' and 5' ends. Some exemplary invab (inverse baseless residues) include, but are not limited to, the following:

[0215] .

[0216] Some embodiments of the SNCA dsRNA compound, the 3' and 5' ends of the sense polynucleotide, and / or the 3' end of the antisense polynucleotide of the present invention comprise at least one modified nucleotide, wherein the at least one modified nucleotide comprises: isomannitol nucleotide or a stereoisomer of said isomannitol nucleotide. Some specific examples of isomannitol nucleotide or stereoisomers of said isomannitol nucleotide include, but are not limited to:

[0217] The phrase "Olig" independently represents a polynucleotide moiety. Some exemplary isomannitol residues (imann) include, but are not limited to, the following:

[0218] .

[0219] In some embodiments, isomannitol nucleotides may also be conjugated to one or more targeting groups or delivery molecules (e.g., the GalNAc moiety).

[0220] Certain embodiments of the SNCA dsRNA compounds and antisense polynucleotides of the present invention comprise at least one modified nucleotide, wherein the at least one modified nucleotide comprises unlocked nucleic acid nucleotide (UNA) and / or glycol nucleic acid nucleotide (GNA). It is known to those skilled in the art that UNA and GNA are thermally unstable chemical modifications that can significantly improve the off-target profile of siRNA compounds (Janas, et al., Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat Commun. 2018;9(1):723. doi:10.1038 / s41467-018-02989-4; Laursen et al., Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo. Mol BioSyst. 2010;6:862–70).

[0221] Certain embodiments of the SNCA dsRNA compounds and antisense polynucleotides of the present invention further comprise a phosphate ester moiety. As used herein, the phosphate ester moiety refers to a phosphate ester group comprising a phosphate ester or phosphate ester mimic linked to the sugar moiety of the nucleotide (e.g., ribose or deoxyribose or an analogue thereof). The nucleotide comprising the phosphate ester mimic may also be defined as a phosphonate-modified nucleotide.

[0222] In some embodiments, the phosphate ester analog is 5'-vinylphosphonate (VP). In some exemplary embodiments, the vinylphosphonate of this disclosure has the following structure:

[0223] .

[0224] The vinylphosphonate of this disclosure may be linked to the antisense or sense strand of the dsRNA of this disclosure. In some preferred embodiments, the vinylphosphonate of this disclosure is linked to the antisense strand of the dsRNA, optionally at the 5' end of the antisense strand of the dsRNA.

[0225] In some embodiments, the vinylphosphonate-modified nucleotides of this disclosure have the structure of formula (IV):

[0226]

[0227] Where X is O or S;

[0228] R is hydrogen, hydroxyl, fluorine, or C. 1-20 Alkoxy (e.g., methoxy or n-hexadecyloxy);

[0229] R5' is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is E-oriented or Z-oriented (e.g., E-oriented); and

[0230] B is a nucleobase or a modified nucleobase, optionally wherein B is adenine, guanine, cytosine, thymine, or uracil.

[0231] In some embodiments, R5' is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is E-oriented. In some embodiments, R is a methoxy group, and R5' is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is E-oriented. In some embodiments, X is S, R is a methoxy group, and R5' is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is E-oriented.

[0232] Vinylphosphonate modification has also been considered for the dsRNA, compositions, and methods of this disclosure. An exemplary vinylphosphonate structure is:

[0233] .

[0234] In some embodiments, the vinylphosphonate-modified nucleotide is VPU*, which has the following structure:

[0235] .

[0236] In some embodiments, the dsRNA contains a phosphate ester or phosphate ester mimic at the 5' end nucleotide of the guide strand, wherein the phosphate ester or phosphate ester mimic fragment of the 5' end nucleotide can be represented by one of the following specific structures or stereoisomers:

[0237]

[0238]

[0239]

[0240] In many cases, protecting groups are used during the preparation of the compounds of the present invention. The term “protected” as used herein means that the indicated portion has a protecting group attached thereto. In some embodiments of the invention, the compound contains one or more protecting groups. A wide variety of protecting groups can be used in the methods of the present invention. Generally, protecting groups inert chemical functional groups to specific reaction conditions and can be attached to a molecule and can remove such functional groups from the molecule without substantially impairing the rest of the molecule. General protecting groups, and especially hydroxyl protecting groups, are well known in the art (Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991).

[0241] Examples of protecting groups (e.g., hydroxyl protecting groups) used in this article include, but are not limited to, methyl, ethyl, benzyl (Bn), phenyl, isopropyl, tert-butyl, acetyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, neopentyl, tert-butoxymethyl, methoxymethyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, allyl, cyclohexyl, 9-fluorenylmethoxycarbonyl (Fmoc), methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, benzyl Acyl, benzoyl carbamate, p-phenylbenzoyl, 4-methoxybenzyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl, trimethoxytriphenylmethyl, 4-chlorobenzyl, 4-nitrobenzyl, 2,4-dinitrophenyl, 4-acyloxybenzyl, 2-methylphenyl, 2,6-dimethylphenyl, 2-chlorophenyl, 2,6-dichlorobenzyl, diphenylmethyl, triphenylmethyl, 4-methylthio-1-butyl, S-acetylthioacetate (SATA), 2- Cyanoethyl, 2-cyano, 1-dimethylethyl (CDM), 4-cyano-2-butenyl, 2-(trimethylsilyl)ethyl (TSE), 2-(phenylthio)ethyl, 2-(triphenylsilyl)ethyl, 2-(benzylsulfonyl)ethyl, 2,2,2-trichloroethyl, 2,2,2-tribromoethyl, 2,3-dibromopropyl, 2,2,2-trifluoroethyl, phenylthio, 2-chloro-4-triphenylmethylphenyl, 2-bromophenyl, 2- [N-Isopropyl-N-(4-Methoxybenzoyl)amino]ethyl, 4-(N-Trifluoroacetamido)butyl, 4-oxopentyl, 4-triphenylmethylaminophenyl, 4-benzylaminophenyl, tetrahydropyranyl, morpholino, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, neopentyloxymethyl (POM), and 9-phenylxanthine-9-yl.

[0242] Examples of amino protecting groups used herein include, but are not limited to, urethane protecting groups such as 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenyl-methoxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups such as formyl, acetyl, neopentyl, trihaloacetyl, benzoyl, and 2-nitrobenzenesulfonyl; and imine and cyclic imide protecting groups such as phthalimide and dithiasuccinoyl. Equivalents of these amino protecting groups are also covered by the compounds and methods of the present invention.

[0243] Some embodiments of SNCA dsRNA agents include at least one lipophilic moiety, said lipophilic moiety comprising, for example, but not limited to, saturated or unsaturated C.16 Hydrocarbon chain (e.g., straight-chain C16 alkyl or alkenyl). This application provides for the inclusion of a lipophilic moiety at any position in the dsRNA agent. In some embodiments, the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or nucleoside internucleotide bond of the double-stranded iRNA agent. For example, C... 16 Some can be conjugated via the 2'-oxygen of ribonucleotides, as shown in the following structure:

[0244]

[0245] As used herein, “lipophile” or “lipophilic moiety” refers to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by means of the octanol-water partition coefficient logKow, where Kow is the ratio of the concentration of the chemical substance in the octanol phase at equilibrium to its concentration in the aqueous phase of the two-phase system. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributed to the structural components of the chemical substance, which are calculated using first-principles or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measurement of this tendency of a substance to prefer a non-aqueous or oily environment rather than water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical substance is lipophilic when its logKow exceeds 0.

[0246] Further modifications to the RNA of certain embodiments of the SNCA dsRNA agent, SNCA antisense polynucleotide, and / or SNCA sense polynucleotide of the present invention include chemically linking one or more ligands, portions, or conjugates that enhance one or more features of the SNCA dsRNA agent, SNCA antisense polynucleotide, and / or SNCA sense polynucleotide to the RNA. Some non-limiting examples of the features that can be enhanced are: SNCA dsRNA agent, SNCA antisense polynucleotide, and / or SNCA sense polynucleotide activity, cellular distribution, delivery of the SNCA dsRNA agent, pharmacokinetic properties of the SNCA dsRNA agent, and cellular uptake of the SNCA dsRNA agent. In some embodiments of the present invention, the SNCA dsRNA agent comprises one or more targeting groups or linking groups that are conjugated to a sense chain in certain embodiments of the SNCA dsRNA agent of the present invention. A non-limiting example of a targeting group is a compound comprising N-acetyl-galactosamine (GalNAc). The terms “targeting group,” “targeting agent,” “linker,” “targeting compound,” “delivery molecule,” “delivery compound,” and “targeting ligand” are used interchangeably herein. In some embodiments of the invention, the SNCA dsRNA agent comprises a targeting compound conjugated to the 5' end of the sense strand. In some embodiments of the invention, the SNCA dsRNA agent comprises a targeting compound conjugated to the 3' end of the sense strand. In some embodiments of the invention, the SNCA dsRNA agent comprises a targeting group containing GalNAc. In some embodiments of the invention, the SCN9A dsRNA agent comprises a targeting group containing a lipophilic moiety. In some embodiments of the invention, the SNCA dsRNA agent does not comprise a targeting compound conjugated to one or both of the 3' and 5' ends of the sense strand. In some embodiments of the invention, the SNCA dsRNA agent does not comprise a GalNAc-containing targeting compound conjugated to one or both of the 5' and 3' ends of the sense strand.

[0247] Other targeting agents and linkers are well known in the art. For example, targeting agents and linkers that can be used in certain embodiments of the present invention include, but are not limited to, lipid moieties, such as: cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556); bile acids (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060); thioethers, such as beryl-S-triphenylmethylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770); and thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 1993, 1994, 1995 ... 20:533-538); aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54); phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycerol-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783); polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973); or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654); palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237); or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0248] Some embodiments of compositions comprising SNCA dsRNA agents, SNCA antisense polynucleotides, and / or SNCA sense polynucleotides may include ligands that alter the distribution, targeting, etc., of the SNCA dsRNA agents. In some embodiments of compositions comprising the SNCA dsRNA agents of the present invention, the ligands enhance affinity for selected targets (e.g., molecules, cells or cell types, compartments such as cell or organ compartments, tissues, organs, or body regions), for example, compared to species without such ligands. Ligands that can be used in the compositions and / or methods of the present invention can be naturally occurring substances, such as: proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, such as synthetic polyamino acids or polyamines. Some examples of polyamino acids are polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) copolymer, diethylene ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Some examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide mimicry polyamines, dendritic polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0249] The ligands included in the compositions and / or methods of the present invention may contain a targeting group, some non-limiting examples of which are cell or tissue targets, such as lectins, glycoproteins, lipids, or proteins, for example, antibodies that bind to specific cell types (e.g., CNS cells, kidney cells, or hepatocytes). The targeting group may be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polylactose, polygalactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptide mimics.

[0250] Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrene), lys-tyr-lys tripeptides, aminoglycosides, guanidine aminoglycosides, artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol (and its thioanalytes), cholic acids, cholanonic acids, lithocholic acids, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono-, di-, or tri-fatty acid esters, e.g., C10 ... 11. C12, C13, C14, C15, C16, C17, C18, C19, or C20 fatty acids and their ethers, such as C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl groups; for example, 1,3-bis-O-(hexadecyl)glycerol, 1,3-bis-O-(octadecyl)glycerol, geraniol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytriphenylmethyl or phenethyl Phosphates and peptide conjugates (e.g., tentacles, Tat peptides), alkylating agents, phosphates / esters, amino groups, thiol groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, diimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraza macrocyclic Eu3+ complexes), dinitrophenyl, HRP, or AP.

[0251] The ligands included in the compositions and / or methods of the present invention may be proteins (e.g., glycoproteins), peptides (e.g., molecules with a specific affinity for a coligand), or antibodies (e.g., antibodies that bind to a specific cell type (e.g., cancer cells, endothelial cells, cardiomyocytes, or osteocytes)). Ligands that can be used in one embodiment of the compositions and / or methods of the present invention may be hormones or hormone receptors. Ligands that can be used in one embodiment of the compositions and / or methods of the present invention may be lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polymannose, or polyvalent fucose. Ligands that can be used in one embodiment of the compositions and / or methods of the present invention may be substances that can, for example, enhance cellular uptake of SNCA dsRNA agents by disrupting the cellular cytoskeleton, such as by disrupting microtubules, microfilaments, and / or intermediate filaments. Some non-limiting examples of this type of substance are: paclitaxel, vincristine, vincaline, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, and myoservin.

[0252] In some embodiments, the ligand linked to the SNCA dsRNA agent of the present invention functions as a pharmacokinetic (PK) modulator. Examples of PK modulators that can be used in the compositions and methods of the present invention include, but are not limited to: lipophils, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, cholesterol, fatty acids, cholic acids, lithocholic acids, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, aptamers that bind to serum proteins, etc. Oligonucleotides comprising numerous thiophosphate bonds are also known to bind to serum proteins; therefore, short oligonucleotides comprising multiple thiophosphate bonds in their backbone, such as oligonucleotides of about 5, 10, 15, or 20 bases, can also be used as ligands in the compositions and / or methods of the present invention.

[0253] SNCA dsRNA pharmaceutical composition

[0254] In some embodiments of the invention, SNCA dsRNA agents are included in the composition. The compositions of the invention may comprise one or more SNCA dsRNA agents and optionally one or more pharmaceutically acceptable carriers, delivery agents, targeting agents, detectable markers, etc. In some embodiments of the method according to the invention, a non-limiting example of a suitable targeting agent is an agent that directs the SNCA dsRNA agent of the invention to and / or into cells to be treated. The selection of the targeting agent will depend on factors such as the nature of the SNCA-related disease or condition and the cell type being targeted. In one non-limiting example, in some embodiments of the invention, it is desirable to target the SNCA dsRNA agent to and / or into hepatocytes. In one non-limiting example, in some embodiments of the invention, it is desirable to target the SNCA dsRNA agent to and / or into brain cells. In one non-limiting example, in some embodiments of the invention, it is desirable to target the SNCA dsRNA agent to and / or into spine cells. It should be understood that in some embodiments of the method of the present invention, the therapeutic agent comprises an SNCA dsRNA agent having only a delivery agent (e.g., a delivery agent comprising N-acetylgalactosamine (GalNAc)) without any additional linking elements. For example, in some aspects of the present invention, the SNCA dsRNA agent may be linked to a delivery compound comprising GalNAc and is contained in a composition containing a pharmaceutically acceptable carrier, and is applied to cells or objects without any detectable markers or targeting agents, etc., linked to the SNCA dsRNA agent.

[0255] When the SNCA dsRNA agent of the present invention is administered and / or linked with one or more delivery agents, targeting agents, labeling agents, etc., those skilled in the art will recognize and be able to select and use suitable agents for the methods of the present invention. Labeling agents can be used in certain methods of the present invention to determine the location of the SNCA dsRNA agent in cells and tissues, and can be used to determine the location of cells, tissues, or organs in which a therapeutic composition containing the SNCA dsRNA agent has been administered in the methods of the present invention. Methods for attachment and use of labeling agents such as enzyme labeling, dyes, radiolabeling, etc., are well known in the art. It should be understood that in some embodiments of the compositions and methods of the present invention, the labeling agent is linked to one or both of the sense polynucleotides and antisense polynucleotides contained in the SNCA dsRNA agent.

[0256] Delivery of SNCA dsRNA and SNCA antisense polynucleotide drugs

[0257] Some embodiments of the method of the present invention include delivering an SNCA dsRNA agent into cells. As used herein, the term "delivery" means promoting or influencing cellular uptake or absorption. Absorption or uptake of the SNCA dsRNA agent can occur through independent diffusion or active cellular processes, or through the use of a delivery agent, targeting agent, etc., that can associate with the SNCA dsRNA agent of the present invention. Delivery means suitable for the method of the present invention include, but are not limited to, in vivo delivery, wherein the SNCA dsRNA agent is injected into a tissue site or administered systemically. In some embodiments of the present invention, the SNCA dsRNA agent is linked to a delivery agent.

[0258] Some non-limiting examples of methods that can be used to deliver SNCA dsRNA agents to cells, tissues, and / or objects include: SNCA dsRNA-GalNAc conjugates, SAMiRNA technology, LNP-based delivery methods, and naked RNA delivery. These and other delivery methods have been successfully used in the art to deliver therapeutic RNAi agents for the treatment of a variety of diseases and conditions, such as, but not limited to: neurodegenerative diseases, liver diseases, acute intermittent porphyria (AIP), hemophilia, pulmonary fibrosis, etc. Details of the various delivery methods can be found in the following publications, such as: Nikam, RR & KR Gore (2018) Nucleic Acid Ther, 28 (4), 209-224 Aug 2018; Springer AD & SF Dowdy (2018) Nucleic Acid Ther. Jun 1; 28(3): 109–118; Lee, K. et al., (2018) Arch Pharm Res, 41(9), 867-874; Nair, JK et al., (2014) J. Am. Chem. Soc. 136:16958-16961; Imran Sajid M. et al., (2023) AdvDrug Deliv Rev. 199:114968; and Padmakumar S. et al., (2022) J Control Release 352:121-145; their contents are incorporated herein by reference.

[0259] Some embodiments of the present invention include the delivery of the SNCA dsRNA agents of the present invention to cells, tissues, and / or subjects using lipid nanoparticles (LNPs). LNPs are commonly used for in vivo delivery of SNCA dsRNA agents, including therapeutic SNCA dsRNA agents. One advantage of using LNPs or other delivery agents is the improved stability of the SNCA RNA agents when delivered to subjects using LNPs or other delivery agents. In some embodiments of the present invention, the LNP comprises a cationic LNP loaded with one or more of the SNCA RNAi molecules of the present invention. When an LNP containing SNCA RNAi molecules is applied to a subject, the LNP and the SNCA RNAi molecules to which they are linked are taken up by the cells via endocytosis, and their presence leads to the release of RNAi-initiating molecules that mediate RNAi.

[0260] Some embodiments of the present invention include using the functional portion to deliver the SNCA dsRNA agent of the present invention to cells, tissues and / or objects.

[0261] The functional portion is a molecule that imparts one or more additional activities to the RNA silencing agent. In some embodiments, the functional portion enhances cellular uptake by target cells (e.g., neurons). Therefore, this disclosure includes RNA silencing agents conjugated or unconjugated (e.g., at their 5' and / or 3' ends) to another portion (e.g., a non-nucleic acid portion, such as a peptide), an organic compound (e.g., a dye), etc. Conjugation can be achieved by methods known in the art, for example, using the following: Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loaded onto polycyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3): 137-43 (1998) (describes nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked to intercalators, hydrophobic groups, polycations, or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describes nucleic acids linked to nanoparticles).

[0262] In one embodiment, the functional portion is a hydrophobic portion. In one embodiment, the hydrophobic portion is selected from fatty acids, steroids, open-ring steroids, lipids, gangliosides and nucleoside analogs, endocannabinoids, and vitamins. In one embodiment, the steroid is selected from cholesterol and lithocholic acid (LCA). In one embodiment, the fatty acid is selected from eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA). In one embodiment, the vitamin is selected from choline, vitamin A, vitamin E, and their derivatives or metabolites. In one embodiment, the vitamin is selected from retinoic acid and α-tocopherol succinate.

[0263] In one embodiment, the RNA silencing agent of this disclosure is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand containing a cationic group. In another embodiment, the lipophilic moiety is attached to one or both strands of the siRNA. In one exemplary embodiment, the lipophilic moiety is attached to one end of the sense strand of the siRNA. In another exemplary embodiment, the lipophilic moiety is attached to the 3' end of the sense strand. In some embodiments, the lipophilic moiety is selected from: cholesterol, vitamin E, vitamin K, vitamin A, folic acid, and cationic dyes (e.g., Cy3). In one exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic moieties include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geraniol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytriphenylmethyl, or phenethyl. Azine.

[0264] In some embodiments, the functional portion may include one or more ligands tethered to the RNA silencer to improve stability, hybridization thermodynamics with the target nucleic acid, targeting of a specific tissue or cell type, or cell permeability, for example, through endocytosis-dependent or endocytosis-independent mechanisms. Ligands and associated modifications may also improve sequence specificity and thereby reduce off-site targeting. The tethering ligand may contain one or more modified bases or sugars that function as intercalators. These may be located in internal regions, such as in the protrusions of the RNA silencer / target duplex. The intercalator may be an aromatic compound, such as a polycyclic aromatic compound or a heterocyclic aromatic compound. Polycyclic intercalators may have stacking capabilities and may comprise a system with 2, 3, or 4 fused rings. The universal bases described herein may be included on the ligand. In one embodiment, the ligand may include a cleaving group that facilitates target gene repression by cleaving the target nucleic acid. The cleavage group can be, for example, bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), polyamine, tripeptide (e.g., lys-tyr-lys tripeptide), or a metal ion chelating group. The metal ion chelating group may include, for example, Lu(III) or EU(III) macrocyclic complexes, Zn(II) 2,9-dimethylphenanthroline derivatives, Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA by free metal ions (e.g., Lu(III)) at the protrusion site. In some embodiments, the peptide ligand may be tethered to an RNA silencing agent to promote the cleavage of the target RNA, for example, in the protrusion region. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be conjugated to a peptide (e.g., via an amino acid derivative) to promote the cleavage of the target RNA. The tethering ligand can be an aminoglycoside ligand, which can impart improved hybridization properties or improved sequence specificity to the RNA silencer. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as Neo-N-acrididine, Neo-S-acrididine, Neo-C-acrididine, Tobra-N-acrididine, and KanaA-N-acrididine. The use of acridine analogs can improve sequence specificity. For example, neomycin B has a high affinity for RNA but low sequence specificity compared to DNA. The acridine analog neo-5-acrididine has an enhanced affinity for HIV Rev-response elements (RREs). In some embodiments, a guanidine analog of the aminoglycoside ligand (guanidinoglycoside) is tethered to the RNA silencer. In guanidinoglycosides, the amino group on an amino acid is exchanged for a guanidino group.The ligation of guanidine analogs can enhance the cellular permeability of RNA silencing agents. Tethering ligands can be polyarginine peptides, peptide-like substances, or peptide mimics, which can enhance the cellular uptake of oligonucleotide agents.

[0265] Exemplary ligands are coupled directly or indirectly to a ligand-conjugated vector via an intervening tether. In some embodiments, coupling occurs via a covalent bond. In some embodiments, the ligand is linked to the vector via an intervening tether. In some embodiments, the ligand alters the distribution, targeting, or lifetime of the RNA silencing agent to which it is incorporated. In some embodiments, for example, compared to the absence of such a ligand, the ligand provides enhanced affinity for selected targets (e.g., molecules, cells or cell types, compartments (e.g., cellular compartments or organ compartments), tissues, organs, or body regions).

[0266] Exemplary ligands can improve transport, hybridization, and specificity properties, and can also improve the nuclease resistance of resulting natural or modified RNA silencers or polymer molecules comprising any combination of monomers and / or natural or modified ribonucleotides described herein. Ligands can generally include therapeutic modifiers, such as those for enhancing uptake; diagnostic compounds or reporter groups, such as those for monitoring distribution; cross-linking agents; nuclease resistance-conferring moieties; and natural or unusual nucleobases. General examples include lipophils, lipids, steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, such as sarsasapogenin, friedelin, lithocholic acid derived from epifriedelanol), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binders, integrin targeting molecules, polycations, peptides, polyamines, and peptide mimics. Ligands may include naturally occurring substances (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pachymannan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); amino acids; or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, like synthetic polyamino acids. Some examples of polyamino acids include: polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Some examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide mimicry polyamines, dendritic polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0267] Ligands may also include targeting groups, such as cell or tissue targets, such as lectins, glycoproteins, lipids, or proteins, such as antibodies, that bind to specific cell types, such as kidney cells. Targeting groups may be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polylactose, polygalactose, N-acetylgalactosamine (GalNAc) or its derivatives, N-acetylglucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, biotin, or RGD peptides or RGD peptide mimics. Other examples of ligands include dyes, intercalating agents (e.g., acridine and substituted acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, Texas porphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrene), lys-tyr-lys tripeptides, aminoglycosides, guanidine aminoglycosides, artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol (and its thioanalytes), cholic acids, cholanonic acids, lithocholic acids, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono-, di-, or tri-fatty acid esters, e.g., C10, C11), etc.), etc. C12, C13, C14, C15, C16, C17, C18, C19, or C20 fatty acids) and their ethers, such as C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl groups; for example, 1,3-bis-O-(hexadecyl)glycerol, 1,3-bis-O-(octadecyl)glycerol, geraniol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, stearic acid (e.g., distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytriphenylmethyl or phenethyl The ligands include azines and peptide conjugates (e.g., tentacles, Tat peptides), alkylating agents, phosphates / esters, amino groups, thiol groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, diimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraza macrocyclic Eu3+ complexes), dinitrophenyl, HRP, or AP. In some embodiments, the ligand is GalNAc or a derivative thereof.

[0268] Ligands can be proteins (e.g., glycoproteins), peptides (e.g., molecules with specific affinity for coligands), or antibodies (e.g., antibodies that bind to specific cell types such as cancer cells, endothelial cells, or osteoblasts). Ligands can also include hormones and hormone receptors. They can also include non-peptide substances such as lipids, lectins, carbohydrates, vitamins, cofactors, polylactose, polygalactose, N-acetylgalactosamine, N-acetylglucosamine, polymannose, or polyfucose.

[0269] In some embodiments, the functional portion is attached to the 5' end and / or the 3' end of the RNA silencing agent of this disclosure. In some embodiments, the functional portion is attached to the 5' end and / or the 3' end of the antisense strand of the RNA silencing agent of this disclosure. In some embodiments, the functional portion is attached to the 5' end and / or the 3' end of the sense strand of the RNA silencing agent of this disclosure. In some embodiments, the functional portion is attached to the 3' end of the sense strand of the RNA silencing agent of this disclosure.

[0270] In some embodiments, the functional portion is linked to an RNA silencing agent via a adapter. In some embodiments, the functional portion is linked to an antisense strand and / or a sense strand via a adapter. In some embodiments, the functional portion is linked to the 3' end of the sense strand via a adapter. In some embodiments, the adapter comprises a divalent or trivalent adapter. In some embodiments, the adapter comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a thiophosphate, an aminophosphate, an amide, a carbamate, or a combination thereof.

[0271] Another non-limiting example of a delivery agent that can be used in embodiments of the present invention to deliver the SNCA dsRNA agent of the present invention to cells, tissues, and / or objects is a GalNAc-containing reagent linked to and delivering the SNCA dsRNA agent of the present invention to cells, tissues, and / or objects. Some examples of certain additional GalNAc-containing delivery agents that can be used in certain embodiments of the methods and compositions of the present invention are disclosed in PCT applications: WO2020191183A1 and WO2023045995 (which are incorporated herein in their entirety). One non-limiting example of a GalNAc targeting ligand that can be used in the compositions and methods of the present invention to deliver the SNCA dsRNA agent to cells is a cluster of targeting ligands. Some examples of targeting ligand clusters presented herein are referred to as: GalNAc ligands having a phosphodiester bond (GLO) and GalNAc ligands having a phosphate thioester bond (GLS). The term "GLX-n" may be used herein to indicate that the linked GalNAc-containing compound is any of the following compounds: GLS-1*, GLS-2*, GLS-3*, GLS-4*, GLS-5*, GLS-6*, GLS-7*, GLS-8*, GLS-9*, GLS-10*, GLS-11*, GLS-12*, GLS-13*, GLS-14*, GLS-15*, GLS-16*. GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16, the structures of which are shown below, wherein the following (below) contains the linking sites of the GalNAc targeting ligand and the RNAi agent of the present invention, which are located on their respective rightmost sides (marked with " "). (As shown). It should be understood that any RNAi and dsRNA molecule of the present invention can be linked to any of the following: GLS-1*, GLS-2*, GLS-3*, GLS-4*, GLS-5*, GLS-6*, GLS-7*, GLS-8*, GLS-9*, GLS-10*, GLS-11*, GLS-12*, GLS-13*, GLS-14*, GLS-15*, GLS-16*, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16. The structures of GLO-1 to GLO-16 and GLS-1* to GLS-16* are shown below.

[0272]

[0273]

[0274]

[0275]

[0276] In some embodiments, the isomannide nucleotide described above may also be conjugated to one or more GalNAc targeting ligands. Specific examples of isomannide nucleotides conjugated to GalNAc targeting ligands include, but are not limited to:

[0277] ,

[0278] ,

[0279] The word "olig" independently represents the polynucleotide portion.

[0280] In some embodiments of the invention, in vivo delivery can also be performed via β-glucan delivery systems, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677 and U.S. Publication No. 2005 / 0281781, which are incorporated herein by reference in their entirety. SNCA RNAi agents can also be introduced into cells in vitro using methods known in the art, such as electroporation and lipid transfection. In some embodiments of the methods of the invention, SNCA dsRNA is delivered without a target agent. These RNAs can be delivered as “naked” RNA molecules. As a non-limiting example, the SNCA dsRNA of the invention can be administered to a subject in a pharmaceutical composition containing an RNAi agent but not a target agent (e.g., a GalNAc targeting compound) to treat SNCA-related diseases or conditions, such as PD.

[0281] In addition to certain delivery methods described herein, it should be understood that RNAi delivery methods (such as, but not limited to, those described herein and those used in the art) may be used in conjunction with embodiments of the SNCA RNAi agents and treatments described herein.

[0282] The SNCA dsRNA agents of the present invention can be administered to subjects in a quantity and manner that effectively reduces the level and activity of SNCA peptides in cells and / or subjects. In some embodiments of the methods of the present invention, one or more SNCA dsRNA agents are administered to cells and / or subjects to treat diseases or conditions associated with SNCA expression and activity. In some embodiments, the methods of the present invention include administering one or more SNCA dsRNA agents to subjects requiring such treatment to alleviate diseases or conditions associated with SNCA expression in the subjects. The SNCA dsRNA agents or SNCA antisense polynucleotide agents of the present invention can be administered to reduce SNCA expression and / or activity in one or more types of cells in vitro, ex vivo, and in vivo.

[0283] In some embodiments of the invention, the level of SNCA peptides in cells is reduced, and thus their activity is decreased, by delivering (e.g., introducing) SNCA dsRNA agents or SNCA antisense polynucleotide agents into cells. Targeting agents and methods can be used to facilitate the delivery of SNCA dsRNA agents or SNCA antisense polynucleotide agents to specific cell types, cell subtypes, organs, spatial regions within a subject, and / or subcellular regions within cells. SNCA dsRNA agents can be administered alone or in combination with one or more other SNCA dsRNA agents in certain methods of the invention. In some embodiments, two, three, four, or more independently selected SNCA dsRNA agents are administered to the subject.

[0284] In some embodiments of the invention, an SNCA dsRNA agent is administered to a subject in combination with one or more other treatment regimens for treating SNCA-related diseases or conditions. Some non-limiting examples of other treatment regimens include: administration of one or more SNCA antisense polynucleotides of the invention, administration of non-SNCA dsRNA therapeutics, and behavioral modification. The other treatment regimens may be administered at one or more of the following times: before, simultaneously with, and after administration of the SNCA dsRNA agent of the invention. It should be understood that "simultaneously" as used herein means within five minutes of time zero, within ten minutes of time zero, within 30 minutes of time zero, within 45 minutes of time zero, and within 60 minutes of time zero, where "time zero" is the time at which the SNCA dsRNA agent of the invention is administered to the subject.Some non-SNCA dsRNA therapeutic agents are: carbidopa-levodopa, levodopa, entacapone, tolcapone, opicapone, pramipexole, ropinirole, apomorphine, rotigotine, selegiline, rasagiline, safinamide, amantadine, istradefylline, trihexyphenidyl, benztropine, rivastigmine, donepezil, galantamine, acetyl-L-carnitine, vinpocetine, and huperzine. A) Alpha-lipoic acid, vitamin E, rhodiola, biotin, reminyl, tacrine (Cognex), selegiline, physostigmine, revistigmin, donepezil, exelon, metrifonate, milameline, xanomeline, saeluzole, idebenone, ENA-713, mermic, quetiapine, neurestrol, idebenone, propentofylline, neuromidal, and memantine, or other agents disclosed herein or otherwise known in the art included for the treatment of SNCA-related neurodegenerative diseases in subjects. Some non-limiting examples of behavior modifications include: physical, occupational, and speech therapies; exercise programs, including cardiopulmonary, resistance, flexibility, and gait and balance exercises; and deep brain stimulation (DBS) involving the implantation of electrodes into target areas of the brain. These and other therapeutic agents and behavior modifiers are known in the art and are used to treat SNCA-related diseases or conditions in subjects, and may be administered to subjects in combination with the administration of one or more of the SNCA dsRNA agents of the present invention to treat SNCA-related diseases or conditions.The SNCA dsRNA agent of the present invention, when applied to cells or objects to treat SNCA-related diseases or conditions, may act synergistically with one or more other therapeutic agents or activities and enhance the effectiveness of one or more therapeutic agents or activities and / or enhance the effectiveness of the SNCA dsRNA agent in treating SNCA-related diseases or conditions.

[0285] The treatment methods of the present invention, including the administration of SNCA dsRNA agents, can be used before the onset of SNCA-related disease or condition and / or while SNCA-related disease or condition is present, including at all times before and after the early, intermediate, and late stages of the disease or condition. The methods of the present invention can also be used to treat subjects who have previously been treated with one or more other therapeutic agents and / or treatment activities for SNCA-related disease or condition, wherein such other therapeutic agents and / or treatment activities were unsuccessful, had minimal success, and / or were no longer successful in the treatment subject's SNCA-related disease or condition.

[0286] vector-encoded dsRNA

[0287] In some embodiments of the invention, a vector can be used to deliver SNCA dsRNA agents into cells. The SNCA dsRNA agent transcription unit may be contained in a DNA or RNA vector. The preparation and use of vectors encoding such transgenes for delivering sequences into cells and / or objects are well known in the art. Vectors can be used in the methods of the invention to result in transient expression of SNCA dsRNA, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks. The length of transient expression can be determined using conventional element-based methods, the elements being, for example, but not limited to, selected specific vector constructs and target cells and / or tissues. Such transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrated or non-integrated vectors. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0288] One or more strands of the SNCA dsRNA agent can be transcribed from a promoter on an expression vector. In cases where two separate strands are to be expressed to produce, for example, dsRNA, methods such as transfection or infection can be used to co-introduce the two separate expression vectors into the cell. In some embodiments, each separate strand of the SNCA dsRNA agent of the present invention can be transcribed from two promoters contained in the same expression vector. In some embodiments of the present invention, the SNCA dsRNA agent is expressed as an inverted repeat polynucleotide linked by a linker polynucleotide sequence, such that the SNCA dsRNA agent has a stem-loop structure.

[0289] Some non-limiting examples of RNA expression vectors are DNA plasmids or viral vectors. Expression vectors that can be used in embodiments of the present invention are compatible with eukaryotic cells. Eukaryotic cell expression vectors are conventionally used in the art and are available from many commercial sources. Delivery of SNCA dsRNA expression vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells explanted from the subject and subsequently reintroduced into the subject, or by any other means that allows introduction into the desired target cells.

[0290] Viral vector systems that may be included in the method embodiments include, but are not limited to: (a) adenovirus vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murineleukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) piconemavirus vectors; (i) poxvirus vectors, such as smallpox, such as vaccinia virus vectors or avipox, such as canary pox or fowl pox; and (j) helper-dependent or gutless adenoviruses. Constructs for recombinant expression of SNCA dsRNA agents may include regulatory elements, such as promoters, enhancers, etc., which may be selected to provide constitutive or regulatory / inducible expression. The use of promoters and enhancers, etc., and viral vector systems are conventional in the art and may be used in conjunction with the methods and compositions described herein.

[0291] Some embodiments of the present invention include the use of a viral vector for delivering SNCA dsRNA agents into cells. Many adenovirus-based delivery systems are conventionally used in the art for delivery to, for example, the lungs, liver, central nervous system, endothelial cells, and muscle. Some non-limiting examples of viral vectors that can be used in the methods of the present invention are: AAV vectors, poxviruses such as vaccinia virus, modified virus Ankara (MVA), NYVAC, fowlpox such as chickenpox, or canarypox.

[0292] Some embodiments of the present invention include a method of delivering an SNCA dsRNA agent into cells using a vector, and such a vector may be a pharmaceutically acceptable carrier, which may, but does not necessarily, contain a slow-release matrix in which a gene delivery carrier is embedded. In some embodiments, the vector for delivering SNCA dsRNA may be generated by recombinant cells, and the pharmaceutical compositions of the present invention may comprise one or more cells that generate an SNCA dsRNA delivery system.

[0293] Pharmaceutical compositions containing SNCA dsRNA or ssRNA agents

[0294] Some embodiments of the present invention include the use of pharmaceutical compositions comprising an SNCA dsRNA agent or an SNCA antisense polynucleotide agent and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising an SNCA dsRNA agent or an SNCA antisense polynucleotide agent can be used in the methods of the present invention to reduce SNCA gene expression and SNCA activity in cells, and can be used to treat SNCA-related diseases or conditions. Such pharmaceutical compositions can be formulated based on a delivery method. Some non-limiting examples of formulations for delivery methods are: compositions formulated for subcutaneous delivery, compositions formulated for intrathecal delivery, compositions formulated for systemic administration via parenteral delivery, compositions formulated for intravenous (IV) delivery, compositions formulated for direct delivery to the brain, etc. The pharmaceutical compositions of the present invention can be administered using one or more means to deliver SNCA dsRNA agents or SNCA antisense polynucleotide agents into cells, such means being: surface (e.g., via a transdermal patch); lung, e.g., by inhalation or blowing in powders or aerosols, including via a nebulizer; intravenous; intratracheal; intranasal; epidermal and transdermal; oral or parenteral administration. Parenteral administration includes intravenous, intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous, e.g., via an implanted device; or intracranial, e.g., via intraparenchymal, intrathecal, or intraventricular administration. SNCA dsRNA agents or SNCA antisense polynucleotide agents can also be delivered directly to target tissues, such as directly to the liver, directly to the kidneys, etc. It should be understood that "delivery of SNCA dsRNA agents" or "delivery of SNCA antisense polynucleotide agents" into cells encompasses the direct delivery of SNCA dsRNA agents or SNCA antisense polynucleotide agents, respectively, as well as the expression of SNCA dsRNA agents in cells from a coding vector delivered to the cells, or by any suitable means of making SNCA dsRNA or SNCA antisense polynucleotide agents present in the cells. The means of delivering repressive RNA, as well as the preparation and use of the formulations, are well known and routinely used in the art.

[0295] As used herein, “pharmaceutical composition” comprises a pharmacologically effective amount of the SNCA dsRNA or SNCA antisense polynucleotide of the present invention, and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextran, water, glycerol, ethanol, and combinations thereof. This term specifically excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, colorants, and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders may include starch and gelatin, while lubricants, if present, are typically magnesium stearate, stearic acid, or talc. Tablets may be coated with materials such as glyceryl monostearate or glyceryl distearate, if desired, to delay absorption in the gastrointestinal tract. Pharmaceutical agents contained in pharmaceutical formulations are further described below.

[0296] As used herein, terms such as “pharmacologically effective amount,” “therapeuticly effective amount,” and “effective amount” refer to the amount by which the SNCA dsRNA agent or SNCA antisense polynucleotide agent of the present invention produces the intended pharmacological, therapeutic, or preventative results. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or condition is reduced by at least 10%, then the therapeutically effective amount of a drug used to treat that disease or condition is the amount that must reduce that parameter by at least 10%. For example, a therapeutically effective amount of the SNCA dsRNA agent or SNCA antisense polynucleotide agent can reduce SNCA protein levels by at least 10%.

[0297] Effective amount

[0298] In some aspects, the methods of the present invention include contacting cells with an effective amount of an SNCA dsRNA agent or an SNCA antisense polynucleotide agent to reduce SNCA gene expression in the contacted cells. Certain embodiments of the methods of the present invention include administering an effective amount of an SNCA dsRNA agent or an SNCA antisense polynucleotide agent to a subject to reduce SNCA gene expression in the subject and to treat SNCA-related diseases or conditions. An “effective amount” used in reducing SNCA expression and / or treating SNCA-related diseases or conditions is an amount necessary to achieve the desired biological effect or an amount sufficient to achieve the desired biological effect. For example, an effective amount of an SNCA dsRNA agent or SNCA antisense polynucleotide agent for treating SNCA-related diseases or conditions may be an amount necessary to: (i) slow or stop the progression of the disease or condition; or (ii) reverse, alleviate, or eliminate one or more symptoms of the disease or condition. In some aspects of the invention, an effective amount is the amount of an SNCA dsRNA agent or SNCA antisense polynucleotide agent that, when administered to a subject requiring treatment for an SNCA-related disease or condition, results in a therapeutic response to prevent and / or treat the disease or condition. According to some aspects of the invention, an effective amount is the amount of the SNCA dsRNA agent or SNCA antisense polynucleotide agent of the invention that, when combined with or co-administered with other therapeutic treatments for an SNCA-related disease or condition, results in a therapeutic response to prevent and / or treat the disease or condition. In some embodiments of the invention, the biological effect of treating a subject with the SNCA dsRNA agent or SNCA antisense polynucleotide agent of the invention may be improvement and / or complete elimination of symptoms caused by the SNCA-related disease or condition. In some embodiments of the invention, the biological effect is the complete elimination of the SNCA-related disease or condition, as demonstrated, for example, by diagnostic tests indicating that the subject has no SNCA-related disease or condition. A non-limiting example of detectable physiological symptoms includes a decrease in SNCA levels in the liver of a subject after administration of the agent of the invention. Other means known in the art for assessing SNCA-related disease or condition status may be used to determine the effect of the agents and / or methods of the present invention on SNCA-related disease or condition.

[0299] Typically, an effective amount of SNCA dsRNA or SNCA antisense polynucleotide agent will be determined in clinical trials to reduce SNCA peptide activity to a level sufficient to treat SNCA-related diseases or conditions, thereby establishing an effective dose for the test population relative to a control population in a blinded study. In some embodiments, the effective amount will be the amount that results in a desired response, for example, the amount that alleviates SNCA-related diseases or conditions in cells, tissues, and / or subjects with the disease or condition. Therefore, an effective amount of SNCA dsRNA or SNCA antisense polynucleotide agent for treating SNCA-related diseases or conditions that can be treated by reducing SNCA peptide activity can be an amount that, when administered, reduces the amount of SNCA peptide activity in the subject to less than the amount present in cells, tissues, and / or subjects without the administration of the SNCA dsRNA or SNCA antisense polynucleotide agent. In some aspects of the invention, the level of SNCA peptide activity and / or SNCA gene expression present in cells, tissues, and / or subjects that have not been exposed to or administered the SNCA dsRNA or SNCA antisense polynucleotide agent of the invention is referred to as a “control” amount. In some embodiments of the method of the present invention, the control level of the subject is the pre-treatment level of the subject; in other words, the level in the subject before administration of the SNCA agent can be the control level of the subject and is compared with the level of SNCA peptide activity and / or SNCA gene expression in the subject after administration of siRNA. In the case of treating SNCA-related diseases or conditions, the desired response can be the reduction or elimination of one or more symptoms of the disease or condition in cells, tissues, and / or the subject. The reduction or elimination can be temporary or permanent. It should be understood that methods for determining SNCA peptide activity, SNCA gene expression, symptom assessment, clinical testing, etc., can be used to monitor the status of SNCA-related diseases or conditions. In some aspects of the present invention, the desired response to the treatment of SNCA-related diseases or conditions is to delay the onset of the disease or condition or even prevent the onset of the disease or condition.

[0300] The effective amount of compounds that reduce SNCA peptide activity can also be determined by assessing the physiological effects of administration of SNCA dsRNA agents or SNCA antisense polynucleotide agents on cells or subjects (e.g., reduction of SNCA-related disease or symptoms after administration). Subject assays and / or symptom monitoring can be used to determine the potency of the SNCA dsRNA agents or SNCA antisense polynucleotide agents of the present invention, which can be administered in the pharmaceutical compounds of the present invention, and to determine the presence or absence of a response to treatment. A non-limiting example is the use of one or more tests known in the art for the levels of SNCA mRNA, α-synuclein, and / or other parameters functionally associated with SNCA expression levels.

[0301] Some embodiments of the present invention include methods for determining the efficacy of the dsRNA agent or SNCA antisense polynucleotide agent of the present invention applied to a subject to treat an SNCA-related disease or condition by assessing and / or monitoring one or more “physiological characteristics” of the SNCA-related disease or condition. Some non-limiting examples of physiological characteristics of an SNCA-related disease or condition are the levels of SNCA mRNA, α-synuclein, or other parameters that are functionally related to SNCA expression levels. Standard means for determining such physiological characteristics are known in the art and include, but are not limited to, blood tests, imaging studies, physical examinations, etc.

[0302] It should be understood that the amount of SNCA dsRNA or SNCA antisense polynucleotide agent administered to the subject may be modified, at least in part, based on such determination of the disease and / or condition and / or physiological characteristics identified in the subject. The therapeutic amount may be varied, for example, by increasing or decreasing the amount of SNCA dsRNA or SNCA antisense polynucleotide agent, by changing the composition of the SNCA dsRNA or SNCA antisense polynucleotide agent administered, by changing the route of administration, by changing the time of administration, etc. The effective amount of SNCA dsRNA or SNCA antisense polynucleotide agent will vary depending on: the specific condition being treated, the age and physical condition of the subject; the severity of the condition, the duration of treatment, the nature of concurrent treatments (if any), the specific route of administration, and other factors within the knowledge and expertise of the health practitioner. For example, the effective amount may depend on the desired level of SNCA peptide activity and / or SNCA gene expression for the effective treatment of SNCA-related diseases or conditions. Those skilled in the art can determine, based on experience, the effective amount of a specific SNCA dsRNA agent or SNCA antisense polynucleotide agent of the present invention for use in the methods of the present invention without excessive experimentation. In conjunction with the teachings provided herein, effective preventative or therapeutic treatment regimens for specific subjects can be planned by selecting from the various SNCA dsRNA agents or SNCA antisense polynucleotide agents of the present invention and by weighing factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred administration method. As used in some embodiments of the present invention, the effective amount of the SNCA dsRNA agent or SNCA antisense polynucleotide agent of the present invention can be the amount that produces the desired biological effect in the cells upon contact with the cells.

[0303] It should be recognized that SNCA gene silencing can be determined in any constitutively or genomically modified SNCA-expressing cells and by any appropriate assay. In some embodiments of the invention, by administration of the SNCA dsRNA agent of the invention, SNCA gene expression is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments of the invention, by administration of the SNCA dsRNA agent of the invention, SNCA gene expression is reduced by 5% to 10%, 5% to 25%, 10% to 50%, 10% to 75%, 25% to 75%, 25% to 100%, or 50% to 100%.

[0304] Dosage

[0305] The SNCA dsRNA agent and the SNCA antisense polynucleotide agent are delivered in the pharmaceutical composition at a dose sufficient to inhibit SNCA gene expression. In some embodiments of the invention, the dose of the SNCA dsRNA agent or the SNCA antisense polynucleotide agent is from 0.01 to 200.0 mg / kg body weight per day, typically from 1 to 50 mg / kg body weight, 5 to 40 mg / kg body weight, 10 to 30 mg / kg body weight, 1 to 20 mg / kg body weight, 1 to 10 mg / kg body weight, 4 to 15 mg / kg body weight, including the endpoints. For example, SNCA dsRNA agents or SNCA antisense polynucleotide agents can be administered in the following amounts: approximately 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / kg, 3.0 mg / kg, 3.1 mg / kg, 3.2 mg / kg per single dose. mg / kg, 3.3mg / kg, 3.4 mg / kg, 3.5 mg / kg, 3.6 mg / kg, 3.7 mg / kg, 3.8 mg / kg, 3.9 mg / kg, 4 mg / kg, 4.1mg / kg, 4.2 mg / kg, 4.3 mg / kg, 4.4 mg / kg, 4.5 mg / kg, 4.6 mg / kg, 4.7 mg / kg, 4.8 mg / kg, 4.9 mg / kg, 5 mg / kg, 5.1 mg / kg, 5.2 mg / kg, 5.3 mg / kg, 5.4 mg / kg, 5.5 mg / kg, 5.6 mg / kg, 5.7 mg / kg, 5.8 mg / kg, 5.9 mg / kg, 6 mg / kg, 6.1 mg / kg, 6.2 mg / kg, 6.3 mg / kg, 6.4mg / kg, 6.5 mg / kg, 6.6 mg / kg, 6.7 mg / kg, 6.8 mg / kg, 6.9 mg / kg, 7 mg / kg, 7.1 mg / kg, 7.2mg / kg, 7.3 mg / kg, 7.4 mg / kg, 7.5 mg / kg, 7.6 mg / kg, 7.7 mg / kg, 7.8 mg / kg, 7.9 mg / kg, 8mg / kg, 8.1 mg / kg, 8.2 mg / kg, 8.3 mg / kg, 8.4 mg / kg, 8.5 mg / kg, 8.6 mg / kg, 8.7 mg / kg, 8.8 mg / kg, 8.9 mg / kg, 9 mg / kg, 9.1 mg / kg, 9.2 mg / kg, 9.3 mg / kg, 9.4 mg / kg, 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22mg / kg, 23mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, to 50 mg / kg body weight. .

[0306] Several factors can be considered when determining the dosage and delivery time of the SNCA dsRNA agent of the present invention. The absolute amount of SNCA dsRNA agent or SNCA antisense polynucleotide agent delivered will depend on a variety of factors, including concurrent treatment, number of doses, and individual subject parameters, including age, physical condition, body size, and weight. These are factors well known to those skilled in the art and can be determined simply through routine experimentation. In some embodiments, a maximum dose may be used, i.e., the highest safe dose based on reasonable medical judgment.

[0307] In some embodiments, the method of the present invention may include administering to a subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of an SNCA dsRNA agent or an SNCA antisense polynucleotide agent. In some cases, the pharmaceutical compound (e.g., a pharmaceutical compound containing an SNCA dsRNA agent or an SNCA antisense polynucleotide agent) may be administered to the subject at least daily, every other day, weekly, every other week, monthly, etc. Doses may be administered once daily or more than once daily, for example, 2, 3, 4, 5 or more times within a 24-hour period. The pharmaceutical composition of the present invention may be administered once daily, or the SNCA dsRNA agent or SNCA antisense polynucleotide agent may be administered throughout the day at appropriate intervals in two, three or more sub-dose, or even using continuous infusion or delivery of a controlled-release formulation. In some embodiments of the method of the present invention, the pharmaceutical composition of the present invention may be administered to the subject once daily or more, once weekly or more, once monthly or more, or once annually or more.

[0308] In some aspects, the method of the present invention includes administering a pharmaceutical compound, alone, in combination with one or more other SNCA dsRNA agents or SNCA antisense polynucleotide agents, and / or in combination with other pharmaceutical treatments or treatment activities or regimens administered to a subject suffering from an SNCA-related disease or condition. The pharmaceutical compound may be administered in a pharmaceutical composition. The pharmaceutical composition used in the method of the present invention may be sterile and contain an amount of SNCA dsRNA agent or SNCA antisense polynucleotide agent that reduces the activity of the SNCA polypeptide to a level sufficient to produce the desired response in units suitable for the weight or volume of the subject. The dose of the pharmaceutical composition containing the SNCA dsRNA agent or SNCA antisense polynucleotide agent that reduces the activity of the SNCA protein, administered to the subject, may be selected based on various parameters, particularly the method of administration used and the condition of the subject. Other factors include the desired duration of treatment. If the response in the subject is insufficient at the initial dose administered, a higher dose may be used (or at an effectively higher dose via a different, more localized delivery route) to the extent permitted by patient tolerance.

[0309] treat

[0310] The terms “SNCA-related diseases,” “SNCA-related diseases and conditions,” and “diseases and conditions caused and / or regulated by SNCA” as used in this article are intended to include any disease associated with the SNCA gene or protein. Such diseases can be caused by, for example, overproduction of the SNCA protein, mutations in the SNCA gene, aberrant cleavage of the SNCA protein, or aberrant interactions between SNCA and other proteins or other endogenous or exogenous substances. Some exemplary SNCA-related diseases include, but are not limited to: Parkinson's disease (PD), multiple system atrophy (MSA), Lewy body dementia (LBD), pure autonomous exhaustion (PAF), Pick's disease, progressive supranuclear palsy, boxing dementia, chromosome 17-related Parkinson's syndrome, Lytico-Bodig disease, tangled dominant dementia, argyrophilic grain disease, silver-philic granulosis, gangliocytoma, meningioma, subacute sclerosing encephalitis, lead poisoning encephalopathy, tuberous sclerosis, Hallewarden-Schpattz disease, lipofuscin deposition, corticobasal degeneration, frontotemporal dementia, frontotemporal lobe degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia and / or Crotzfeldt-Jab disease, or other conditions associated with SNCA expression.

[0311] In some aspects of the invention, the SNCA dsRNA agent or SNCA antisense polynucleotide agent of the invention may be administered to a subject at one or more times before or after the diagnosis of an SNCA-related disease or condition. In some aspects of the invention, the subject is at risk of having or developing an SNCA-related disease or condition. A subject at risk of developing an SNCA-related disease or condition is a subject with an increased likelihood of developing an SNCA-related disease or condition compared to a control risk level. In some embodiments of the invention, the risk level may be statistically significant compared to a control risk level. Subjects at risk may include, for example, subjects who are or will be: subjects with pre-existing diseases and / or genetic abnormalities that make them more susceptible to SNCA-related diseases or conditions than control subjects without pre-existing diseases or genetic abnormalities; subjects with a family and / or personal history of SNCA-related diseases or conditions; and subjects who have previously been treated for SNCA-related diseases or conditions. It should be understood that a pre-existing disease and / or genetic abnormality that makes a subject more susceptible to SNCA-related diseases or conditions can be a disease or genetic abnormality that has been previously identified as being associated with a higher likelihood of developing SNCA-related diseases or conditions when present.

[0312] It should be understood that SNCA dsRNA agents or SNCA antisense polynucleotide agents can be administered to subjects based on their individual medical conditions. For example, healthcare provided to a subject may assess SNCA levels measured in samples obtained from the subject and determine whether it is expected that the subject's SNCA levels can be reduced by administering the SNCA dsRNA agent or SNCA antisense polynucleotide agent of the present invention. In this example, SNCA levels can be considered a physiological characteristic of SNCA-related conditions, even if the subject has not been diagnosed with an SNCA-related disease (such as one disclosed herein). Healthcare providers may monitor changes in the subject's SNCA levels as a measure of the efficacy of the administered SNCA dsRNA agent or SNCA antisense polynucleotide agent of the present invention. In a non-limiting example, a biological sample, such as a blood or tissue sample, can be obtained from the subject, and the subject's SNCA levels can be determined in the sample. The SNCA dsRNA agent or SNCA antisense polynucleotide agent is administered to the subject, and a blood sample is obtained from the subject after administration, and the SNCA level is determined using the sample, with the result compared to the result determined in a sample prior to (before) administration to the subject. The decrease in SNCA levels in subjects in the later-stage samples compared to pre-application levels indicates the efficacy of the applied SNCA dsRNA agent or SNCA antisense polynucleotide agent in reducing SNCA levels in subjects.

[0313] Some embodiments of the method of the present invention include modulating treatment, which involves administering the dsRNA agent or SNCA antisense polynucleotide agent of the present invention to a subject based at least in part on an assessment of changes in one or more physiological characteristics of a subject’s SNCA-related disease or condition induced by the treatment. For example, in some embodiments of the present invention, the effect of the administered dsRNA agent or SNCA antisense polynucleotide agent of the present invention on the subject may be determined and used to help modulate the amount of the dsRNA agent or SNCA antisense polynucleotide agent of the present invention subsequently administered to the subject. In a non-limiting example, the dsRNA agent or SNCA antisense polynucleotide agent of the present invention is administered to the subject, the subject’s SNCA level is determined after administration, and at least in part based on the determined level, a higher amount of the dsRNA agent or SNCA antisense polynucleotide agent is determined to be desirable to enhance the physiological effect of the administered agent, such as reducing or further reducing the subject’s SNCA level. In another non-limiting example, the dsRNA agent or SNCA antisense polynucleotide agent of the present invention is administered to a subject, the subject's SNCA level is determined after administration, and based at least in part on the determined level, a lower amount of the dsRNA agent or SNCA antisense polynucleotide agent is desired to be administered to the subject.

[0314] Therefore, some embodiments of the present invention include assessing changes in one or more physiological characteristics induced by prior treatment of the subject to adjust the amount of the dsRNA agent or SNCA antisense polynucleotide agent of the present invention subsequently administered to the subject. Some embodiments of the method of the present invention include determining, 1, 2, 3, 4, 5, 6 or more physiological characteristics of an SNCA-related disease or condition to assess and / or monitor the efficacy of the administered SNCA dsRNA agent or SNCA antisense polynucleotide agent of the present invention, and optionally using said determination to adjust one or more of the following: the dosage, administration regimen, and / or frequency of the dsRNA agent or SNCA antisense polynucleotide agent of the present invention; thereby treating the SNCA-related disease or condition in the subject. In some embodiments of the method of the present invention, the desired result of administering an effective amount of the dsRNA agent or SNCA antisense polynucleotide agent of the present invention to the subject is a reduction in the levels of the subject's SNCA mRNA, α-synuclein, or other parameters functionally associated with SNCA expression levels compared to previous levels determined for the subject or to control levels.

[0315] As used herein, the terms “treatment,” “treated,” or “under treatment” can refer to preventive treatment that reduces the likelihood of a subject developing an SNCA-related disease or condition, or to treatment that occurs after a subject has developed an SNCA-related disease or condition, in order to eliminate or reduce the level of the SNCA-related disease or condition in the subject, prevent the SNCA-related disease or condition from becoming more advanced (e.g., more severe), and / or slow the progression of the SNCA-related disease or condition, compared to a subject without treatment that reduces the activity of the SNCA peptide in the subject.

[0316] Certain embodiments of the agents, compositions, and methods of the present invention can be used to inhibit SNCA gene expression. The terms “inhibit,” “silence,” “reduction,” “downregulation,” and “knockdown” as used herein refer to SNCA gene expression, respectively, when cells, cell populations, tissues, organs, or objects are contacted (e.g., treated with) the SNCA dsRNA agent or SNCA antisense polynucleotide agent of the present invention, compared to control levels of RNA transcribed from the SNCA gene, the level of activity of expressed SNCA, or the level of SNCA translated from mRNA, as measured by one or more of the following: the level of RNA transcribed from the gene, the level of activity of expressed SNCA, and the level of SNCA polypeptide, protein, or protein subunit translated from mRNA. In some embodiments, the control level is the level in cells, tissues, organs, or objects that have not been contacted (e.g., treated with) the SNCA dsRNA agent or SNCA antisense polynucleotide agent.

[0317] Application method

[0318] Various routes of administration of SNCA dsRNA agents or SNCA antisense polynucleotide agents can be used in the methods of the present invention. The specific delivery method chosen will depend at least in part on the specific condition being treated and the dose required for therapeutic efficacy. Generally, the methods of the present invention can be implemented using any medically acceptable route of administration, meaning any method that produces an effective therapeutic level against SNCA-related diseases or conditions without causing clinically unacceptable adverse effects. The siRNA molecules of this disclosure can be delivered directly to the CNS or neurons of the recipient requiring SNCA silencing, for example by intrathecal, intraventricular, striatal, or intraparenchymal injection; direct injection into specific nerves or ganglia (e.g., the trigeminal or dorsal root ganglia); intracerebellomedullary cistern injection, for example by catheter insertion, intravenous injection, subcutaneous injection, or intramuscular injection. In some embodiments of the present invention, SNCA dsRNA agents or SNCA antisense polynucleotide agents can be administered via oral, enteric, mucosal, subcutaneous, and / or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intrathecal, intramuscular, intraperitoneal, and intrasternal injection, or infusion techniques. Other routes include, but are not limited to, nasal (e.g., via a gastronasal tube), percutaneous, vaginal, rectal, sublingual, and inhalation. Delivery routes of the present invention may include intrathecal, intraventricular, intracerebral (ICV), striatum, intraparenchymal, cerebellomedullary cistern, or intracranial.

[0319] Some implementations of this method include intrathecal injection or injection via the cerebellomedullary cistern, performed via catheter insertion. Other implementations include direct injection into a specific nerve or ganglion (e.g., the trigeminal or dorsal root ganglion).

[0320] Intrathecal injection involves direct injection into the spinal cord or subarachnoid space. Through direct injection into the spinal CSF, the siRNA molecules of this disclosure can directly enter cells in the spinal cord (e.g., neurons and glial cells) and can also enter brain cells by bypassing the blood-brain barrier, or enter the cell bodies of neurons outside the blood-brain barrier.

[0321] Intraventricular ventricle (ICV) injection is a method of direct injection into the brain's ventricles' cranial fluid (CSF). Similar to intrathecal injection, ICV is an injection method that bypasses the blood-brain barrier. Using ICV offers the advantage of reaching cells in the brain and spinal cord without the risk of therapeutic agent degradation in the bloodstream.

[0322] Intrastriatal injection involves direct injection into the striatum, a region in the subcortical basal ganglia of the brain. Injection into the striatum bypasses the blood-brain barrier and the pharmacokinetic challenges of injection into the bloodstream, allowing direct access to brain cells.

[0323] Intraplasmic administration involves direct injection into the parenchyma (e.g., brain parenchyma). Injection into the brain parenchyma allows direct injection into brain regions affected by disease or condition, while bypassing the blood-brain barrier.

[0324] Intracerebellomedullary cistern injection via catheter insertion involves direct injection into the cerebellomedullary cistern, a brain region located between the cerebellum and the dorsal surface of the medulla oblongata. Injection into the cerebellomedullary cistern allows for more direct delivery to cells of the cerebellum, brainstem, and spinal cord. In some embodiments of the methods described herein, the therapeutic composition may be delivered to the subject via systemic administration (e.g., intravenous, intramuscular, or subcutaneous).

[0325] Intravenous (IV) injection is a method of direct injection into the bloodstream of the subject. IV administration can be in the form of a bolus dose, or by continuous infusion, or by any other method of treatment composition intolerance.

[0326] Intramuscular (IM) injection is administered into the muscles of the recipient, such as the deltoid or gluteal muscles. IM allows for rapid absorption of the therapeutic composition.

[0327] In some embodiments of the invention, SNCA dsRNA agents or SNCA antisense polynucleotide agents may be placed in a slow-release matrix and administered by placing the matrix into a target. In some aspects of the invention, SNCA dsRNA agents or SNCA antisense polynucleotide agents may be delivered to target cells using nanoparticles coated with a delivery agent targeting specific cells or organelles. Various delivery means, methods, and agents are known in the art. Some non-limiting examples of delivery methods and agents are provided elsewhere herein. In some aspects of this invention, the term "delivery" as used with reference to an SNCA dsRNA agent or an SNCA antisense polynucleotide agent may mean administering one or more "naked" SNCA dsRNA agent or SNCA antisense polynucleotide agent sequences to a cell or object, and in other aspects of this invention, "delivery" means administration to a cell or object via transfection, delivery of a cell containing an SNCA dsRNA agent or an SNCA antisense polynucleotide agent to a object, delivery of a vector encoding an SNCA dsRNA agent or an SNCA antisense polynucleotide agent to a cell and / or object, etc. Delivery of SNCA dsRNA agents or SNCA antisense polynucleotide agents via transfection may include administration of a vector to a cell and / or object.

[0328] In some methods of the present invention, one or more SNCA dsRNA agents or SNCA antisense polynucleotide agents may be administered in a formulation, which may be administered as a pharmaceutically acceptable solution and may conventionally contain a pharmaceutically acceptable concentration of salts, buffers, preservatives, a compatible carrier, excipients, and optionally other therapeutic ingredients. In some embodiments of the present invention, the SNCA dsRNA agent or SNCA antisense polynucleotide agent may be formulated together with other therapeutic agents for simultaneous administration. According to the methods of the present invention, the SNCA dsRNA agent or SNCA antisense polynucleotide agent may be administered in a pharmaceutical composition. Generally, the pharmaceutical composition comprises an SNCA dsRNA agent or SNCA antisense polynucleotide agent and optionally a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art. As used herein, a pharmaceutically acceptable carrier means a non-toxic material that does not interfere with the bioactivity and effectiveness of the active ingredient (e.g., the ability of the SNCA dsRNA agent or SNCA antisense polynucleotide agent to inhibit SNCA gene expression in cells or subjects). Many methods for administering and delivering dsRNA agents or SNCA antisense polynucleotide agents for therapeutic purposes are known in the art and can be used in the methods of the present invention.

[0329] Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Patent No. 5,211,657, and others are known to those skilled in the art. Such formulations may conventionally contain salts, buffers, preservatives, compatibility carriers, and optional other therapeutic agents. When used in pharmaceuticals, the salts should be pharmaceutically acceptable, but non-pharmaceutical salts may be conveniently used to prepare their pharmaceutically acceptable form and are not excluded from the scope of this invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Moreover, pharmaceutically acceptable salts may be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium, or calcium salts.

[0330] Some embodiments of the method of the present invention include direct administration of one or more SNCA dsRNA agents or SNCA antisense polynucleotide agents to tissues. In some embodiments, the tissue to which the compound is administered is a tissue in which SNCA-related disease or condition is present or may occur, some non-limiting examples being the brain. Direct tissue administration can be achieved by direct injection or other means. Many orally delivered compounds naturally reach and pass through the liver and kidneys, and some embodiments of the treatment methods of the present invention include oral administration of one or more SNCA dsRNA agents to the subject. SNCA dsRNA agents or SNCA antisense polynucleotide agents, alone or in combination with other therapeutic agents, can be administered once, or alternatively, they can be administered in multiple administrations. If multiple administrations are performed, the SNCA dsRNA agents or SNCA antisense polynucleotide agents can be administered via different routes. For example, but not intended to be limiting, the first (or first few) administrations can be performed subcutaneously, and one or more additional administrations can be oral and / or systemic.

[0331] For some embodiments of the present invention where systemic administration of SNCA dsRNA or SNCA antisense polynucleotide agents is desired, the SNCA dsRNA or SNCA antisense polynucleotide agents may be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be present in unit dosage forms, with or without added preservatives, such as in ampoules or multi-dose containers. SNCA dsRNA agent formulations (also referred to as pharmaceutical compositions) may be in the form of suspensions, solutions, or emulsions, for example, in oily or aqueous carriers, and may contain formulations such as suspending agents, stabilizers, and / or dispersants.

[0332] Preparations intended for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Some examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Aqueous carriers include water, alcohol / water solutions, emulsions, or suspensions, including saline and buffer media. Parenteral carriers include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or non-volatile oils. Intravenous carriers include fluids and nutritional supplements, electrolyte supplements (e.g., those based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases. Lower doses will be obtained by other forms of administration, such as intravenous administration. If the patient's response is insufficient at the initial dose, a higher dose may be used (or at an effective higher dose via a different, more localized delivery route) to the extent that patient tolerance allows. Multiple daily doses may be used as needed to achieve appropriate systemic or local levels of one or more SNCA dsRNA agents or SNCA antisense polynucleotide agents and to achieve an appropriate reduction in SNCA protein activity.

[0333] In other embodiments, the method of the present invention includes using a delivery carrier suitable for implantation into a recipient (e.g., a subject), such as biocompatible microparticles, nanoparticles, or implants. Exemplary bioerodible implants available according to this method are described in PCT Publication No. WO 95 / 24929 (incorporated herein by reference), which describes a biocompatible, biodegradable polymer matrix comprising biomacromolecules.

[0334] In the methods of this invention, both non-biodegradable and biodegradable polymer matrices can be used to deliver one or more SNCA dsRNA agents or SNCA antisense polynucleotide agents to a target. In some embodiments, the matrix may be biodegradable. The matrix polymer may be a natural or synthetic polymer. The polymer may be selected based on the desired release time period, typically from about a few hours to a year or longer. Generally, release over a period of several hours to three to twelve months is possible. The polymer is optionally in the form of a hydrogel in which it can absorb up to about 90% of its weight in water, and is also optionally crosslinked with multivalent ions or other polymers.

[0335] Generally, in some embodiments of the invention, biodegradable implants can be used to deliver SNCA dsRNA agents or SNCA antisense polynucleotide agents via diffusion or degradation of a polymer matrix. Exemplary synthetic polymers for such uses are well known in the art. Biodegradable and non-biodegradable polymers can be used for the delivery of SNCA dsRNA agents or SNCA antisense polynucleotide agents using methods known in the art. Bioadhesive polymers (e.g., biodegradable hydrogels) (see HS Sawhney, CP Pathak and JA Hubell in Macromolecules, 1993, 26, 581-587, whose teachings are incorporated herein by reference) can also be used to deliver SNCA dsRNA agents or SNCA antisense polynucleotide agents for the treatment of SNCA-related diseases or conditions. Other suitable delivery systems may include timed-release, delayed-release, or sustained-release delivery systems. Such systems avoid repeated administration of SNCA dsRNA agents or SNCA antisense polynucleotide agents, increasing convenience for subjects and healthcare professionals. Many types of release delivery systems are available and are known to those skilled in the art. (See, for example: U.S. Patent Nos. 5,075,109; 4,452,775; 4,675,189; 5,736,152; 3,854,480; 5,133,974; and 5,407,686 (the teachings of which are incorporated herein by reference). Additionally, pump-based hardware delivery systems are available, some of which are suitable for implantation.

[0336] The use of long-term sustained-release implants is suitable for prophylactic treatment subjects and subjects at risk of developing recurrent SNCA-related diseases or conditions. As used herein, long-term release means that the implant is constructed and positioned to deliver therapeutic levels of SNCA dsRNA or SNCA antisense polynucleotide agents for at least 10 days, 20 days, 30 days, 60 days, 90 days, six months, one year, or longer. Long-term sustained-release implants are well known to those skilled in the art and include some of the aforementioned release systems.

[0337] Therapeutic formulations of SNCA dsRNA or SNCA antisense polynucleotide drugs can be prepared for storage by mixing molecules or compounds of desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers [Remington's Pharmaceutical Sciences, 21st edition, (2006)] in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dose and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethylamine chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); low molecular weight (less than about 10). (Residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN. ® PLURONICS ® Or polyethylene glycol (PEG).

[0338] Cells, subjects, and controls

[0339] The methods of this invention can be used in conjunction with cells, tissues, organs, and / or objects. In some aspects of this invention, the object is a human or vertebrate mammal, including but not limited to dogs, cats, horses, cattle, sheep, mice, rats, and primates such as monkeys. Therefore, this invention can be used to treat SNCA-related diseases or conditions in both human and non-human objects. In some aspects of this invention, the object can be a farm animal, zoo animal, domesticated animal, or non-domesticated animal, and the methods of this invention can be used in veterinary prevention and treatment programs. In some embodiments of this invention, the object is a human, and the methods of this invention can be used in human prevention and treatment programs.

[0340] Some non-limiting examples of subjects to whom this invention can be applied are those diagnosed with, suspected of having, or at risk of having a disease or condition associated with higher-than-expected SNCA expression and / or activity (also referred to as "elevated SNCA expression levels"). Some non-limiting examples of diseases and conditions associated with higher-than-expected SNCA expression and / or activity levels are described elsewhere herein. The methods of this invention can be applied to subjects who have been diagnosed with a disease or condition associated with higher-than-expected SNCA expression and / or activity at the time of treatment, or who are considered to be at risk of having or developing a disease or condition associated with higher-than-expected SNCA expression and / or activity levels. In some aspects of this invention, the disease or condition associated with higher-than-expected SNCA expression and / or activity levels is an acute disease or condition, and in other aspects of this invention, the disease or condition associated with higher-than-expected SNCA expression and / or activity levels is a chronic disease or condition.

[0341] In one non-limiting example, the SNCA dsRNA agent of the present invention is administered to a subject diagnosed with, suspected of having, or at risk of having symptoms of SNCA-related neurodegenerative disease, which is a disease for which it is desired to reduce SNCA expression. The method of the present invention can be applied to subjects who have been diagnosed with the disease or condition at the time of treatment, or who are considered to be at risk of having or developing the disease or condition.

[0342] In another non-limiting example, the SNCA dsRNA agent of the present invention is administered to subjects diagnosed with, suspected of having, or at risk of having symptoms of PD, wherein PD is a disease for which it is desired to reduce SNCA expression. The method of the present invention can be applied to subjects who have been diagnosed with the disease or condition at the time of treatment, or who are considered to be at risk of having or developing the disease or condition.

[0343] Cells applicable to the methods of the present invention include cells that are in vitro, in vivo, or isolated. Cells may be in the object, in a culture, and / or suspension, or in any other suitable state or condition. Cells to which the methods of the present invention are applicable may be liver cells, hepatocytes, brain cells, prickle cells, cardiomyocytes, pancreatic cells, cardiovascular cells, kidney cells, or other types of vertebrate cells, including human and non-human mammalian cells. In some embodiments, the cells are neurons. In some embodiments, the neurons or tissues are peripheral sensory neurons, such as peripheral sensory neurons in the dorsal root ganglion, or nociceptive neurons, such as A-δ fibers or C-type fibers. In some aspects of the invention, the cells to which the methods of the present invention are applicable are healthy, normal cells known not to be diseased cells. In some embodiments of the invention, the cells to which the methods and compositions of the present invention are applied are: liver cells, hepatocytes, brain cells, prickle cells, cardiomyocytes, pancreatic cells, cardiovascular cells, and / or kidney cells. In some aspects of the invention, control cells are normal cells, but it should be understood that cells with disease or symptom may also be used as control cells in certain circumstances, for example, to compare the results of treated cells with disease or symptom relative to untreated cells with disease or symptom, etc.

[0344] According to the method of the present invention, the level of SNCA peptide activity can be determined and compared with a control level of SNCA peptide activity. The control can be a predetermined value, which can take various forms. It can be a single cutoff value, such as a median or average. It can be established based on a comparison group, such as a group with normal levels of SNCA peptide and / or SNCA peptide activity and a group with elevated levels of SNCA peptide and / or SNCA peptide activity. Another non-limiting example of a comparison group can be a group with one or more symptoms of or diagnosed with an SNCA-related disease or condition; a group without one or more symptoms of or not diagnosed with the disease or condition; a group of subjects treated with the siRNA of the present invention; or a group of subjects not treated with the siRNA of the present invention. Generally, the control can be based on clearly healthy normal individuals or clearly healthy cells within an appropriate age range. It should be understood that, in addition to predetermined values, the control according to the present invention can also be a sample of material tested in parallel with the experimental material. Some examples include samples from a control population to be tested in parallel with the experimental sample or control samples generated by manufacturing. In some embodiments of the present invention, the control may include cells or objects that have not been contacted with or treated with the SNCA dsRNA agent of the present invention, and in such cases, the control level of SNCA peptide and / or SNCA peptide activity may be compared with the level of SNCA peptide and / or SNCA peptide activity in cells or objects that have been contacted with the SNCA dsRNA agent or SNCA antisense polynucleotide agent of the present invention.

[0345] In some embodiments of the invention, the SNCA peptide level determined for a subject may be a control level for comparing SNCA peptide levels determined for the same subject at different times. In a non-limiting example, the SNCA level is determined in a biological sample obtained from a subject who has not received SNCA treatment according to the invention. In some embodiments, the biological sample is a tissue sample. The SNCA peptide level determined in the sample obtained from the subject may be used as a baseline or control for the subject. In the treatment methods of the invention, after one or more administrations of the SNCA dsRNA agent to the subject, one or more additional tissue samples may be obtained from the subject, and the SNCA peptide levels in the subsequent one or more samples may be compared with the subject's control / baseline level. Such comparisons can be used to assess the onset, progression, or regression of SNCA-related disease or condition in the subject. For example, a higher level of SNCA peptide in a baseline sample obtained from the subject than the level obtained from the same subject after administration of the SNCA dsRNA agent or SNCA antisense polynucleotide agent of the invention indicates regression of the SNCA-related disease or condition and indicates the efficacy of the administered SNCA dsRNA agent of the invention in treating the SNCA-related disease or condition.

[0346] In some aspects of the invention, the value of one or more of the SNCA peptide and / or SNCA peptide activity levels determined for a subject can be used as a control value for later comparison of SNCA peptide and / or SNCA activity levels in the same subject, thus allowing assessment of changes in SNCA peptide activity in the subject relative to a “baseline.” Therefore, an initial SNCA peptide level and / or an initial SNCA peptide activity level may be present in and / or determined in the subject, and the methods and compounds of the invention can be used to reduce the level of SNCA peptide and / or SNCA peptide activity in a subject, wherein the initial level is used as a control level for that subject.

[0347] Using the method of the present invention, the SNCA dsRNA agent and / or SNCA antisense polynucleotide agent of the present invention can be administered to a subject. The efficacy of administration and treatment of the present invention can be evaluated when the SNCA peptide level in the tissue sample obtained from the subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more compared to the pre-administration level of the SNCA peptide in the tissue sample obtained from the subject at a previous time point, or compared to a control level (e.g., the SNCA peptide level in a control tissue sample) without prior exposure. It should be understood that both the SNCA peptide level and the SNCA peptide activity level are related to the SNCA gene expression level. Some embodiments of the method of the present invention include administering the SNCA dsRNA and / or SNCA antisense agent of the present invention to the subject in an amount that effectively inhibits SNCA gene expression, thereby reducing the SNCA peptide level and the SNCA peptide activity level in the subject.

[0348] Some embodiments of the present invention include determining the presence, absence, and / or amount (also referred to herein as level) of SNCA peptides in one or more biological samples obtained from one or more subjects. This determination can be used to evaluate the efficacy of the treatment methods of the present invention. For example, the methods and compositions of the present invention can be used to determine the level of SNCA peptides in biological samples obtained from subjects previously treated by administration of the SNCA dsRNA agent and / or SNCA antisense agent of the present invention. A reduction in the level of SNCA peptides determined in tissue samples obtained from treated subjects by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more compared to the pre-treatment level of the SNCA peptides determined for the subject, or compared to the level in unexposed control biological samples, indicates the efficacy level of the treatment administered to the subject.

[0349] In some embodiments of the invention, the physiological characteristics of SNCA-related disease or condition identified in a subject may be determined by a control determination comparing the determination of physiological characteristics in the same subject at different times. In a non-limiting example, the level of physiological characteristics, such as SNCA mRNA, α-synuclein, or other parameters functionally associated with SNCA expression levels in plasma or tissue samples, is determined in biological samples (e.g., tissue samples) obtained from a subject who has not received the SNCA treatment of the present invention. The SNCA mRNA levels (and / or other physiological characteristics of SNCA disease or condition) determined in samples obtained from the subject may be used as a baseline or control for the subject. In the treatment methods of the present invention, after one or more administrations of an SNCA dsRNA agent to a subject, one or more additional tissue samples may be obtained from the subject, and the levels of SNCA mRNA, α-synuclein, or other parameters functionally associated with SNCA expression levels in the subsequent one or more samples are compared, respectively, to the control / baseline levels and / or ratios of the subject. Such comparisons can be used to assess the onset, progression, or remission of SNCA-related disease or condition in the subject. For example, if the SNCA mRNA level in a baseline sample obtained from a subject is higher than the SNCA mRNA level determined in a sample obtained from the same subject after administration of the SNCA dsRNA agent or SNCA antisense polynucleotide agent of the present invention to the subject, it indicates the regression of SNCA-related disease or condition and demonstrates the efficacy of the applied SNCA dsRNA agent of the present invention in treating SNCA-related disease or condition.

[0350] In some aspects of the invention, values ​​of one or more of the physiological characteristics of an SNCA-related disease or condition identified for a subject can be used as control values ​​for later comparison of physiological characteristics in the same subject, thus allowing assessment of changes in physiological characteristics relative to a “baseline” in the subject. Therefore, initial physiological characteristics may be present in and / or identified in the subject, and the methods and compounds of the invention can be used to reduce the level of SNCA peptides and / or SNCA peptide activity in a subject, wherein the initial physiological characteristics identified are used as a control for that subject.

[0351] Using the method of the present invention, the SNCA dsRNA agent and / or SNCA antisense polynucleotide agent of the present invention can be administered to a subject in an effective amount to treat SNCA disease or condition. The efficacy of the administration and treatment of the present invention can be assessed by determining changes in one or more physiological characteristics of the SNCA disease or condition. In a non-limiting example, the SNCA mRNA level in the tissue sample obtained from the subject was reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the pre-administration SNCA mRNA level in a tissue sample obtained from the subject at a previous time point, or compared to the level of a control that has not been exposed (e.g., the SNCA mRNA level in a control tissue sample). It should be understood that the levels of SNCA mRNA, α-synuclein, or other parameters functionally associated with SNCA expression levels in plasma or tissue samples are each correlated with SNCA gene expression levels. Some embodiments of the method of the present invention include administering the SNCA dsRNA and / or SNCA antisense agent of the present invention to a subject in an amount that effectively inhibits SNCA gene expression, thereby reducing the levels of SNCA mRNA, α-synuclein, or other parameters that are functionally associated with SNCA expression levels in the subject, or otherwise positively influencing the physiological characteristics of SNCA-related diseases or conditions in the subject.

[0352] Some embodiments of the present invention include using, for example but not limited to, the following methods to determine the presence, absence, and / or variation of physiological characteristics of SNCA-related diseases or conditions: (1) assessing the physiological characteristics of one or more biological samples obtained from one or more subjects; (2) imaging the subjects (e.g., but not limited to obtaining liver imaging); and (3) or performing a physical examination on the subjects. This determination can be used to evaluate the efficacy of the treatment methods of the present invention.

[0353] medicine box

[0354] The kit is also within the scope of this invention, comprising one or more SNCA dsRNA agents and / or SNCA antisense polynucleotide agents, along with instructions for their use in the methods of this invention. The kit of this invention may comprise one or more of SNCA dsRNA agents, SNCA sense polynucleotides, and SNCA antisense polynucleotide agents that can be used to treat SNCA-related diseases or conditions. Kits comprising one or more SNCA dsRNA agents, SNCA sense polynucleotides, and SNCA antisense polynucleotide agents can be prepared for use in the treatment methods of this invention. The components of the kit of this invention may be packaged in an aqueous medium or in lyophilized form. The kit of this invention may comprise a carrier divided into sections that tightly seal and contain one or more container devices or a series of container devices, such as test tubes, vials, flasks, bottles, syringes, etc. The first container device or series of container devices may contain one or more compounds, such as SNCA dsRNA agents and / or SNCA sense or antisense polynucleotide agents. The second container device or a series of container devices may contain a targeting agent, a labeling agent, a delivery agent, etc., which, in one embodiment of the treatment method of the present invention, may be included as part of an SNCA dsRNA agent to be administered and / or an SNCA antisense polynucleotide.

[0355] The medicine box of the present invention may also include instructions. The instructions will generally be in written form and will provide guidance for implementing the treatment achieved through the medicine box and for making decisions based on the treatment.

[0356] The following examples are provided to illustrate specific instances of the practice of the invention and are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that the invention will be implemented in a variety of compositions and methods.

[0357] Example

[0358] Example 1. Phosphoramide compound 1

[0359]

[0360] Compound D (607 mg, 3.34 mmol, 3.0 equivalent) and DIEA (432 mg, 3.34 mmol, 582 μL, 3.0 equivalent) were added to a solution of compound B (500 mg, 1.11 mmol, 1.0 equivalent) in DCM (5.0 mL) at 0–5 °C under a N2 atmosphere. The mixture was stirred at 25 °C for 1.0 h. LC-MS showed complete consumption of compound B, with several new peaks appearing and approximately 70.9% of the desired compound detected. The resulting reaction mixture was cooled to -20 °C and poured into a cold (0–5 °C) saturated NaHCO3 (5.0 mL) solution. Extraction was performed with DCM (5.0 mL * 2). The combined organic layers were washed with cold (0–5 °C) saturated NaHCO3 / saline solution at a 1:1 ratio (5.0 mL / 5.0 mL), dried over Na2SO4, and concentrated under vacuum to give the residue (approximately 5 mL). The residue was purified by column chromatography (basic Al2O3, petroleum ether / ethyl acetate = 10 / 1 to 5 / 1, 0.1% Et3N) to give compound 1 as a white solid (280 mg, 471 μmol, 42.3% yield).

[0361]

[0362]

[0363] Example 2. Phosphoramide compound 2

[0364]

[0365] DMTrCl (232 g, 684 mmol, 1.0 equivalent) in pyridine (400 mL) was added to a solution of isomannitol compound A (100 g, 684 mmol, 1.0 equivalent) in pyridine (600 mL), and the mixture was stirred at 25 °C for 12 h. LC-MS showed complete consumption of compound A and a main peak with the desired mass was detected. The resulting reaction mixture was diluted with water (500 mL), extracted with DCM (500 mL * 2), and the combined organic phases were washed with brine (500 mL), dried over Na2SO4, and concentrated under vacuum to give the residue. The residue was purified by column chromatography (DCM / MeOH = 100 / 1 to 50 / 1, 0.1% Et3N) to give compound B (150 g, 48.9% yield) as a yellow solid.

[0366]

[0367] Under a nitrogen atmosphere, 2H-tetrazole (0.45 M, 436 mL, 1.1 equivalents) was added dropwise to a solution of compound B (80.0 g, 178 mmol, 1.0 equivalents) in DCM (800 mL) at 25 °C, followed by the dropwise addition of compound C (80.6 g, 267 mmol, 85.0 mL, 1.5 equivalents) in DCM (200 mL). The reaction mixture was stirred at 25 °C for 1.0 h. LC-MS showed complete consumption of compound B and a main peak with the desired mass was detected. The resulting reaction mixture was cooled to -20 °C and poured into ice-cold saturated NaHCO3 (500 mL), extracted with DCM (500 mL * 3), and the combined organic layers were washed with saturated NaHCO3 / salt water at a ratio of 1:1 (300 mL / 300 mL), dried over Na2SO4, and concentrated under vacuum (35 °C) to give the residue (100 mL). The residue was purified by column chromatography (Al2O3, DCM / MeOH = 100 / 1 to 50 / 1, 0.1% Et3N) to give compound 2 as a white solid (77 g, 119 mmol, 66.5% yield).

[0368]

[0369] Other phosphoramides can be prepared according to the procedures described herein and / or existing techniques (e.g., but not limited to US426,220 and WO02 / 36743).

[0370] Example 3. Preparation of a solid support containing the phosphorus amide monomer of the present invention.

[0371]

[0372] This refers to the carrier portion of amine methyl polyethylene macroporous resin.

[0373] Under nitrogen protection, dichloromethane (19.50 kg) was added to a 50 L glass reactor and stirring was initiated. The temperature was maintained at 20–30 °C, and DMT Trimann (1.47 kg), triethylamine (1.50 kg), 4-dimethylaminopyridine (0.164 kg), and succinic anhydride (1.34 kg) were added to the reactor. The system was maintained at 20–30 °C for 18 hours, a sample was obtained, and the reaction was terminated. A saturated sodium bicarbonate solution (22.50 kg) was added to the reaction system, and the mixture was stirred for 10–20 minutes, allowing for phase separation. The organic phase was separated, and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give 1.83 kg of residue forming a gray to off-white solid.

[0374] N,N-Dimethylformamide (23.50 kg) was added to a 100 L glass reactor and stirred. The temperature was maintained at 20 to 30 °C. Under nitrogen protection, the products from the previous step, O-benzotriazole tetramethylurea hexafluorophosphate (0.33 kg) and N,N-diisopropylethylamine (0.13 kg), were added to the aforementioned 100 L glass reactor through a solid feeding funnel and stirred for 10 to 30 minutes. The mixture was then discharged into a 50 L zinc barrel for use. Macroporous aminomethyl resin (3.25 kg) (purchased from Tianjin Nankai Hecheng Science and Technology Co., Ltd., batch number HA2X1209, loading capacity 0.48 mmol / g) was added to the aforementioned 100 L solid-phase synthesis reactor through a solid feed funnel. The temperature was controlled at 20 to 30 °C. N,N-dimethylformamide (21.00 kg + 21.00 kg) and the reaction solution from the zinc tank in the previous step were added to the solid-phase synthesis reactor. The system underwent a thermal insulation reaction, and the solid loading was tracked to ≥ 250 μmol / g, and the loading was detected by UV. The system was filtered under nitrogen pressure, and the filter cake was washed three times with N,N-dimethylformamide (26.00 kg + 26.10 kg + 26.00 kg), and the filter cake was left in the reactor. CAP.A (50% acetonitrile and 50% acetic anhydride, 4.40 kg + 4.42 kg + 4.30 kg) and CAP.B (20% pyridine and 30% N-methylimidazole and 50% acetonitrile, 4.40 kg + 4.40 kg + 4.47 kg) were added to an 80 L glass reactor and stirred for 3 to 8 minutes before use. This operation was repeated three times to cap the mixture, and acetonitrile (18.00 kg + 18.00 kg + 18.00 kg + 17.50 kg + 17.50 kg) was added to a solid-phase synthesis reactor. After bubbling with nitrogen for 10 to 30 minutes, the mixture was filter-pressed. This operation was repeated four times, and the filter cake was purged with nitrogen in the solid-phase synthesis reactor for 2 to 4 hours, and then transferred to a 50 L filter press. The temperature was controlled at 15 to 30 °C and the mixture was continuously dried, yielding a yellow to white solid product weighing 3.516 kg.

[0375] Isomannitol residues can be added to the 5' or 3' end of the oligonucleotide chain and further added to the target group using methods known to those skilled in the art, such as the invab method.

[0376] Example 4. Preparation of 5'-phosphate mimic phosphoramidite

[0377]

[0378] Enantiomers of phosphoramidite-15-1 and phosphoramidite-15-2

[0379] Benzoyl chloride (126 g, 893 mmol, 104 mL) was added to a solution containing 50.0 g, 446 mmol of uracil in acetonitrile (1.50 L) and pyridine (735 g, 9.29 mol, 750 mL). The reaction solution was stirred at 20 to 25 °C for 12.0 h, and TLC showed complete consumption of the compound uracil. The reaction mixture was concentrated under vacuum to obtain a residue. The residue was diluted with cold water (1.0 L) and extracted with ethyl acetate (1.0 L * 3). The combined organic layers were washed with brine (500 mL) and dried over anhydrous sodium sulfate to obtain a residue. The residue was purified by column chromatography (SiO2, ethyl acetate / petroleum ether = 1 / 10 to 1 / 1) to obtain a white solid Phos-15-1A (63 g, 65.3% yield).

[0380]

[0381] Triphenylphosphine (11.5 g, 43.9 mmol) and diethyl azodicarbonate (7.64 g, 43.9 mmol, 7.98 mL) were added to a solution of Phos-15-SM2 (4.0 g, 47.6 mmol) and compound Phos-15-1A (7.91 g, 36.6 mmol) in tetrahydrofuran (80 mL). The mixture was stirred at 20–25 °C for 16 h. LC-MS showed complete consumption of compound Phos-15-1A. The reaction mixture was concentrated under reduced pressure to remove tetrahydrofuran. The residue was diluted with water (80 mL) and subsequently extracted with ethyl acetate (80 mL * 3). The combined organic phases were washed with brine (80 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, MeOH / DCM = 0 / 10 to 1 / 10) to obtain compound Phos-15-1B (14 g, crude product) as a white solid.

[0382] Under nitrogen protection, a mixture of compound Phos-15-1B (7.0 g, 9.30 mmol) and m-chloroperoxybenzoic acid (2.27 g, 11.1 mmol, 85% purity) in dichloromethane (70 mL) was heated at 0 to 5 °C. After 16 hours of reaction, TLC showed complete consumption of compound Phos-15-1B and detection of a major new spot with lower polarity. The pH of the reaction mixture was slowly adjusted to 7 to 8 with a saturated solution of NaHSO3 and NaHCO3 (1:1), followed by extraction with ethyl acetate (70 mL * 3) and washing of the combined organic phases with brine (700 mL). The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (100 to 200 mesh silica gel) and eluted with ethyl acetate:petroleum ether (1:30 to 1:1) to give a white solid compound Phos-15-1C (1.2 g, crude product).

[0383] KSAc (1.81 g, 15.8 mmol) and tetrabutylammonium iodide (TBAI, 531.4 mg, 1.44 mmol) were added to a solution of compound Phos-15-SM3 (4.0 g, 14.4 mmol) in tetrahydrofuran (24.0 mL), and the mixture was stirred at 70 °C for 4.0 h. LC-MS showed complete consumption of the starting material Phos-15-SM3 and detected a main peak with the desired target molecular weight. The reaction mixture was cooled and concentrated under reduced pressure. The solid residue was removed by filtration through a short silica gel pad and washed with ethyl acetate. The filtrate was concentrated under vacuum to obtain compound Phos-15-1D (3.50 g, 98.5% yield) as a brown oil. Compound Phos-15-1D was ready for use in the next step without further purification.

[0384]

[0385] Potassium carbonate (1.11 g, 8.05 mmol) and compound Phos-15-1D (1.91 g, 8.45 mmol) were added to a solution of compound Phos-15-1C (1.20 g, 4.02 mmol) in ethanol (15.0 mL), and the mixture was stirred at 20–25 °C for 3.0 h. TLC showed complete consumption of compound Phos-15-1C and the detection of a major new spot with high polarity. The resulting reaction mixture was filtered, diluted with water (20 mL), extracted with dichloromethane (20 mL x 3), the combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the residue. The residue was purified by column chromatography (SiO2, MeOH / DCM = 1 / 100 to 10 / 100) to obtain compound Phos-15-1E as a brown oil (1.00 g, 65.7% yield, a 1:1 mixture of enantiomers -1E-1 and -1E-2).

[0386]

[0387] Compound Phos-15-1E can be obtained as enantiomers Phos-15-1E-1 and Phos-15-1E-2 by chiral resolution. Resolution conditions: DAICELCHIRALPAK AD 40 mm column, 140 mL / min, ethanol:carbon dioxide = 35:75. It should be understood that when enantiomers phosphoramidite-15-1 or phosphoramidite-15-2 are desired, they can be obtained simply by using the corresponding enantiomers Phos-15-1E-1 or Phos-15-1E-2 as starting materials and reacting them with a phosphorus reagent.

[0388] At room temperature, under a nitrogen atmosphere, a solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (P reagent, 956 mg, 3.17 mmol, 1.01 mL) in dichloromethane (0.5 mL) was added to a solution of compound Phos-15-1E (400 mg, 1.06 mmol) and diisopropylamine-tetrazole salt (199 mg, 1.16 mmol) in dichloromethane (4.0 mL), and the mixture was stirred at 40 °C for 1.0 h. LC-MS showed complete consumption of compound Phos-15-1E, several new peaks appeared on the LC-MS, and approximately 80% of the desired compound was detected. The resulting reaction mixture was cooled to -20°C and poured into a cold (0 to 5°C) saturated sodium bicarbonate aqueous solution (10 mL). Extraction was performed with dichloromethane (10 mL x 2), and the combined organic layers were washed with a cold (0 to 5°C) saturated sodium bicarbonate aqueous solution / saline solution (5 mL x 5 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain a residue (approximately 2.0 mL). The residue was purified by column chromatography (basic Al₂O₃, MeOH / DCM = 1 / 80 to 1 / 40, 0.1% Et₃N) to give phosphoramidite-15 as a colorless oil (350 mg, 0.6 mmol, 57.2% yield, a 1:1 mixture of enantiophosphoramidite-15-1 and enantiophosphoramidite-15-2).

[0389] Enantiophosphoramidite-15-1 or enantiophosphoramidite-15-2 can be obtained from the corresponding Phos-15-1E-1 or Phos-15-1E-2 obtained by SFC separation and purification, according to the same method as described above.

[0390]

[0391] The specific preparation methods for Phos-15-1E-1 or Phos-15-1E-2 chiral compounds are as follows:

[0392] System: Waters SFC 150

[0393] Column Name: DAICELCHIRALCEL® AD

[0394] Column model: 250*50 mm 10 m

[0395] Mobile phase A: Supercritical CO2

[0396] Mobile phase B: EtOH

[0397] Wavelength: 214 nm

[0398] Flow rate: 140 mL / min

[0399] Column temperature: room temperature (RT)

[0400] Injection volume: 7.0 mL; Cycle time: 10.0 min

[0401] Solvents: Supercritical CO2: Food grade; EtOH: Redistilled grade.

[0402] Preparation of phosphoramide-43:

[0403]

[0404] (3aR, 6aR)-2,2-dimethyltetrahydro-3aH-cyclopentane[d][1,3]dioxacyclopenten-4(6aH)-one (phos-43-SM1, 16.2 g, 105 mmol, 1.0 equivalent), diethyl(mercaptomethyl)phosphonate (19.3 g, 105 mmol, 1.0 equivalent), and dichloromethane (200 mL) were added to a 500 mL flask. The flask was stirred and cooled to 0–5 °C under nitrogen protection, and then triethylamine (1.06 g, 10.5 mmol, 0.1 equivalent) was added dropwise. After the addition was complete, the temperature was restored to 25 °C, and the mixture was stirred overnight under nitrogen protection. The reaction was detected as complete by LCMS, and the reaction solution was concentrated under vacuum to obtain the crude product. The crude product was purified by rapid column chromatography using an eluent of (EA: DCM = 0% to 15%) to elute the product. The product was concentrated under vacuum to obtain 25 g of Phos-43-1A as a pale yellow oil, in 70.3% yield.

[0405] LCMS: M+H=339.5.

[0406] Phos-43-1A (25 g, 73.9 mmol, 1.0 equivalent) and ethanol (250 mL) were added to a 500 mL flask. The flask was stirred and cooled to 0–5 °C under nitrogen protection, and sodium borohydride (3.1 g, 81.3 mmol, 1.1 equivalent) was added in batches. After the addition was complete, the mixture was stirred at 0–5 °C for 0.5 h. The reaction was confirmed to be complete by LCMS. Ice water (200 mL) was added dropwise to the reaction solution and stirred for 10 min. The mixture was then extracted twice with dichloromethane (500 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under vacuum to obtain a crude product. The crude product was purified by rapid column chromatography using a solution of MeOH: DCM = 0%–5% to elute the product. The product was concentrated under vacuum to obtain 24.5 g of Phos-43-1B as a pale yellow oil. The yield was 97.4%.

[0407] LCMS:M+H=341.5.

[0408]

[0409] Phos-43-1B (10 g, 29.4 mmol, 1.0 equivalent), pyridine (7 g, 88.1 mmol, 3.0 equivalent), and dichloromethane (100 mL) were added to a 250 mL flask. The flask was stirred and cooled to -78 °C under nitrogen protection, followed by dropwise addition of trifluoromethanesulfonic anhydride (12.4 g, 44.1 mmol, 1.5 equivalent). After the dropwise addition was complete, the flask was maintained at -78 °C and stirred under nitrogen protection for 3 hours. The reaction was confirmed to be complete by LC-MS. The reaction solution was poured into 50 mL of ice water and subsequently extracted twice with dichloromethane (100 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under vacuum to obtain the crude product Phos-43-1C, which was used directly in the next step.

[0410] LCMS:M+H=473.4.

[0411] Phos-43-1C (16 g, 33.8 mmol, 1.0 equivalent), 3-benzoyluracil (8.8 g, 40.6 mmol, 1.2 equivalent), cesium carbonate (22 g, 67.7 mmol), and acetonitrile (200 mL) were added to a 500 mL flask. The flask was stirred overnight at 25 °C under nitrogen protection. The reaction was confirmed by LC-MS. The reaction solution was filtered, and the filtrate was concentrated under vacuum to obtain a crude product. The crude product was purified by rapid column chromatography using MeOH:DCM = 0% to 5% as eluent. The product was concentrated under vacuum to obtain 18 g of Phos-43-1D as a brown oil, with a yield of 98.7%.

[0412] LCMS:M+H=539.4.

[0413] Phos-43-1D (18 g, 33.4 mmol, 1.0 equivalent) and methanol (180 mL) were added to a 500 mL flask. Ammonia-methanol (180 mL) was added dropwise to the flask under nitrogen protection. After the dropwise addition was complete, the mixture was stirred at 25 °C for 5 hours under nitrogen protection. The reaction was detected as complete by LCMS, and the reaction solution was concentrated under vacuum to obtain the crude product Phos-43-1E, which was used directly in the next step.

[0414] LCMS:M+H=435.4.

[0415] Phos-43-1E (14.5 g, 33.4 mmol, 1.0 equivalent) and dioxane (180 mL) were added to a 500 mL flask. Dioxane hydrochloride (4 M, 180 mL) was added dropwise, and the flask was stirred overnight at 25 °C under nitrogen protection. The reaction was detected as complete by LC-MS, and the reaction solution was concentrated under vacuum to obtain the crude product. The crude product was purified by rapid column chromatography using MeOH:DCM = 0% to 10% as eluent. The product was concentrated under vacuum to obtain 5.2 g of Phos-43-1F as a white solid, in a yield of 39.5%.

[0416] LCMS:M+H=395.4.

[0417] Phos-43-1F (5.2 g, 13.2 mmol, 1.0 equivalent), toluene (100 mL), and acetonitrile (20 mL) were added to a 250 mL flask, followed by the addition of cyanomethylenetri-n-butylphosphine (6.4 g, 26.5 mmol, 2.0 equivalent). The flask was stirred at 90 °C for 48 hours under nitrogen protection. The reaction was confirmed by LC-MS, and the reaction solution was concentrated under vacuum to obtain the crude product. The crude product was purified by rapid column chromatography using eluent (MeOH:DCM = 0% to 10%). The product was concentrated under vacuum to obtain 3.5 g of Phos-43-1G as a white solid, with a yield of 70.5%.

[0418] LCMS:M+H=377.3.

[0419] Phos-43-1G (2.0 g, 5.3 mmol, 1.0 equivalent), anhydrous methanol (20 mL), trimethyl borate (1.1 g, 10.6 mmol, 2.0 equivalent), methyl orthoformate (0.56 g, 5.3 mmol, 1.0 equivalent), and sodium bicarbonate (44.5 mg, 0.52 mmol, 0.2 equivalent) were added to a 250 mL stuffy jar. The mixture was heated to 120 °C and stirred for 48 hours. The stuffy jar was then cooled to room temperature. The reaction was detected as complete by LCMS, and the reaction solution was concentrated under vacuum to obtain a crude product. The crude product was purified by rapid column chromatography using an eluent (MeOH:DCM = 0% to 10%). The product was concentrated under vacuum to obtain 1.3 g of Phos-43-1H as a white solid, in a yield of 60%.

[0420] LCMS:M+H=409.4.

[0421] Phos-43-1H (0.6 g, 1.47 mmol, 1.0 equivalent) and anhydrous dichloromethane (10 mL) were added to a 50 mL flask, followed by the sequential addition of tetrazolium (0.13 g, 1.76 mmol, 1.2 equivalent) and bis(diisopropyl)(2-cyanoethoxy)phosphine (0.66 g, 2.2 mmol, 1.5 equivalent). The mixture was stirred at 25 °C for 1 hour under nitrogen protection. The reaction was confirmed to be complete by LC-MS. The reaction solution was poured into an aqueous sodium bicarbonate solution and extracted twice with dichloromethane (20 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under vacuum to obtain a crude product. The crude product was purified by rapid silica gel column chromatography, and the product was eluted with eluent (DCM:MeOH:TEA = 0% to 5% + 0.2% TEA). Concentrated under vacuum at 35°C, phosphoramidite-43 (0.89 g, 100% yield) was obtained as a colorless oil.

[0422] LCMS:M+H=609.6.

[0423]

[0424] The preparation method of phosphoramide-47 is the same as that of phosphoramide-43, except that the starting material 5-methyluracil is used as the nucleobase.

[0425] Preparation of phosphoramide-45

[0426]

[0427] Magnesium chip (0.26 g, 10.6 mmol, 10.0 equivalent) and anhydrous ethanol (40 mL) were added to a 100 mL airtight container, and the mixture was heated to 90 °C and stirred for 18 hours. The container was cooled to room temperature, and Phos-43-1G (0.4 g, 1.06 mmol, 1.0 equivalent) was added. The mixture was heated to 90 °C and stirred for 18 hours. The container was then cooled to room temperature. LC-MS analysis revealed incomplete reaction, with approximately 50% converted to Phos-45-1A. The reaction solution was concentrated under vacuum to obtain a crude product. The crude product was purified by rapid column chromatography using an eluent (MeOH:DCM = 0% to 8%). The product was concentrated under vacuum to obtain 0.13 g of Phos-45-1A as a pale yellow oil, in a yield of 29%.

[0428] LCMS:M+H=423.4.

[0429] Phos-45-1A (0.11 g, 0.26 mmol, 1.0 equivalent) and anhydrous dichloromethane (3 mL) were added to a 50 mL flask, followed by the sequential addition of tetrazolium (22 mg, 0.31 mmol, 1.2 equivalent) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.12 g, 0.4 mmol, 1.5 equivalent). The mixture was stirred at 25 °C for 1 hour under nitrogen protection. The reaction was confirmed by LCMS. The reaction solution was poured into an aqueous sodium bicarbonate solution and extracted twice with dichloromethane (10 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under vacuum to obtain a crude product. The crude product was purified by rapid silica gel column chromatography and eluted with eluent (DCM:MeOH:TEA = 0% to 3% + 0.2% TEA). Concentrated under vacuum at 35°C, to obtain phosphorus amide yellow oil Phos-45 (0.1 g, yield 61.7%).

[0430] LCMS: M+H=623.5

[0431]

[0432] Preparation of phosphoramide-46:

[0433]

[0434] Magnesium flakes (0.48 g, 20.0 mmol, 15.0 equivalent) and anhydrous ethylene glycol monomethyl ether (50 mL) were added to a 100 mL aerator-free vessel, and the mixture was heated to 90 °C and stirred for 1 hour. The aerator-free vessel was cooled to room temperature, and Phos-43-1G (0.5 g, 1.33 mmol, 1.0 equivalent) was added. The mixture was heated to 90 °C and stirred for 18 hours. The aerator-free vessel was cooled to room temperature, and LC-MS showed complete disappearance of the reactants. The reaction solution was transferred to a flask, and 0.5 N dilute hydrochloric acid was added dropwise at 0 °C to adjust the pH to 6. The mixture was extracted five times with dichloromethane (50 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under vacuum to obtain a crude product. The crude product was purified by rapid column chromatography using an eluent (MeOH:DCM = 0% to 10%). The product was concentrated under vacuum to obtain 0.13 g of Phos-46-1A as a pale yellow oil, with a yield of 20%.

[0435] LCMS: M+H=513.4.

[0436] Phos-46-1A (0.1 g, 0.19 mmol, 1.0 equivalent) and anhydrous dichloromethane (3 mL) were added to a 50 mL flask, followed by the sequential addition of tetrazolium (16 mg, 0.23 mmol, 1.2 equivalent) and bis(diisopropyl)(2-cyanoethoxy)phosphine (0.09 g, 0.3 mmol, 1.5 equivalent). The mixture was stirred at 25 °C for 1 hour under nitrogen protection. The reaction was confirmed by LCMS. The reaction solution was poured into an aqueous sodium bicarbonate solution and extracted twice with dichloromethane (10 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under vacuum to obtain a crude product. The crude product was purified by rapid silica gel column chromatography and eluted with eluent (DCM:MeOH:TEA = 0% to 5% + 0.2% TEA). The phosphoramidite was concentrated under vacuum at 35°C to obtain Phos-46 (93 mg, yield 66.9%), a yellow oily substance.

[0437] LCMS: M+H=713.6.

[0438]

[0439] The 5'-phosphonate-modified nucleoside analogs described in this article can be prepared using similar methods or synthetic routes known in the art.

[0440] The phosphoramidite compounds described herein are coupled to the 5' end of oligonucleotides to produce 5'-terminal nucleotides, as described in CN110072530A and CN103154014A, wherein each phosphonate group has a hydroxyl protecting atom, for example, containing two methyl or ethyl protected oxygen atoms, one or both methyl or ethyl atoms being removed depending on the deprotection step used. In some embodiments, a nitrile:trimethylsilyl iodide:pyridine = 50:2:2 (v / v / v) deethylation solution is used to remove ethyl protection.

[0441] Preparation of phosphoramide-53

[0442]

[0443] BzCl (31.34 g, 5.0 equivalent) was added dropwise to a mixture of Phos-53-SM1 (25.00 g, 1.0 equivalent) and DMAP (2.72 g, 0.5 equivalent) in pyridine (250 mL) at 0 °C, and the mixture was stirred at 24 °C for 18 h. LC MS showed that Phos-53-1A was produced and Phos-53-SM1 was consumed. The mixture was quenched with MeOH and evaporated under reduced pressure to give a crude product, which was used directly for the next step. Analytical data: MS: [MH] -=768.

[0444] TFA (15 mL) was added to a solution of Phos-53-1A (100 g, crude) in DCM (500 mL), and the mixture was stirred at 24 °C for 2 h. LCMS showed that Phos-53-1A was consumed and Phos-53-1B was produced. The mixture was quenched with MeOH and evaporated under reduced pressure to obtain the crude product, which was purified by silica gel chromatography using a gradient of MeOH in DCM (0 to 50%) to obtain Phos-53-1B (20 g, 96% after two steps). Analytical data: [M+H] + =467.

[0445] 4-OHTEMPO (2.95 g, 0.4 equivalent) and DIB (27.62 g, 2.0 equivalent) were added to a solution of Phos-53-1B (20 g, 1.0 equivalent) in ACN (210 mL) and H2O (140 mL), and the mixture was stirred at 24 °C for 18 hours. 4-OHTEMPO (1.5 g, 0.2 equivalent) and DIB (13 g, 0.95 equivalent) were added to the mixture, and the mixture was stirred at 24 °C for 4 hours. The mixture was diluted with ethyl acetate. The organic layer was separated, washed with water and brine, dried over ...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of synuclein α (SNCA), wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides differing from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides differing from the nucleotide sequence of SEQ ID NO: 2 by no more than 3 nucleotides, wherein the sense strand and the antisense strand may be partially, substantially, or completely complementary to each other, and optionally contain a targeting ligand.

2. The dsRNA agent of claim 1, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by no more than 3 nucleotides, and the sense strand is complementary to the at least 15 consecutive nucleotides in the antisense strand.

3. The dsRNA of any one of claims 1 to 2, wherein the dsRNA agent comprises at least one modified nucleotide.

4. The dsRNA agent according to any one of claims 1 to 3, wherein all or substantially all nucleotides of the sense strand and / or the antisense strand are modified nucleotides.

5. A double-stranded RNA (dsRNA) agent for inhibiting the expression of synuclein α (SNCA), wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand is complementary to the antisense strand, wherein the antisense strand comprises a region partially complementary to mRNA encoding SNCA, wherein each strand is about 15 to about 30 nucleotides in length, wherein the sense strand sequence is represented by formula (I): in: Each N' F This refers to nucleotides modified with 2'-fluorine. N' N1 、N' N2 、N' N3 、N' N4 、N' N5 and N' N6 Each can be used independently to represent a modified or unmodified nucleotide; Each N' L Independently represents modified or unmodified nucleotides, but not nucleotides with 2'-fluorine modification; Furthermore, m' and n' are each an independent integer from 0 to 7.

6. A double-stranded RNA (dsRNA) agent for inhibiting the expression of synuclein α (SNCA), wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand is complementary to the antisense strand, wherein the antisense strand comprises a region partially complementary to mRNA encoding SNCA, wherein each strand is about 18 to about 30 nucleotides in length, wherein the sequence of the antisense strand is represented by formula (II): in: Each N F This refers to nucleotides modified with 2'-fluorine. N M1 N M2 N M3 N M4 N M5 N M6 N M7 N M8 and N M9 Each can be used independently to represent a modified or unmodified nucleotide; N L and N Z Each can be used independently to represent a modified or unmodified nucleotide, but not a nucleotide with 2'-fluorine modification. Optional, N Z This indicates a 5' terminal nucleotide containing a phosphate ester mimic, preferably N. Z It is a vinylphosphonate-modified nucleotide, more preferably, N Z It is Vpu*, which has a structure ,or, Optional, N Z It is selected from any of the following or their stereoisomers or racemates: ， And n is an integer from 0 to 7.

7. A double-stranded RNA (dsRNA) agent for inhibiting synuclein α (SNCA) expression, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a dsRNA duplex, wherein the sense strand is complementary to the antisense strand, wherein the antisense strand comprises a region complementary to mRNA encoding SNCA, wherein the complementary region comprises at least 15 consecutive nucleotides, wherein the dsRNA duplex is represented by formula (III): in: Each strand is independently about 17 to about 30 nucleotides in length; N F and N' F Each can independently represent a nucleotide modified with 2'-fluorine; N M1 N M2 N M3 N M4 N M5 N M6 N M7 N M8 N M9 、N' N1 、N' N2 、N' N3 、N' N4 、N' N5 and N' N6 Each can be used independently to represent a modified or unmodified nucleotide; N Z N L and N' L Each can be used independently to represent a modified or unmodified nucleotide, but not a nucleotide with 2'-fluorine modification; Optional, N Z This indicates a 5' terminal nucleotide containing a phosphate ester mimic, preferably N. Z It is a vinylphosphonate-modified nucleotide, more preferably, N Z It is Vpu*, which has a structure ,or, Optional, N Z It is selected from any of the following or their stereoisomers or racemates: ， Furthermore, m', n', and n are each independent integers from 0 to 7.

8. The dsRNA agent according to any one of claims 1 to 7, wherein the antisense strand comprises a region complementary to the mRNA encoding SNCA, which comprises the region of SEQ ID NO:

1. At least 15, 16, 17, 18, 19, or 20 consecutive nucleotides whose complementary sequences differ by no more than 0, 1, 2, or 3 nucleotides: 776-796, 699-719, 628-648, 614-634, 913-933, 828-848, 238-258, 325-345, 326-346, 337-357, 397-417, 587-607, 591-611, 201-221, 202-222, 203-223, 204-224, 206-226, 207-227, 209-229, 214-234, 215- 235, 216-236, 217-237, 220-240, 223-243, 224-244, 225-245, 226-246, 227-247, 228-248, 230-250, 232-252, 235-255, 236-256, 238-258, 239-259, 241-261, 268-288, 275-295, 277-297, 278-298, 306-326, 307-327, 308-328, 322-342, 324-344, 326-346, 327-347, 328-348, 338-3 58, 378-398, 386-406, 392-412, 394-414, 396-416, 398-418, 399-419, 400-420, 401-421, 402-422, 403-423, 405-425, 406-426, 409-429, 506-526, 513-533, 580-600, 583-603, 584-604, 585-605, 587-607, 594-614, 595-615, 598-618, 616-636, 617-637, 620-640, 623-643, 635-65 5. 638-658, 640-660, 642-662, 644-664, 654-674, 657-677, 664-684, 667-687, 697-717, 700-720, 704-724, 705-725, 719-739, 722-742, 726-746, 728-748, 729-749, 731-751, 735-755, 737-757, 771-791, 773-793, 775-795, 776-796, 778-798, 781-801, 782-802, 783-803, 822-842825-845、826-846、831-851、832-852、835-855、837-857、839-859、856-876、864-884、867-887、869-889、872-892、873-893、875-895、876-896、906-926、911-931、912-932、915-935、919-939、920-940、921-941、923-943、925-945、926-946、929-949、938-958、943-963、945-965、948-968、949-969、957-977、962-982、963-983、967-987、968-988、970-990、971-991、976-996、979-999、980-1000、1003-1023、1005-1025、1006-1026、1010-1030、1011-1031、1012-1032、1014-1034、1016-1036、1017-1037、1023-1043、1026-1046、1028-1048、1049-1069、1066-1086、1081-1101、1083-1103、1085-1105、1086-1106、1088-1108、1089-1109、1090-1110、1091-1111、1092-1112、1097-1117、1099-1119、1105-1125、1106-1126、1107-1127、1166-1186、1168-1188、778-796、701-719、630-648、616-634、915-933、830-848、240-258、327-345、328-346、339-357、399-417、589-607、593-611、203-221、204-222、205-223、206-224、208-226、209-227、211-229、216-234、217-235、218-236、219-237、222-240、225-243、226-244、227-245、228-246、229-247、230-248、232-250、234-252、237-255、238-256、240-258、241-259、243-261、270-288、277-295、279-297、280-298、308-326、309-327、310-328、324-342、326-344、328-346、329-347、330-348、340-358、380-398、388-406、394-412、396-414、398-416、400-418、401-419、402-420、403-421、404-422、405-423、407-425、408-426、411-429、508-526、515-533、582-600、585-603、586-604、587-605、589-607、596-614、597-615、600-618、618-636、619-637、622-640、625-643、637-655、640-658、642-660、644-662、646-664、656-674、659-677、666-684、669-687、699-717、702-720、706-724、707-725、721-739、724-742、728-746、730-748、731-749、733-751、737-755、739-757、773-791、775-793、777-795、778-796、780-798、783-801、784-802、785-803、824-842、827-845、828-846、833-851、834-852、837-855、839-857、841-859、858-876、866-884、869-887、871-889、874-892、875-893、877-895、878-896、908-926、913-931、914-932、917-935、921-939、922-940、923-941、925-943、927-945、928-946、931-949、940-958、945-963、947-965、950-968、951-969、959-977、964-982、965-983、969-987、970-988、972-990、973-991、978-996、981-999、982-1000、1005-1023、1007-1025、1008-1026、1012-1030、1013-1031、1014-1032、1016-1034、1018-1036、1019-1037、1025-1043、1028-1046、1030-1048、1051-1069、1068-1086、1083-1101、1085-1103、1087-1105、1088-1106、1090-1108、1091-1109、1092-1110、1093-1111、1094-1112、1099-1117、1101-1119、1107-1125、1108-1126、1109-1127、1168-1186、1170-1188、777-795、700-718、629-647、615-633、914-932、829-847、239-257、326-344、327-345、338-356、398-416、588-606、592-610、202-220、203-221、204-222、205-223、207-225、208-226、210-228、215-233、216-234、217-235、218-236、221-239、224-242、225-243、226-244、227-245、228-246、229-247、231-249、233-251、236-254、237-255、239-257、240-258、242-260、269-287、276-294、278-296、279-297、307-325、308-326、309-327、323-341、325-343、327-345、328-346、329-347、339-357、379-397、387-405、393-411、395-413、397-415、399-417、400-418、401-419、402-420、403-421、404-422、406-424、407-425、410-428、507-525、514-532、581-599、584-602、585-603、586-604、588-606、595-613、596-614、599-617、617-635、618-636、621-639、624-642、636-654、639-657、641-659、643-661、645-663、655-673、658-676、665-683、668-686、698-716、701-719、705-723、706-724、720-738、723-741、727-745、729-747、730-748、732-750、736-754、738-756、772-790、774-792、776-794、777-795、779-797、782-800、783-801、784-802、823-841、826-844、827-845、832-850、833-851、836-854、838-856、840-858、857-875、865-883、868-886、870-888、873-891、874-892、876-894、877-895、907-925、912-930、913-931、916-934、920-938、921-939、922-940、924-942、926-944、927-945、930-948、939-957、944-962、946-964、949-967、950-968、958-976、963-981、964-982、968-986、969-987、971-989、972-990、977-995、980-998、981-999、1004-1022、1006-1024、1007-1025、1011-1029、1012-1030、1013-1031、1015-1033、1017-1035、1018-1036、1024-1042、1027-1045、1029-1047、1050-1068、1067-1085、1082-1100、1084-1102、1086-1104、1087-1105、1089-1107、1090-1108、1091-1109、1092-1110、1093-1111、1098-1116、1100-1118、1106-1124、1107-1125、1108-1126、1167-1185、1169-1187、777-793、700-716、629-645、615-631、914-930、829-845、239-255、326-342、327-343、338-354、398-414、588-604、592-608、202-218、203-219、204-220、205-221、207-223、208-224、210-226、215-231、216-232、217-233、218-234、221-237、224-240、225-241、226-242、227-243、228-244、229-245、231-247、233-249、236-252、237-253、239-255、240-256、242-258、269-285、276-292、278-294、279-295、307-323、308-324、309-325、323-339、325-341、327-343、328-344、329-345、339-355、379-395、387-403、393-409、395-411、397-413、399-415、400-416、401-417、402-418、403-419、404-420、406-422、407-423、410-426、507-523、514-530、581-597、584-600、585-601、586-602、588-604、595-611、596-612、599-615、617-633、618-634、621-637、624-640、636-652、639-655、641-657、643-659、645-661、655-671、658-674、665-681、668-684、698-714、701-717、705-721、706-722、720-736、723-739、727-743、729-745、730-746、732-748、736-752、738-754、772-788、774-790、776-792、777-793、779-795、782-798、783-799、784-800、823-839、826-842、827-843、832-848、833-849、836-852、838-854、840-856、857-873、865-881、868-884、870-886、873-889、874-890、876-892、877-893、907-923、912-928、913-929、916-932、920-936、921-937、922-938、924-940、926-942、927-943、930-946、939-955、944-960、946-962、949-965、950-966、958-974、963-979、964-980、968-984、969-985、971-987、972-988、977-993、980-996、981-997、1004-1020、1006-1022、1007-1023、1011-1027、1012-1028、1013-1029、1015-1031、1017-1033、1018-1034、1024-1040、1027-1043、1029-1045、1050-1066、1067-1083、1082-1098、1084-1100, 1086-1102, 1087-1103, 1089-1105, 1090-1106, 1091-1107, 1092-1108, 1093-1109, 1098-1114, 1100-1116, 1106-1122, 1107-1123, 1108-1124, 1167-1183 or 1169-1185.

9. The dsRNA agent of any one of claims 1 to 8, wherein the antisense strand comprises a region complementary to the mRNA encoding SNCA, comprising at least 15, 16, 17, 18, or 19 consecutive nucleotides differing from any of the antisense sequences listed in any one of Tables 1 to 3 by no more than 1, 2, or 3 nucleotides.

10. The dsRNA agent of any one of claims 1 to 9, wherein the antisense strand comprises a region complementary to the mRNA encoding SNCA, comprising at least 15, 16, 17, 18, or 19 consecutive nucleotides from any of the antisense sequences listed in any one of Tables 1 to 3.

11. The dsRNA agent of any one of claims 1 to 10, wherein the antisense strand of the dsRNA is substantially or completely complementary to any of the target regions of SEQ ID NO: 1, and preferably, the dsRNA agent comprises the antisense strand sequence shown in any one of Tables 1 to 3.

12. The dsRNA agent of any one of claims 1 to 11, wherein the sense sequence of the dsRNA agent is at least substantially complementary to or completely complementary to the antisense sequence, preferably wherein the dsRNA agent comprises a sense sequence shown in any one of Tables 1 to 3.

13. The dsRNA agent according to any one of claims 1 to 12, wherein the dsRNA agent comprises a sequence shown as a double-stranded sequence in any one of Tables 1 to 3.

14. The dsRNA agent according to any one of claims 1 to 13, wherein one or more of the modified nucleotides are independently selected from: 2'-O-methyl nucleotides, 2'-fluoronucleotides, 2'-deoxynucleotides, 2'3'-seco nucleotide mimics, locked nucleotides, unlocked nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, abase-free nucleotides, ribitol, reverse nucleotides, reverse abase-free nucleotides, isomannitol nucleotides, reverse 2'-Ome nucleotides, reverse 2'-deoxynucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholinonucleotides, 3'-OMe nucleotides, modified nucleotides modified with 5'-phosphonates or 5'-phosphate mimics, nucleotides containing a 5'-thiophosphate group, terminal nucleotides linked to a cholesterol derivative or a dodecanoic acid bis(decylamide) group, 2'-amino-modified nucleotides, aminophosphates, or nucleotides containing a non-natural base.

15. The dsRNA agent according to any one of claims 1 to 14, wherein the 5' end of the guide strand comprises an E-vinylphosphonate nucleotide, or the 5' end of the antisense strand comprises a 5'-phosphate mimic nucleotide represented by formula (VIII) or a stereoisomer or racemate thereof: in: Q8 is O, S, SO, SO2, PR 16 R 17 or NR 11 ;R 16 and R 17 Independently, it is (=O), (=S), OH, SH, C1-C6 alkyl, NR 18 R 19 Ra and Rc are each independently selected from hydroxyl or protected hydroxyl, mercapto or protected mercapto, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, protected or optionally substituted amino, native or modified nucleoside; and R b Is it O, S, or NR? 12 R 12 It is protected by hydrogen, C1-C6 alkyl, and amino groups; Q1 and Q2 are each independently H, halogen, -CN, or optionally substituted C1-C6 alkyl; The substituents in the substituted amino group are selected from: optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, sulfinyl, sulfonyl, acetyl; R 11 R 18 and R 19 Independently, it is H, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, methanesulfonyl, and sulfonic acid groups; Z is a nucleoside containing a sugar or a sugar-substituted portion; T3 is an internucleotide linker that links the 5' terminal nucleotide of formula (VIII) or its stereoisomer to the remaining portion of the 5' end of the guide strand; Each substituent contains one or more substituents optionally selected independently from the following: halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl mercapto, CN.

16. The dsRNA agent according to any one of claims 1 to 15, wherein the dsRNA agent comprises at least one phosphate thioester nucleoside linker.

17. The dsRNA agent of any one of claims 1 to 15, wherein the sense strand comprises at least one phosphate thioester nucleoside linker.

18. The dsRNA agent according to any one of claims 1 to 15, wherein the antisense strand comprises at least one phosphate thioester nucleoside linker.

19. The dsRNA agent according to any one of claims 1 to 15, wherein the sense strand comprises 1, 2, 3, 4, 5 or 6 phosphate thioester nucleosides linked together.

20. The dsRNA agent according to any one of claims 1 to 15, wherein the antisense strand comprises 1, 2, 3, 4, 5 or 6 thiophosphate nucleosides linked together.

21. The dsRNA agent according to any one of claims 1 to 20, wherein the modified sense strand is a modified sense strand sequence shown in one of Tables 2 to 3.

22. The dsRNA agent according to any one of claims 1 to 20, wherein the modified antisense strand is a modified antisense strand sequence shown in one of Tables 2 to 3.

23. The dsRNA agent according to any one of claims 1 to 22, wherein the sense strand is complementary or substantially complementary to the antisense strand, and the length of the complementary region is 16 to 23 nucleotides.

24. The dsRNA agent according to any one of claims 1 to 23, wherein the length of the complementary region is 19 to 21 nucleotides.

25. The dsRNA agent according to any one of claims 1 to 24, wherein the length of each strand does not exceed 30 nucleotides.

26. The dsRNA agent according to any one of claims 1 to 24, wherein the length of each strand does not exceed 25 nucleotides.

27. The dsRNA agent according to any one of claims 1 to 24, wherein the length of each strand does not exceed 23 nucleotides.

28. The dsRNA agent of any one of claims 1 to 27, wherein the dsRNA agent comprises at least one modified nucleotide and further comprises one or more targeting groups or linking groups.

29. The dsRNA agent of claim 28, wherein one or more targeting groups or linking groups are conjugated to the sense strand.

30. The dsRNA agent of claim 28 or 29, wherein the targeting group or linking group comprises N-acetyl-galactosamine (GalNAc).

31. The dsRNA agent of claim 28 or 29, wherein the targeting group has the following structure: 。 32. The dsRNA agent of any one of claims 1 to 31, wherein the dsRNA agent comprises a targeting group conjugated to the 5' end of the sense strand.

33. The dsRNA agent of any one of claims 1 to 31, wherein the dsRNA agent comprises a targeting group conjugated to the 3' end of the sense strand.

34. The dsRNA agent according to any one of claims 1 to 31, wherein the antisense strand comprises a reverse abase-free residue at its 3' end.

35. The dsRNA agent according to any one of claims 1 to 31, wherein the sense strand comprises one or two reverse abase-free residues or imann residues at the 3' and / or 5' ends.

36. The dsRNA agent according to any one of claims 1 to 35, wherein the dsRNA agent has two blunt ends.

37. The dsRNA agent according to any one of claims 1 to 35, wherein at least one strand comprises a 3' overhang having at least one nucleotide.

38. The dsRNA agent according to any one of claims 1 to 35, wherein at least one strand comprises a 3' overhang having at least 2 nucleotides.

39. A composition comprising the dsRNA agent according to any one of claims 1 to 38.

40. The composition of claim 39, further comprising a pharmaceutically acceptable carrier.

41. The composition of claim 40, further comprising one or more additional therapeutic agents.

42. The composition of claim 41, wherein the composition is packaged in a box, container, packaging, dispenser, pre-filled syringe, or vial.

43. The composition of any one of claims 39 to 42, wherein the composition is formulated for subcutaneous administration, for intrathecal administration, or for intravenous (IV) administration.

44. A cell comprising the dsRNA agent according to any one of claims 1 to 38, wherein the cell is optionally a mammalian cell, optionally a human cell, and optionally a neuron.

45. A method for inhibiting the expression of SNCA genes in cells, the method comprising: (i) Preparing cells containing an effective amount of the double-stranded ribonucleic acid (dsRNA) agent of any one of claims 1 to 38 or the composition of any one of claims 39 to 43.

46. ​​The method of claim 45, further comprising: (ii) The cells prepared in claim 45(i) are maintained for a time sufficient to allow degradation of the mRNA transcript of the SNCA gene, thereby inhibiting the expression of the SNCA gene in the cells.

47. The method of any one of claims 45 to 46, wherein the cells are in the subject, and the dsRNA agent is administered subcutaneously to the subject.

48. The method of any one of claims 45 to 46, wherein the cells are in the subject, and the dsRNA agent is administered to the subject intrathecally, intracranially, intraventricularly, or intracerebrally.

49. The method of any one of claims 45 to 46, wherein the cells are in the subject, and the dsRNA agent is administered to the subject via IV administration.

50. The method of any one of claims 47 to 49, further comprising evaluating the inhibition of the SNCA gene after administering the dsRNA agent to the subject, wherein the means for evaluation include: (i) Identify one or more physiological characteristics of SNCA-related diseases or conditions in the subject, and (ii) Compare the identified physiological characteristics with the baseline physiological characteristics of the SNCA-related disease or condition before treatment and / or with the control physiological characteristics of the SNCA-related disease or condition. The comparisons described therein indicate one or more instances of the presence or absence of inhibition of SNCA gene expression in the object.

51. The method of claim 50, wherein the determined physiological characteristic is one or more of the following: the levels of SNCA mRNA, α-synuclein, and other parameters functionally related to SNCA expression levels in the subject.

52. The method of claim 51, wherein a decrease in one or more of the levels of SNCA mRNA, SNCA protein, and other parameters functionally related to SNCA expression levels in the object indicates a decrease in SNCA gene expression in the object.

53. A method for inhibiting the expression of the SNCA gene in a subject, the method comprising administering to the subject an effective amount of a double-stranded ribonucleic acid (dsRNA) agent as described in any one of claims 1 to 38 or a composition as described in any one of claims 39 to 43.

54. The method of claim 53, wherein the dsRNA agent is administered subcutaneously to the subject.

55. The method of claim 53, wherein the dsRNA agent is administered intrathecally, intracranially, intraventricularly, or intracerebrally to the subject.

56. The method of claim 53, wherein the dsRNA agent is administered to the subject via intravenous (IV) administration.

57. The method of any one of claims 54 to 56, further comprising evaluating the inhibition of the SNCA gene after administration of the dsRNA agent, wherein the means for said evaluation include: (i) Identify one or more physiological characteristics of SNCA-related diseases or conditions in the subject, and (ii) Compare the identified physiological characteristics with the baseline physiological characteristics of the SNCA-related disease or condition before treatment and / or with the control physiological characteristics of the SNCA-related disease or condition. The comparisons described therein indicate one or more instances of the presence or absence of inhibition of SNCA gene expression in the object.

58. The method of claim 57, wherein the identified physiological characteristic is one or more of the following: the level of SNCA mRNA, α-synuclein, or other parameters that are functionally related to SNCA expression levels.

59. The method of claim 58, wherein a decrease in one or more of the levels of SNCA mRNA, α-synuclein, or other parameters functionally related to SNCA expression levels in the object indicates a decrease in SNCA gene expression in the object.

60. A method for treating a disease or condition associated with the presence of SNCA protein, the method comprising administering to a subject an effective amount of a double-stranded ribonucleic acid (dsRNA) agent as described in any one of claims 1 to 38 or a composition as described in any one of claims 39 to 43 to inhibit SNCA gene expression.

61. The method of claim 60, wherein the disease or condition is an SNCA-related disease.

62. The method of claim 61, wherein the SNCA-related disease is a synucleinosis.

63. The method of claim 61, wherein the SNCA-related disease is Parkinson's disease (PD), multiple system atrophy (MSA), Lewy body dementia (LBD), pure autonomic failure (PAF), Pick's disease, progressive supranuclear palsy, boxing dementia, chromosomal-related Parkinson's syndrome, Lytico-Bodig disease, tangled dominant dementia, aurophilic granuloma, aurophilic granulosome disease, gangliocytoma, meningioma, subacute sclerosing encephalitis, lead poisoning encephalopathy, tuberous sclerosis, Hallewarden-Schpattz disease, lipofuscin deposition, corticobasal degeneration, frontotemporal dementia, frontotemporal lobe degeneration, Alzheimer's disease, Huntington's disease, Down syndrome, psychosis, schizophrenia, and / or Crotzfeldt-Jab disease.

64. The method of any one of claims 60 to 63, further comprising administering an additional treatment to the subject.

65. The method of claim 64, wherein the additional treatment option comprises: The application of one or more of the SNCA antisense polynucleotides of the present invention to the subject, the application of a non-SNCA dsRNA therapeutic agent to the subject, and behavioral changes in the subject.

66. The method of claim 65, wherein the additional treatment regimen is one or more of the following: an antipruritic agent, an astringent, a local anesthetic, an anti-inflammatory agent, a cholinesterase inhibitor, a muscarinic agonist, an antioxidant or anti-inflammatory drug, or any combination thereof; preferably, the additional treatment regimen is one or more of the following: carbidopa-levodopa, levodopa, entacapone, tocapone, octopone, pramipexole, ropinirole, apomorphine, rotigotine, selegiline, rasagiline, safenamide, amantadine, itratheline, trihexyphenidyl, benzoline, rivastigmine, rivastigmine, donepezil, galantamine, acetyl-L-carnitine, vinpocetine, huperzine A, alpha-lipoic acid, vitamin B1, etc. E, Rhodiola rosea, Biotin, Reminyl galantamine, Cognex, Selegiline, Physostigmine, Revistigmin, Donepezil, Exelon, Metridone, Milamarin, Xanomelide, Saeluzole, Idebenone, ENA-713, Mermic, Quetiapine, Neurostrol, Idebenone, Propanfil, Neuromidal, and Memantine, as well as physical, occupational, and speech therapy, including movement programs for cardiopulmonary, resistance, flexibility, and gait and balance movements, and deep brain stimulation (DBS) involving the implantation of electrodes into target brain regions.

67. The method of any one of claims 60 to 66, wherein the dsRNA agent is administered subcutaneously to the subject.

68. The method of any one of claims 60 to 66, wherein the dsRNA agent is administered intrathecally, intracranially, intraventricularly, or intracerebrally to the subject.

69. The method of any one of claims 60 to 66, wherein the dsRNA agent is administered to the subject via intravenous (IV) administration.

70. The method of any one of claims 60 to 66, further comprising determining the efficacy of the applied double-stranded ribonucleic acid (dsRNA) agent in the subject.

71. The method of claim 70, wherein the means of determining the efficacy of the treatment in the subject comprises: (i) Identify one or more physiological characteristics of SNCA-related diseases or conditions in the subject, and (ii) Compare the identified physiological characteristics with the baseline physiological characteristics prior to treatment for the SNCA-related disease or condition. The comparisons described therein indicate one or more of the presence, absence, and level of efficacy of the double-stranded ribonucleic acid (dsRNA) agent when applied to the subject.

72. The method of claim 71, wherein the identified physiological characteristics are: the levels of SNCA mRNA, α-synuclein, and other parameters that are functionally related to SNCA expression levels.

73. The method of claim 71, wherein a decrease in the levels of one or more of the SNCA mRNA, α-synuclein, and other parameters functionally related to SNCA expression levels indicates the presence of efficacy of the double-stranded ribonucleic acid (dsRNA) agent administered to the subject.

74. A method for reducing the level of SNCA protein in a subject compared to a pre-treatment baseline level of SNCA protein in the subject, the method comprising administering to the subject an effective amount of a double-stranded ribonucleic acid (dsRNA) agent according to any one of claims 1 to 38 or a composition according to any one of claims 39 to 43 to reduce the level of SNCA gene expression.

75. The method of claim 74, wherein the dsRNA agent is administered subcutaneously, intracranially, intrathecally, intraventricularly, or intracerebrally to the subject, or administered via intravenous infusion.

76. A method of altering the physiological characteristics of an SNCA-related disease or condition in a subject compared to a pre-treatment baseline physiological characteristic, the method comprising administering to the subject an effective amount of a double-stranded ribonucleic acid (dsRNA) agent of any one of claims 1 to 38 or a composition of any one of claims 39 to 43 to alter the physiological characteristics of the SNCA-related disease or condition in the subject.

77. The method of claim 76, wherein the dsRNA agent is administered subcutaneously, intrathecally, intraventricularly, or intracerebrally to the subject, or administered via intravenous infusion.

78. The method of any one of claims 76 to 77, wherein the physiological characteristic is one or more of the following: the level of SNCA mRNA, α-synuclein, and other parameters that are functionally related to the expression level of SNCA.

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