Amino adipate semialdehyde synthase (aass) irna compositions and methods of use thereof

Abstract

This invention relates to a double-stranded ribonucleic acid (dsRNA) composition targeting the AASS gene and the use of such dsRNA. s Methods and treatments for inhibiting AASS expression using RNA compositions that would benefit subjects who would benefit from a reduction in AASS expression, such as subjects with AASS-related diseases, conditions, or illnesses.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 672,129, filed July 16, 2024, and U.S. Provisional Application No. 63 / 517,811, filed August 4, 2023. The entire contents of the foregoing applications are hereby incorporated herein by reference. Technical Field

[0002] This disclosure generally relates to dsRNA agents targeting aminoadiate semialdehyde synthase (AASS) and methods of using them.

[0003] sequence list This application includes a sequence list, which has been submitted electronically in eXtensible Markup Language (XML) format and incorporated herein in its entirety by reference. The XML copy was created on August 1, 2024, named A108868_1730WO_SL.xml, and has a size of 63,031,180 bytes. Background Technology

[0004] Lysine is an essential amino acid involved in protein synthesis, carnitine production, and enzyme catalysis. The most common lysine catabolism pathway is the saccharopine pathway, which primarily occurs in liver mitochondria and leads to the production of acetyl-CoA. The first two reactions of the saccharopine pathway are catalyzed by aminoadipic acid semialdehyde synthase (AASS), a bifunctional enzyme with lysine-ketoglutarate reductase and saccharopine dehydrogenase activities, resulting in the production of glutaryl-CoA (glutaryl-CoA). Glutamicyl-CoA dehydrogenase (GCDH) catalyzes the conversion of glutaryl-CoA to crotonyl-CoA. Inhibition of the conversion of glutaryl-CoA to crotonyl-CoA leads to the production and accumulation of toxic catabolites such as glutaric acid and 3-hydroxyglutaric acid.

[0005] An alternative lysine catabolism pathway is the pipecolate pathway, which occurs primarily in the adult brain and merges with the yeast lysine pathway at a step catalyzed by the enzyme α-aminoadipic acid dehydrogenase (ALDH7A1) into a common degradation pathway.

[0006] Disruptions in the lysine catabolism pathway can lead to a variety of disorders. At least two serious neurometabolic disorders, glutaric aciduria type 1 (GA1) and pyridoxine-dependent epilepsy (PDE), are associated with mutations in lysine catabolism enzymes. GA1 is caused by a deficiency of the GCDH enzyme, while PDE is caused by a mutation in the ALDH7A1 enzyme. GCDH deficiency inhibits the conversion of glutaryl-CoA to crotonyl-CoA, leading to the production and accumulation of toxic catabolites such as glutaric acid and 3-hydroxyglutaric acid. The accumulation of these catabolites can cause encephalopathy crises, striatal brain damage, and severe motor disorders. ALDH7A1 deficiency leads to the accumulation of L-piperidinic acid, resulting in infancy-related seizures, intellectual disability, and neurological abnormalities. In the lysine catabolism pathway, the AASS enzyme is upstream of both GCDH and ALDH7A1 enzymes and is a potential therapeutic target for lysine catabolism disorders.

[0007] Therefore, there is a need for methods to treat lysine catabolism disorders (such as GA1 and PDE), including agents that can selectively and effectively inhibit the AASS gene. Summary of the Invention

[0008] Improved approaches are needed to treat lysine catabolism disorders, such as type 1 glutaric aciduria (GA1) and pyridoxine-dependent epilepsy (PDE), including agents that can selectively and effectively inhibit the AASS gene. Current standard care for subjects with lysine catabolism disorders includes a low-lysine diet, carnitine supplementation, emergency treatment during illness (ET), fasting, and / or pyridoxine administration.

[0009] This invention provides an iRNA composition that achieves cleavage of the para-aminoadipic acid semialdehyde synthase (AASS) gene RNA transcript mediated by an RNA-induced silencing complex (RISC). The AASS gene is intracellular, for example, within the cells of a subject (such as a human). This invention also provides methods for using the iRNA composition of this invention to inhibit AASS gene expression and / or treat subjects who would benefit from inhibiting or reducing AASS gene expression, for example, subjects who have or are susceptible to AASS-related diseases (e.g., lysine catabolism disorders).

[0010] Therefore, in one aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of adipic acid semialdehyde synthase (AASS) in cells. 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 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 by no more than 1, 2, or 3 nucleotides. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence of SEQ ID NO: 2.

[0011] In another aspect, the present invention provides a double-stranded RNA (dsRNA) agent for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells. The dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand contains a region complementary to the mRNA encoding AASS, comprising at least 15 consecutive nucleotides differing from any antisense sequence listed in any of Tables 3-6 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 contains a region complementary to the mRNA encoding AASS, comprising at least 15 consecutive nucleotides from any antisense sequence listed in any of Tables 3-6.

[0012] In another embodiment, the complementary region comprises at least 15 consecutive nucleotides, which are related to SEQ ID NO. 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3039、1494-1516、1147-1169、1148-1170、1150-1172、2968-2990、3329-3351、584-606、795-817、3603-3625、2750-2772、3299-3321、3300-3322、3222-3244、3532-3554、3533-3555、1601-1623、1467-1489、2153-2175、3661-3683、3662-3684、257-279、1472-1494、1473-1495、3286-3308、3287-3309、741-763、742-764、826-848、3550-3572、3205-3227、2521-2543、3204-3226、3470-3492、3471-3493、863-885、864-886、2756-2778、3213-3235、651-673、3535-3557、1166-1188、1167-1189、963-985、77-99、2257-2279、437-459、1144-1166、313-335、316-338、2291-2313、1838-1860、1841-1863、1260-1282、340-362、2090-2112、2858-2880、343-365、3165-3187、2880-2902、851-873、2606-2628、1273-1295、1546-1568、933-955、481-503、401-423、402-424、403-425、404-426、407-429、1398-1420、3472-3494、3490-3512、3491-3513、3492-3514、3548-3570、3549-3571、3550-3572、3551-3573、2301-2323、2302-2324、2303-2325、2304-2326、2305-2327、3408-3430、3290-3312、1865-1887、3525-3547、3293-3315、3527-3549、3156-3178、244-266、246-268、248-270、3054-3076、3055-3077、2937-2959、1850-1872、1851-1873, 1852-1874, 1853-1875, 1854-1876, 1855-1877, 1856-1878, 2199-2221, 2200-2222, 997-1019, 3157-3179, 3158-3180, 589-611, 1197-1219, 1198-1220, 1200-1222, 2608-2630, 1202-1224, 2971-2993, 3347 -3369, 2246-2268, 42-64, 1317-1339, 2499-2521, 3316-3338, 3317-3339, 3110-3132, 1697-1719, 1698-1720, 3132-3154, 1154-1176, 1155-1177, 3556-3578, 3558-3580, 3560-3582, 1485-1507, 589-611, 2205-2227, 3 540-3562, 2717-2739, 3280-3302, 3282-3304, 3555-3577, 3556-3578, 2851-2873, 2853-2875, 3087-3109, 3088-3110, 3089-3111, 1346-1368, 3347-3369, 1348-1370, 597-619, 598-620, 599-621, 2188-2210, 3007-302 9. Any one of SEQ ID NO: 3008-3030, 3009-3031, 2722-2744, 2157-2179, 2724-2746, 2725-2747, 2726-2748, 2727-2749, 1194-1216, 1195-1217, 1196-1218, 1197-1219, 1199-1221, 1200-1222, 2150-2172, or 2151-2173 differs by no more than 1, 2, or 3 nucleotides. In some embodiments, the complementary region contains from SEQ ID NO: 3008-3030, 3009-3031, 2722-2744, 2157-2179, 2724-2746, 2725-2747, 2726-2748, 2727-2749, 1194-1216, 1195-1217, 1196-1218, 1197-1219, 1199-1221, 1200-1222, 2150-2172, or 2151-2173. The nucleotides of NO:1 are 381-403, 746-768, 747-769, 263-285, 2465-2487, 248-270, 249-271, 251-273, 254-276, 255-277, 256-278, 258-280, 260-282, 263-285, and 266. -288, 272-294, 273-295, 274-296, 275-297, 276-298, 278-300, 279-301, 280-302, 281-303, 284-306, 285-307, 288-310, 289-311, 291-313, 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2849-2871、2850-2872、2851-2873、2852-2874、2853-2875、2854-2876、2855-2877、1487-1509、2227-2249、2858-2880、2859-2881、3451-3473、2861-2883、2862-2884、2863-2885、2864-2886、2865-2887、2866-2888、2869-2891、2870-2892、2871-2893、2872-2894、2873-2895、2874-2896、2875-2897、2876-2898、2877-2899、2878-2900、2879-2901、2880-2902、2881-2903、2882-2904、2884-2906、2885-2907、2886-2908、2887-2909、2888-2910、2889-2911、2180-2202、440-462、2045-2067、2910-2932、2914-2936、2915-2937、2916-2938、2917-2939、2918-2940、2022-2044、2023-2045、103-125、467-489、1160-1182、759-781、1238-1260、1869-1891、1577-1599、1871-1893、940-962、2614-2636、2615-2637、2933-2955、2934-2956、2884-2906、2885-2907、2886-2908、519-541、1029-1051、726-748、2312-2334、2313-2335、2943-2965、3250-3272、2316-2338、2946-2968、2947-2969、2948-2970、2950-2972、2951-2973、2952-2974、2004-2026、3172-3194、3344-3366、3345-3367、3325-3347、3347-3369、3348-3370、3349-3371、3316-3338、2839-2861、2958-2980、2962-2984、2963-2985、2964-2986、2965-2987、2982-3004、2858-2880、2859-2881、2088-2110、1034-1056、3135-3157、3234-3256、3091-3113、3092-3114、3004-3026、3006-3028、3007-3029、3008-3030、716-738、492-514、3153-3175、854-876、2343-2365、3070-3092、3071-3093、3072-3094、3073-3095、3079-3101、3080-3102、1425-1447、3082-3104、3281-3303、3257-3279、417-439、2129-2151、3113-3135、3114-3136、407-429、2879-2901、2999-3021、3000-3022、3002-3024、3003-3025、3313-3335、3314-3336、3315-3337、3232-3254、3233-3255、1176-1198、1178-1200、1416-1438、10-32、10-32、12-34、2149-2171、13-35、762-784、2150-2172、2151-2173、1027-1049、3601-3623、1253-1275、1254-1276、3169-3191、3172-3194、3174-3196、3175-3197、3176-3198、3177-3199、3178-3200、3179-3201、3180-3202、3258-3280、820-842、822-844、3214-3236、43-65、2031-2053、2868-2890、2176-2198、1023-1045、979-1001、1108-1130、3001-3023、2762-2784、641-663、1200-1222、2588-2610、3501-3523、3502-3524、3309-3331、3504-3526、3316-3338、3318-3340、3319-3341、3320-3342、3321-3343、3322-3344、3323-3345、3570-3592、3230-3252、3361-3383、3362-3384、3363-3385、3364-3386、3365-3387、3366-3388、3324-3346、1829-1851、3399-3421、3024-3046、3648-3670、3068-3090、3426-3448、2161-2183、3073-3095、3429-3451、3430-3452、440-462、441-463、442-464、3434-3456、3198-3220、3199-3221、3200-3222、259-281、260-282、261-283、262-284、2194-2216、1620-1642、1621-1643、1622-1644、3042-3064、3043-3065、3044-3066、2885-2907、3046-3068、3490-3512、3491-3513、2604-2626、879-901、2691-2713、2692-2714、3212-3234、2695-2717、2529-2551、2151-2173、2152-2174、3613-3635、3616-3638、1290-1312、2170-2192、3219-3241、934-956、3569-3591、2219-2241、3538-3560、3539-3561、3540-3562、3541-3563、3542-3564、3543-3565、3545-3567、2767-2789、1377-1399、3565-3587、3327-3349、1608-1630、1126-1148、803-825、364-386、366-388、367-389、619-641、620-642、3312-3334、3187-3209、2453-2475、2046-2068、1011-1033、2054-2076、1013-1035、1014-1036、1015-1037、1016-1038、1017-1039、3177-3199、3178-3200、48-70、1503-1525、3641-3663、3651-3673、1670-1692、3514-3536、3154-3176、3516-3538、3199-3221、3200-3222、59-81、2408-2430、3491-3513、3494-3516、1627-1649、3479-3501、3444-3466、2992-3014、2993-3015、24-46、3049-3071、3050-3072、3051-3073、787-809、790-812、791-813、2274-2296、2275-2297、2812-2834、1613-1635、2814-2836、1615-1637、1618-1640、1619-1641、1620-1642、2659-2681、1495-1517、1496-1518、3483-3505、3484-3506、3485-3507、2297-2319、2781-2803、2782-2804、2783-2805、2784-2806、3497-3519、2303-2325、2968-2990、2969-2991、2970-2992、3325-3347、3326-3348、3327-3349、3328-3350、3329-3351、583-605、584-606、669-691、922-944、284-306、2836-2858、2837-2859、2223-2245、2224-2246、2225-2247、2226-2248、2227-2249、2228-2250、2229-2251、2230-2252、2231-2253、2232-2254、2233-2255、3528-3550、3568-3590、1902-1924、2451-2473、3224-3246、1599-1621、1600-1622、3237-3259、1530-1552、3159-3181、3030-3052、3033-3055、2185-2207、152-174、84-106、1492-1514、1493-1515、3282-3304、3200-3222、3204-3226、3206-3228、1144-1166、3207-3229、3208-3230、3643-3665、815-837、2282-2304、2284-2306、1829-1851、1830-1852、1915-1937、148-170、3610-3632、3611-3633、2574-2596、2575-2597、2576-2598、2577-2599、3198-3220、3199-3221、1315-1337、345-367、2870-2892、2871-2893、2223-2245、2224-2246、2476-2498、2478-2500、1481-1503、1482-1504、1927-1949、1928-1950、1929-1951、1930-1952、1931-1953、3539-3561、2332-2354、912-934、3285-3307、3268-3290、1468-1490、3270-3292、549-571、1166-1188、3310-3332、822-844、3312-3334、3313-3335、3314-3336、3351-3373、3524-3546、3378-3400、3354-3376、3380-3402、2087-2109、2088-2110、2811-2833、1272-1294、1540-1562、3387-3409、1308-1330、99-121、3659-3681、989-1011、1022-1044、596-618、1805-1827、487-509、488-510、2603-2625、2604-2626、2605-2627、1488-1510、3214-3236、1653-1675、3491-3513、3456-3478、3457-3479、3459-3481、3460-3482、2961-2983、3521-3543、1029-1051、2770-2792、2771-2793、2772-2794、2328-2350、751-773、2400-2422、2402-2424、2403-2425、2222-2244、2816-2838、2224-2246、2235-2257、2283-2305、2884-2906、2885-2907、3293-3315、3294-3316、3295-3317、48-70、52-74、53-75、2820-2842、1610-1632、1812-1834、371-393、712-734、1910-1932、1911-1933、3647-3669、492-514、711-733、3651-3673、3358-3380、3327-3349、3328-3350、3329-3351、1628-1650、170-192、3137-3159、3138-3160、786-808、20-42、771-793、745-767、462-484、322-344、3522-3544、3523-3545、3524-3546、1670-1692、1411-1433、1505-1527、1413-1435、1507-1529、1044-1066、3650-3672、3207-3229、1514-1536、3226-3248、3128-3150、3340-3362、3231-3253、3232-3254、2855-2877、2856-2878、2857-2879、2456-2478、2457-2479、2658-2680、1953-1975、1954-1976、368-390、1135-1157、3560-3582、1623-1645、2858-2880、848-870、3284-3306、52-74、2559-2581、2575-2597、393-415、1507-1529、3154-3176、3408-3430、3494-3516、3412-3434、3111-3133、3569-3591、483-505、2575-2597、1402-1424、3060-3082、1630-1652、172-194、2102-2124、1850-1872、3014-3036、3015-3037、1866-1888、1867-1889、2808-2830、2809-2831、2810-2832、2811-2833、2812-2834、2813-2835、2814-2836、808-830、269-291、810-832、2277-2299、2278-2300、2279-2301、814-836、2281-2303、2282-2304、721-743、354-376、355-377、3063-3085、2858-2880、2860-2882、1487-1509、3225-3247、2033-2055、1488-1510、1111-1133、1554-1576、2171-2193、2047-2069、3206-3228、3283-3305、3284-3306、3286-3308、3287-3309、3017-3039、1494-1516、1147-1169、1148-1170、1150-1172、2968-2990、3329-3351、584-606、795-817、3603-3625、2750-2772、3299-3321、3300-3322、3222-3244、3532-3554、3533-3555、1601-1623、1467-1489、2153-2175、3661-3683、3662-3684、257-279、1472-1494、1473-1495、3286-3308、3287-3309、741-763、742-764、826-848、3550-3572、3205-3227、2521-2543、3204-3226、3470-3492、3471-3493、863-885、864-886、2756-2778、3213-3235、651-673、3535-3557、1166-1188、1167-1189、963-985、77-99、2257-2279、437-459、1144-1166、313-335、316-338、2291-2313、1838-1860、1841-1863、1260-1282、340-362、2090-2112、2858-2880、343-365、3165-3187、2880-2902、851-873、2606-2628、1273-1295、1546-1568、933-955、481-503、401-423、402-424、403-425、404-426、407-429、1398-1420、3472-3494、3490-3512、3491-3513、3492-3514、3548-3570、3549-3571、3550-3572、3551-3573、2301-2323、2302-2324、2303-2325、2304-2326、2305-2327、3408-3430、3290-3312、1865-1887、3525-3547、3293-3315、3527-3549、3156-3178、244-266、246-268、248-270、3054-3076、3055-3077、2937-2959、1850-1872、1851-1873、1852-1874、1853-1875、1854-1876、1855-1877、1856-1878、2199-2221、2200-2222、997-1019、3157-3179、3158-3180、589-611、1197-1219、1198-1220、1200-1222、2608-2630、1202-1224、2971-2993、3347-3369、2246-2268、42-64、1317-1339、2499-2521、3316-3338、3317-3339、3110-3132、1697-1719、1698-1720、3132-3154、1154-1176、1155-1177、3556-3578、3558-3580、3560-3582, 1485-1507, 589-611, 2205-2227, 3540-3562, 2717-2739, 3280-3302, 3282-3304, 3555-3577, 3556-3578, 2851-2873, 2853-2875, 3087-3109, 3088-3110, 3089-3111, 1346-1368, 3347-3369, 1348-1370, 597-619, 598-620, 599-6 21. At least 15 consecutive nucleotides of any one of 2188-2210, 3007-3029, 3008-3030, 3009-3031, 2722-2744, 2157-2179, 2724-2746, 2725-2747, 2726-2748, 2727-2749, 1194-1216, 1195-1217, 1196-1218, 1197-1219, 1199-1221, 1200-1222, 2150-2172, or 2151-2173.

[0013] In one embodiment, the antisense strand comprises at least 15 consecutive nucleotides selected from AD-2320680, AD-2320683, AD-2320684, AD-2320739, AD-2320769, AD-2320870, AD-2320871, AD-2320873, AD-2320876, AD-2320877, AD-2320878, AD-2320880, AD-2320882, AD-2320885, AD-2320938, AD-2320944, AD-2320945, AD-2320946, AD-2320947, AD-23209... A D-2320985, AD-2320986, AD-2321037, AD-2321038, AD-2321039, AD-2321040, AD-2321041, AD-2321042, AD-2321043, AD-2321044, AD-2321045, AD-23 21046, AD-2321047, AD-2321048, AD-2321049, AD-2321050, AD-2321051, AD-2321052, AD-2321055, AD-2321056, AD-2321058, AD-2321059, AD-232106 1. AD-2321070, AD-2321071, AD-2321073, AD-2321076, AD-2321077, AD-2321079, AD-2321080, AD-2321081, AD-2321082, AD-2321083, AD-2321084, AD -2321085, AD-2321086, AD-2321137, AD-2321138, AD-2321139, AD-2321140, AD-2321141, AD-2321142, AD-2321143, AD-2321144, AD-2321145, AD-232 1146, AD-2321147, AD-2321148, AD-2321149, AD-2321150, AD-2321153, AD-2321155, AD-2321156, AD-2321157, AD-2321158, AD-2321159, 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3154、AD-2323155、AD-2323156、AD-2323157、AD-2323158、AD-2323159、AD-2323160、AD-2323163、AD-2323179、AD-2323181、AD-2323182、AD-2323183、AD-2323185、AD-2323186、AD-2323237、AD-2323238、AD-2323241、AD-2323242、AD-2323248、AD-2323249、AD-2323253、AD-2323254、AD-2323255、AD-2323256、AD-2323257、AD-2323258、AD-2323259、AD-2323260、AD-2323277、AD-2323278、AD-2323279、AD-2323280、AD-2323281、AD-2323282、AD-2323283、AD-2323284、AD-2323357、AD-2323360、AD-2323364、AD-2323366、AD-2323369、AD-2323370、AD-2323382、AD-2323438、AD-2323439、AD-2323440、AD-2323441、AD-2323444、AD-2323445、AD-2323451、AD-2323452、AD-2323454、AD-2323455、AD-2323458、AD-2323465、AD-2323466、AD-2323467、AD-2323468、AD-2323472、AD-2323473、AD-2323476、AD-2323477、AD-2323478、AD-2323479、AD-2323480、AD-2323481、AD-2323482、AD-2323544、AD-2323546、AD-2323547、AD-2323548、AD-2323549、AD-2323550、AD-2323551、AD-2323552、AD-2323553、AD-2323554、AD-2323555、AD-2323556、AD-2323557、AD-2323558、AD-2323559、AD-2323560、AD-2323561、AD-2323565、AD-2323566、AD-2323570、AD-2323571、AD-2323573、AD-2323574、AD-2323575、AD-2323577、AD-2323578、AD-2323579、AD-2323580、AD-2323581、AD-2323582、AD-2323583、AD-2323584、AD-2323585、AD-2323586、AD-2323637、AD-2323638、AD-2323639、AD-2323641、AD-2323642、AD-2323648、AD-2323651、AD-2323652、AD-2323653、AD-2323654、AD-2323655、AD-2323656、AD-2323657、AD-2323658、AD-2323681、AD-2323682、AD-2323683、AD-2323684、AD-2323685、AD-2323686、AD-2323737、AD-2323738、AD-2323739、AD-2323740、AD-2323741、AD-2323742、AD-2323743、AD-2323744、AD-2323745、AD-2323746、AD-2323747、AD-2323748、AD-2323749、AD-2323751、AD-2323752、AD-2323771、AD-2323772、AD-2323779、AD-2323781、AD-2323784、AD-2323785、AD-2323786、AD-2323837、AD-2323840、AD-2323842、AD-2323843、AD-2323845、AD-2323847、AD-2323848、AD-2323850、AD-2323851、AD-2323853、AD-2323854、AD-2323855、AD-2323856、AD-2323858、AD-2323859、AD-2323886、AD-2323940、AD-2323941、AD-2323946、AD-2323947、AD-2323948、AD-2323949、AD-2323950、AD-2323952、AD-2323953、AD-2323955、AD-2323956、AD-2323958、AD-2323961、AD-2323965、AD-2323970、AD-2323972、AD-2323973、AD-2323976、AD-2323982、AD-2323984、AD-2323985、AD-2323986、AD-2324037、AD-2324038、AD-2324039、AD-2324040、AD-2324041、AD-2324042、AD-2324043、AD-2324044、AD-2324045、AD-2324046、AD-2324047、AD-2324048、AD-2324049、AD-2324050、AD-2324051、AD-2324052、AD-2324053、AD-2324054、AD-2324055、AD-2324056、AD-2324057、AD-2324058、AD-2324059、AD-2324060、AD-2324066、AD-2324069、AD-2324070、AD-2324073、AD-2324074、AD-2324075、AD-2324077、AD-2324080、AD-2324082、AD-2324083、AD-2324084、AD-2324139、AD-2324140、AD-2324141、AD-2324144、AD-2324162、AD-2324163、AD-2324164、AD-2324165、AD-2324166、AD-2324167、AD-2324168、AD-2324169、AD-2324170、AD-2324171、AD-2324172、AD-2324173、AD-2324174、AD-2324175、AD-2324176、AD-2324177、AD-2324178、AD-2324179、AD-2324180、AD-2324181、AD-2324182、AD-2324183、AD-2324184、AD-2324185、AD-2324186、AD-2324237、AD-2324238、AD-2324239、AD-2324240、AD-2324241、AD-2324242、AD-2324243、AD-2324244、AD-2324245、AD-2324246、AD-2324247、AD-2324267、AD-2324268、AD-2324269、AD-2324270、AD-2324271、AD-2324272、AD-2324273、AD-2324274、AD-2324275、AD-2324276、AD-2324277、AD-2324278、AD-2324280、AD-2324281、AD-2324282、AD-2324347、AD-2324348、AD-2324349、AD-2324350、AD-2324351、AD-2324352、AD-2324353、AD-2324354、AD-2324355、AD-2324356、AD-2324357、AD-2324358、AD-2324365、AD-2324366、AD-2324367、AD-2324368、AD-2324369、AD-2324370、AD-2324371、AD-2324374、AD-2324440、AD-2324441、AD-2324442、AD-2324443、AD-2324445、AD-2324446、AD-2324447、AD-2324449、AD-2324450、AD-2324452、AD-2324455、AD-2324473、AD-2324486、AD-2324538、AD-2324539、AD-2324540、AD-2324542、AD-2324545、AD-2324572、AD-2324586、AD-2324639、AD-2324640、AD-2324646、AD-2324647、AD-2324648、AD-2324649、AD-2324650、AD-2324654、AD-2324656、AD-2324657、AD-2324658、AD-2324659、AD-2324660、AD-2324661、AD-2324662、AD-2324667、AD-2324668、AD-2324670、AD-2324672、AD-2324673、AD-2324674、AD-2324677、AD-2324678、AD-2324681、AD-2324682、AD-2324684、AD-2324685、AD-2324737、AD-2324738、AD-2324739、AD-2324740、AD-2324742、AD-2324743、AD-2324744、AD-2324745、AD-2324746、AD-2324747、AD-2324748、AD-2324752、AD-2324753、AD-2324754、AD-2324755、AD-2324756、AD-2324757、AD-2324758、AD-2324760、AD-2324761、AD-2324765、AD-2324766、AD-2324770、AD-2324771、AD-2324772、AD-2324775、AD-2324777、AD-2324778、AD-2324779、AD-2324780、AD-2324781、AD-2324782、AD-2324783、AD-2324784、AD-2324786、AD-2324837、AD-2324839、AD-2324841、AD-2324842、AD-2324845、AD-2324846、AD-2324847、AD-2324848、AD-2324849、AD-2324850、AD-2324851、AD-2324852、AD-2324853、AD-2324854、AD-2324855、AD-2324856、AD-2324857、AD-2324858、AD-2324859、AD-2324863、AD-2324864、AD-2324865、AD-2324866、AD-2324867、AD-2324868、AD-2324877、AD-2324878、AD-2324879、AD-2324881、AD-2324882、AD-2324883、AD-2324884、AD-2324886、AD-2324941、AD-2324942、AD-2324946、AD-2324947、AD-2324985、AD-2325037、AD-2325038、AD-2325040、AD-2325041、AD-2325049、AD-2325050、AD-2325052、AD-2325053、AD-2325054、AD-2325055、AD-2325058、AD-2325059、AD-2325066、AD-2325077、AD-2325080、AD-2325081、AD-2325082、AD-2325083、AD-2325084、AD-2325085、AD-2325139、AD-2325142、AD-2325145、AD-2325146、AD-2325147、AD-2325148、AD-2325149、AD-2325150、AD-2325151、AD-2325152、AD-2325154、AD-2325172、AD-2325237、AD-2325248、AD-2325250、AD-2325277、AD-2325349、AD-2325350、AD-2325353、AD-2325354、AD-2325356、AD-2325357、AD-2325366、AD-2325369、AD-2325370、AD-2325373、AD-2325374、AD-2325375、AD-2325376、AD-2325377、AD-2325378、AD-2325379、AD-2325380、AD-2325439、AD-2325442、AD-2325446、AD-2325448、AD-2325450、AD-2325451、AD-2325452、AD-2325453、AD-2325454、AD-2325455、AD-2325457、AD-2325458、AD-2325459、AD-2325460、AD-2325462、AD-2325463、AD-2325468、AD-2325470、AD-2325471、AD-2325473、AD-2325474、AD-2325477、AD-2325479、AD-2325480、AD-2325481、AD-2325482、AD-2325483、AD-2325484、AD-2325485、AD-2325486、AD-2325538、AD-2325543、AD-2325544、AD-2325545、AD-2325546、AD-2325547、AD-2325552、AD-2325553、AD-2325557、AD-2325558、AD-2325559、AD-2325567、AD-2325568、AD-2325640、AD-2325642、AD-2325643、AD-2325644、AD-2325645、AD-2325646、AD-2325647、AD-2325648、AD-2325650、AD-2325651、AD-2325653、AD-2325654、AD-2325657、AD-2325658、AD-2325659、AD-2325660、AD-2325671、AD-2325672、AD-2325673、AD-2325674、AD-2325676、AD-2325677、AD-2325678、AD-2325679、AD-2325680、AD-2325681、AD-2325683、AD-2325684、AD-2325685、AD-2325686、AD-2325737、AD-2325738、AD-2325739、AD-2325740、AD-2325741、AD-2325742、AD-2325743、AD-2325744、AD-2325745、AD-2325746、AD-2325747、AD-2325748、AD-2325749、AD-2325750、AD-2325751、AD-2325752、AD-2325753、AD-2325754、AD-2325758、AD-2325759、AD-2325760、AD-2325761、AD-2325762、AD-2325763、AD-2325764、AD-2325765、AD-2325766、AD-2325767、AD-2325768、AD-2325769、AD-2325770、AD-2325771、AD-2325772、AD-2325773、AD-2325774、AD-2325775、AD-2325776、AD-2325777、AD-2325780、AD-2325781、AD-2325782、AD-2325783、AD-2325784、AD-2325785、AD-2325786、AD-2325837、AD-2325838、AD-2325839、AD-2325840、AD-2325841、AD-2325842、AD-2325843、AD-2325845、AD-2325846、AD-2325847、AD-2325848、AD-2325849、AD-2325850、AD-2325874、AD-2325881、AD-2325882、AD-2325883、AD-2325937、AD-2325938、AD-2325939、AD-2325940、AD-2325941、AD-2325942、AD-2325943、AD-2325944、AD-2325945、AD-2325946、AD-2325947、AD-2325948、AD-2325949、AD-2325950、AD-2325951、AD-2325952、AD-2325953、AD-2325954、AD-2325955、AD-2325956、AD-2325957、AD-2325958、AD-2325959、AD-2325960、AD-2325961、AD-2325962、AD-2325963、AD-2325964、AD-2325965、AD-2325966、AD-2325967、AD-2325968、AD-2325969、AD-2325970、AD-2325972、AD-2325973、AD-2325974、AD-2325975、AD-2325976、AD-2325977、AD-2325978、AD-2325979、AD-2325980、AD-2325981、AD-2325982、AD-2325983、AD-2325984、AD-2325985、AD-2326039、AD-2326040、AD-2326041、AD-2326042、AD-2326059、AD-2326060、AD-2326061、AD-2326067、AD-2326069、AD-2326070、AD-2326072、AD-2326073、AD-2326074、AD-2326075、AD-2326077、AD-2326078、AD-2326079、AD-2326080、AD-2326170、AD-2326242、AD-2326243、AD-2326244、AD-2326245、AD-2326246、AD-2326247、AD-2326248、AD-2326254、AD-2326255、AD-2326256、AD-2326257、AD-2326273、AD-2326274、AD-2326275、AD-2326286、AD-2326340、AD-2326341、AD-2326342、AD-2326348、AD-2326349、AD-2326350、AD-2326352、AD-2326353、AD-2326354、AD-2326355、AD-2326356、AD-2326769、AD-2326770、AD-2326781、AD-2326783、AD-2326785、AD-2326846、AD-2326847、AD-2326848、AD-2326849、AD-2326850、AD-2326851、AD-2326852、AD-2326853、AD-2326858、AD-2326876、AD-2326881、AD-2326882、AD-2326885、AD-2326938、AD-2326940、AD-2326941、AD-2326942、AD-2326943、AD-2326944、AD-2326945、AD-2326946、AD-2327065、AD-2327067、AD-2327069、AD-2327070、AD-2327071、AD-2327072、AD-2327073、AD-2327076、AD-2327077、AD-2327079、AD-2327080、AD-2327154、AD-2327157、AD-2327158、AD-2327159、AD-2327160、AD-2327161、AD-2327162、AD-2327163、AD-2327164、AD-2327172、AD-2327174、AD-2327175、AD-2327176、AD-2327177、AD-2327178、AD-2327179、AD-2327262、AD-2327263、AD-2327264、AD-2327265、AD-2327266、AD-2327267、AD-2327268、AD-2327269、AD-2327345、AD-2327371、AD-2327372、AD-2327374、AD-2327379、AD-2327461、AD-2327464、AD-2327465、AD-2327466、AD-2327467、AD-2327468、AD-2327469、AD-2327470、AD-2327471、AD-2327472、AD-2327473、AD-2327474、AD-2327475、AD-2327551、AD-2327552、AD-2327553、AD-2327554、AD-2327555、AD-2327556、AD-2327557、AD-2327558、AD-2327559、AD-2327560、AD-2327561、AD-2327562、AD-2327563、AD-2327564、AD-2327565、AD-2327566、AD-2327567、AD-2327568、AD-2327569、AD-2327571、AD-2327572、AD-2327573、AD-2327574、AD-2327575、AD-2327576、AD-2327579、AD-2327580、AD-2327658、AD-2327659、AD-2327660、AD-2327662、AD-2327665、AD-2327670、AD-2327671、AD-2327672、AD-2327673、AD-2327674、AD-2327675、AD-2327677、AD-2327679、AD-2327684、AD-2327738、AD-2327742、AD-2327747、AD-2327753、AD-2327755、AD-2327756、AD-2327758、AD-2327759、AD-2327761、AD-2327762、AD-2327771、AD-2327774、AD-2327775、AD-2327776、AD-2327777、AD-2327778、AD-2327779、AD-2327780、AD-2327781、AD-2327782、AD-2327783、AD-2327784、AD-2327785、AD-2327846、AD-2327849、AD-2327856、AD-2327866、AD-2327867、AD-2327868、AD-2327869、AD-2327870、AD-2327871、AD-2327872、AD-2327940、AD-2327943、AD-2327944、AD-2327947、AD-2327960、AD-2327961、AD-2327962、AD-2328051、AD-2328052、AD-2328053、AD-2328054、AD-2328055、AD-2328056、AD-2328057、AD-2328060、AD-2328061、AD-2328065、AD-2328066、AD-2328067、AD-2328068、AD-2328069、AD-2328070、AD-2328071、AD-2328072、AD-2328073、AD-2328074、AD-2328075、AD-2328076、AD-2328077、AD-2328078、AD-2328079、AD-2328080、AD-2328081、AD-2328082、AD-2328083、AD-2328084、AD-2328085、AD-2328086、AD-2328137、AD-2328138、AD-2328139、AD-2328140、AD-2328141、AD-2328142、AD-2328143、AD-2328144、AD-2328145、AD-2328146、AD-2328171、AD-2328172、AD-2328179、AD-2328182、AD-2328183、AD-2328184、AD-2328185、AD-2328186、AD-2328237、AD-2328238、AD-2328239、AD-2328240、AD-2328241、AD-2328242、AD-2328243、AD-2328244、AD-2328246、AD-2328251、AD-2328252、AD-2328253、AD-2328254、AD-2328255、AD-2328256、AD-2328257、AD-2328258、AD-2328266、AD-2328374、AD-2328377、AD-2328576、AD-2328578、AD-2328579、AD-2328582、AD-2328583、AD-2328585、AD-2328586、AD-2328637、AD-2328639、AD-2328640、AD-2328669、AD-2328670、AD-2328839、AD-2328843、AD-2328844、AD-2328846、AD-2328859、AD-2328860、AD-2328937、AD-2328942、AD-2328943、AD-2328944、AD-2328945、AD-2328946、AD-2328947、AD-2328948、AD-2328949、AD-2328950、AD-2328979、AD-2329038、AD-2329044、AD-2329045、AD-2329046、AD-2329047、AD-2329048、AD-2329050、AD-2329051、AD-2329052、AD-2329080、AD-2329081、AD-2329082、AD-2329083、AD-2329084、AD-2329085、AD-2329143、AD-2329144、AD-2329145、AD-2329147、AD-2329148、AD-2329149、AD-2329150、AD-2329151、AD-2329152、AD-2329153、AD-2329154、AD-2329155、AD-2329156、AD-2329157、AD-2329158、AD-2329159、AD-2329160、AD-2329161、AD-2329162、AD-2329163、AD-2329164、AD-2329165、AD-2329166、AD-2329168、AD-2329173、AD-2329175、AD-2329176、AD-2329179、AD-2329180、AD-2329244、AD-2329245、AD-2329248、AD-2329249、AD-2329253、AD-2329254、AD-2329255、AD-2329259、AD-2329260、AD-2329263、AD-2329265、AD-2329268、AD-2329269、AD-2329271、AD-2329272、AD-2329273、AD-2329274、AD-2329275、AD-2329276、AD-2329277、AD-2329278、AD-2329284、AD-2329285、AD-2329373、AD-2329375、AD-2329376、AD-2329378、AD-2329379、AD-2329380、AD-2329381、AD-2329382、AD-2329385、AD-2329386、AD-2329437、AD-2329438、AD-2329439、AD-2329440、AD-2329444、AD-2329445、AD-2329446、AD-2329471、AD-2329475、AD-2329545、AD-2329546、AD-2329550、AD-2329551、AD-2329554、AD-2329555、AD-2329557、AD-2329558、AD-2329562、AD-2329563、AD-2329564、AD-2329565、AD-2329638、AD-2329639、AD-2329738、AD-2329739、AD-2329740、AD-2329743、AD-2329744、AD-2329751、AD-2329752、AD-2329753、AD-2329754、AD-2329755、AD-2329756、AD-2329757、AD-2329758、AD-2329759、AD-2329760、AD-2329761、AD-2329762、AD-2329763、AD-2329769、AD-2329772、AD-2329839、AD-2329840、AD-2329841、AD-2329878、AD-2329879、AD-2329880、AD-2329881、AD-2329882、AD-2329883、AD-2329884、AD-2329885、AD-2329951、AD-2329952、AD-2330058、AD-2330059、AD-2330060、AD-2330061、AD-2330062、AD-2330063、AD-2330065、AD-2330066、AD-2330071、AD-2330072、AD-2330075、AD-2330077、AD-2330079、AD-2330156、AD-2330159、AD-2330160、AD-2330163、AD-2330164、AD-2330165、AD-2330167、AD-2330168、AD-2330173、AD-2330176、AD-2330177、AD-2330179、AD-2330180、AD-2330244、AD-2330245、AD-2330246、AD-2330247、AD-2330248、AD-2330249、AD-2330250、AD-2330251、AD-2330252、AD-2330253、AD-2330254、AD-2330255、AD-2330256、AD-2330257、AD-2330258、AD-2330259、AD-2330260、AD-2330261、AD-2330262、AD-2330263、AD-2330267、AD-2330268、AD-2330269、AD-2330270、AD-2330271、AD-2330273、AD-2330342、AD-2330343、AD-2330344、AD-2330345、AD-2330365、AD-2330368、AD-2330484、AD-2330510、AD-2330515、AD-2330516、AD-2330517、AD-2330519、AD-2330520、AD-2330521、AD-2330532、AD-2330533、AD-2330534、AD-2330536、AD-2330560、AD-2330563、AD-2330565、AD-2330573、AD-2330574、AD-2330575、AD-2330596、AD-2330597、AD-2330598、AD-2330599、AD-2330600、AD-2330601、AD-2330602、AD-2330603、AD-2330604、AD-2330605、AD-2330606、AD-2330607、AD-2330608、AD-2330612、AD-2330613、AD-2330614、AD-2330615、AD-2330616、AD-2330618、AD-2330619、AD-2330620、AD-2330623、AD-2330624、AD-2330625、AD-2330626、AD-2330627、AD-2330628、AD-2330629、AD-2330630、AD-2330631、AD-2330632、AD-2330633、AD-2330634、AD-2330638、AD-2330639、AD-2330640、AD-2330641、AD-2330644、AD-2330647、AD-2330648、AD-2330649、AD-2330652、AD-2330653、AD-2330654、AD-2330655、AD-2330656、AD-2330657、AD-2330658、AD-2330659、AD-2330671、AD-2330672、AD-2330673、AD-2330674、AD-2330675、AD-2330679、AD-2330680、AD-2330681、AD-2330682、AD-2330683、AD-2330686、AD-2330691、AD-2330698、AD-2330699、AD-2330700、AD-2330701、AD-2330702、AD-2330703、AD-2330704、AD-2330705、AD-2330706、AD-2330707、AD-2330708、AD-2330709、AD-2330710、AD-2330711、AD-2330712、AD-2330713、AD-2330714、AD-2330715、AD-2330716、AD-2330717、AD-2330720、AD-2330722、AD-2330724、AD-2330736、AD-2330737、AD-2330738、AD-2330739、AD-2330740、AD-2330741、AD-2330742、AD-2330743、AD-2330744、AD-2330745、AD-2330746、AD-2330747、AD-2330752、AD-2330753、AD-2330754、AD-2330755、AD-2330756、AD-2330757、AD-2330759、AD-2330760、AD-2330761、AD-2330762、AD-2330763、AD-2330764、AD-2330783、AD-2330785、AD-2330821、AD-2330843、AD-2330844、AD-2330847、AD-2330849、AD-2330850、AD-2330851、AD-2330855, AD-2330856, AD-2330873, AD-2330875, AD-2330877, AD-2330878, AD-2330880, AD-2330881, AD-2330882, AD-2330883, AD-2330885, AD-233 0887, AD-2330891, AD-2330892, AD-2330897, AD-2330899, AD-2330901, AD- 2330902, AD-2330903, AD-2330904, AD-2330905, AD-2330906, AD-2330907, A The antisense strand nucleotide sequences of the duplexes of D-2330908, AD-2330909, AD-2330915, AD-2330916, AD-2330917, AD-2330918, AD-2330919, AD-2330920, AD-2330921, AD-2330922, AD-2330923, AD-2330924, AD-2330933, AD-2330934, AD-2330935, AD-2330936, AD-2330938, AD-2330939, AD-2330940, and AD-2330941 differ by no more than 0, 1, 2, or 3 nucleotides.

[0014] In one embodiment, the dsRNA agent is selected from AD-2320882.1, AD-2320938.1, AD-2321352.1, AD-2321573.1, AD-2321665.1, AD-2321839.1, AD-2321863.1, AD-2322178.1, AD-2322257.1, AD-2322371.1, AD-2322980.1, AD-2323248.1, AD-2323253.1, AD-2323445.1, AD-2323482.1, AD-2323584.1, AD-2323585.1, AD-2323784.1, AD-2323856.1, AD-2323858.1, AD-2323886.1, AD-2323961.1, AD-2323965.1, AD-2323970.1, AD-2323972.1, AD-2323976.1, AD-2324043.1, AD-2324044.1, AD-2324046.1, AD-2324047.1, AD-2324276.1, AD-2324670.1, AD-2325059.1, AD-2325148.1, AD-2325356.1, AD-2325357.1, AD-2325369.1, AD-2325373.1, AD-2325448.1, AD-2325450.1, AD-2325473.1, AD-2325474.1, AD-2325477.1, AD-2325546.1, AD-2325552.1, AD-2325646.1, AD-2325648.1, AD-2325674.1, AD-2325677.1, AD-2325678.1, AD-2325679.1, AD-2325681.1, AD-2325744.1, AD-2325745.1, AD-2325746.1, AD-2325748.1, AD-2325752.1, AD-2325762.1, AD-2325763.1, AD-2325764.1, AD-2325765.1, AD-2325769.1, AD-2325780.1, AD-2325781.1, AD-2325782.1, AD-2325784.1, AD-2325785.1, AD-2325786.1, AD-2325837.1, AD-2325838.1, AD-2325839.1, AD-2325840.1, AD-2325847.1, AD-1. AD-2325959.1, AD-2325968.1, AD-2325969.1, AD-2325973.1, AD-2325976.1, AD-2325979.1, AD-2325984.1, AD-23 26042.1, AD-2326067.1, AD-2326170.1, AD-2328140.1, AD-2328186.1, AD-2328257.1, AD-2328266.1 phase AD-2328843.1. .

[0015] In one embodiment, the dsRNA agent comprises at least one modified nucleotide.

[0016] In one embodiment, substantially all or all nucleotides of the sense strand contain a modification. In another embodiment, substantially all or all nucleotides of the antisense strand contain a modification. In yet another embodiment, substantially all or all nucleotides of both the sense and antisense strands contain modifications.

[0017] In one aspect, the present invention provides a double-stranded RNA (dsRNA) agent for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells. The dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand contains at least 15 consecutive nucleotides differing from the nucleotide sequence of SEQ ID NO: 1 by no more than 1, 2, or 3 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides differing from the nucleotide sequence of SEQ ID NO: 2 by no more than 1, 2, or 3 nucleotides, wherein substantially all or all nucleotides of the sense strand and substantially all or all nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a linked ligand at its 3'-end. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence of SEQ ID NO: 2, wherein substantially all or all nucleotides of the sense strand and substantially all or all nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a linked ligand at the 3'-end.

[0018] In one embodiment, all nucleotides of the sense strand contain a modification. In another embodiment, all nucleotides of the antisense strand contain a modification. In yet another embodiment, all nucleotides of both the sense and antisense strands contain a modification.

[0019] In one embodiment, at least one of the modified nucleotides is selected from deoxynucleotides, 3′-terminal deoxythymidine (dT) nucleotides, 2′-O-methyl modified nucleotides, 2′-fluoro modified nucleotides, 2′-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, debased nucleotides, 2′-amino modified nucleotides, 2′-O-allyl modified nucleotides, 2′-C-alkyl modified nucleotides, 2′-hydroxy modified nucleotides, 2′-methoxyethyl modified nucleotides, 2′-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, tetrahydropyran modified nucleotides, 1,5-dehydrated hexadiol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5′-phosphate, nucleotides containing 5′-phosphate mimics, diol modified nucleotides, and 2-O-(N-methylacetamide) modified nucleotides, and combinations thereof.

[0020] In one embodiment, the nucleotide modification is a 2′-O-methyl and / or a 2′-fluoro modification.

[0021] The length of the complementary region can be at least 17 nucleotides; 19 to 30 nucleotides; 19 to 25 nucleotides; or 21 to 23 nucleotides.

[0022] The length of each sense and antisense strand can not exceed 30 nucleotides. For example, the length of each sense and antisense strand can be 19-30 nucleotides independently; the length of each sense and antisense strand can be 19-25 nucleotides independently; or the length of each sense and antisense strand can be 21-23 nucleotides independently.

[0023] dsRNA may include: at least one of the sense strand or antisense strand having a 3' overhang of at least one nucleotide; or at least one of the sense strand or antisense strand having a 3' overhang of at least two nucleotides.

[0024] In some implementations, the dsRNA agent also contains a ligand.

[0025] In one implementation, the ligand is conjugated to the 3' end of the positive strand of the dsRNA agent.

[0026] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0027] In one implementation, the ligand is

[0028] In one embodiment, the dsRNA agent is conjugated with the ligand as illustrated below. Furthermore, X is either O or S.

[0029] In one implementation, X is O.

[0030] In one implementation, the complementary region contains any of the antisense sequences in Tables 3-6.

[0031] In one aspect, the present invention provides a double-stranded RNA (dsRNA) agent for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells. The dsRNA agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand contains a region complementary to a portion of the mRNA encoding AASS, wherein each strand is about 14 to about 30 nucleotides in length, and wherein the dsRNA agent is represented by formula (Ij): Justice: 5'n p -N a -(XX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3′ Antonym: 3′n p ′-N a ′-(X′X′X′) k -N b ′-Y′Y′Y′-N b ′-(Z′Z′Z′) l -N a ′-n q ′5′ (Ij) in: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0-6; Each N a and N a ′ Independently represents an oligonucleotide sequence comprising 0-25 nucleotides, wherein the nucleotides are modified, unmodified, or a combination thereof, and each sequence comprises at least two differently modified nucleotides; Each N b and N b ′ Independently represents an oligonucleotide sequence comprising 0-10 nucleotides, wherein the nucleotides are modified, unmodified, or a combination thereof; Each n p n p ′、n q and n qEach of the '' symbols may or may not be present, and each independently represents a nucleotide at the overhang. XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent a motif with three identical modifications on three consecutive nucleotides; N b The modifiers on N are different from those on Y, N b The modifications on ' are different from those on Y'; and The justice chain is conjugated with at least one ligand.

[0032] In one implementation, i is 0; j is 0; i is 1; j is 1; i and j are both 0; or i and j are both 1. In another implementation, k is 0; l is 0; k is 1; l is 1; k and 1 are both 0; or k and l are both 1.

[0033] In one implementation, XXX is complementary to X′X′X′, YYY is complementary to Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′.

[0034] In one implementation, the YYY motif is located at or near the cleavage site of the sense chain, for example, the Y′Y′Y′ motif is located at positions 11, 12, and 13 of the antisense chain, counting from the 5' end.

[0035] In one implementation, equation (Ij) is represented by equation (Ik): Justice: 5'n p -N a -YYY-N a -n q 3′ Antonym: 3′n p′ -N a′ -Y′Y′Y′-N a′ -n q′ 5′ (Ik).

[0036] In another implementation, equation (Ij) is represented by equation (II): Justice: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3′ Antonym: 3′n p′ -N a′ -Y′Y′Y′-N b′ -Z′Z′Z′-N a′ -n q′ 5′ (Il) Where each N b and Nb ′ represents an oligonucleotide sequence containing 1-5 modified nucleotides independently.

[0037] In another embodiment, equation (Ij) is represented by equation (Im): Justice: 5'n p -N a -XXX-N b -YYY-N a -n q 3′ Antonym: 3′n p′ -N a′ -X′X′X′-N b′ -Y′Y′Y′-N a′ -n q′ 5′ (Im) Where each N b and N b ′ represents an oligonucleotide sequence containing 1-5 modified nucleotides independently.

[0038] In another implementation, equation (Ij) is represented by equation (In): Justice: 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3′ Antonym: 3′n p′ -N a′ -X′X′X′-N b′ -Y′Y′Y′-N b′ -Z′Z′Z′-N a′ -n q′ 5′ (In) Where each N b and N b ′ independently represents an oligonucleotide sequence containing 1-5 modified nucleotides and each N a and N a ′ Independently represents an oligonucleotide sequence containing 2-10 modified nucleotides.

[0039] The length of the complementary region can be at least 17 nucleotides; 19 to 30 nucleotides; 19 to 25 nucleotides; or 21 to 23 nucleotides.

[0040] Each strand may be no more than 30 nucleotides long; for example, each strand may be 19-30 nucleotides long independently.

[0041] In one embodiment, the modification on the nucleotide is selected from LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluorine, 2'-O-methyl, 2'-deoxy, 2'-hydroxy, and combinations thereof.

[0042] In one embodiment, the modification on the nucleotide is a 2'-O-methyl or 2'-fluoro modification.

[0043] In one embodiment, Y′ is a 2′-O-methyl or 2′-fluoromodified nucleotide.

[0044] In one embodiment, at least one of the sense strand or antisense strand of the dsRNA agent may include a 3' overhang having at least one nucleotide; or a 3' overhang having at least two nucleotides.

[0045] In one embodiment, the dsRNA agent further comprises at least one phosphate thioester or methylphosphonate nucleotide linker.

[0046] In one embodiment, the thiophosphate or methylphosphonate nucleotides are linked at the 3' end of one strand. In one embodiment, this strand is an antisense strand. In another embodiment, this strand is a sense strand.

[0047] In one embodiment, the thiophosphate or methylphosphonate nucleotides are linked at the 5' end of one strand. In one embodiment, this strand is an antisense strand. In another embodiment, this strand is a sense strand.

[0048] In one implementation, the chain is an antisense chain. In another implementation, the chain is a justice chain.

[0049] In one embodiment, the thiophosphate or methylphosphonate nucleotides are linked together at both the 5'- and 3'-termini of a single chain.

[0050] In one embodiment, the base pair at position 1 of the 5'-end of the antisense strand of the duplex is an AU base pair.

[0051] In one implementation, p′ > 0. In another implementation, p′ = 2.

[0052] In one implementation, q' = 0, p = 0, q = 0, and the p' overhang nucleotide is complementary to the target mRNA. In another implementation, q' = 0, p = 0, q = 0, and the p' overhang nucleotide is not complementary to the target mRNA.

[0053] In one implementation, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0054] In one implementation, at least n p It is linked to adjacent nucleotides via a phosphate thioester bond. In another embodiment, all n p It is linked to an adjacent nucleotide via a thiophosphate bond.

[0055] In one implementation, all nucleotides of the sense strand and all nucleotides of the antisense strand contain modifications.

[0056] In one implementation, the ligand is conjugated to the 3' end of the positive strand of the dsRNA agent.

[0057] In one embodiment, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives linked by monovalent, divalent, or trivalent branching linkers.

[0058] In one implementation, the ligand is

[0059] In one embodiment, the dsRNA agent is conjugated with the ligand as illustrated below. Furthermore, X is either O or S.

[0060] In one implementation, X is O.

[0061] In one aspect, the present invention provides a double-stranded RNA (dsRNA) agent for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells. The dsRNA agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand contains a region complementary to a portion of the mRNA encoding AASS, wherein each strand is about 14 to about 30 nucleotides in length, and wherein the dsRNA agent is represented by formula (Ij): Justice: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3′ Antonym: 3′n p ′-N a ′-(X′X′X′) k -N b ′-Y′Y′Y′-N b ′-(Z′Z′Z′) l -N a ′-n q ′5′ (Ij) in: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0-6; Each N a and N a ′ Independently represents an oligonucleotide sequence comprising 0-25 nucleotides, wherein the nucleotides are modified, unmodified, or a combination thereof, and each sequence comprises at least two differently modified nucleotides; Each N b and N b ′ Independently represents an oligonucleotide sequence comprising 0-10 nucleotides, wherein the nucleotides are modified, unmodified, or a combination thereof; Each n p n p ′、n q and n q Each of the '' symbols may or may not be present, and each independently represents a nucleotide at the overhang. XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent a motif having three identical modifications on three consecutive nucleotides, wherein the modification is a 2'-O-methyl or a 2'-fluoro modification; N b The modifiers on N are different from those on Y, N b The modifications on ' are different from those on Y'; and The justice chain is conjugated with at least one ligand.

[0062] In one aspect, the present invention provides a double-stranded RNA (dsRNA) agent for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells. The dsRNA agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand contains a region complementary to a portion of the mRNA encoding AASS, wherein each strand is about 14 to about 30 nucleotides in length, and wherein the dsRNA agent is represented by formula (Ij): Justice: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3′ Antonym: 3′n p ′-N a ′-(X′X′X′) k -N b ′-Y′Y′Y′-Nb ′-(Z′Z′Z′) l -N a ′-n q ′5′ (Ij) in: i, j, k, and l are each independently 0 or 1; Each n p n q and n q Each of the '' symbols may or may not be present, and each independently represents a nucleotide at the overhang. p, q, and q′ are each independently 0-6; n p ′>0 and at least one n p It is linked to an adjacent nucleotide via a phosphate thioester bond; Each N a and N a ′ Independently represents an oligonucleotide sequence comprising 0-25 nucleotides, wherein the nucleotides are modified, unmodified, or a combination thereof, and each sequence comprises at least two differently modified nucleotides; Each N b and N b ′ Independently represents an oligonucleotide sequence comprising 0-10 nucleotides, wherein the nucleotides are modified, unmodified, or a combination thereof; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent a motif having three identical modifications on three consecutive nucleotides, wherein the modification is a 2'-O-methyl or 2'-fluoro modification; N b The modifiers on N are different from those on Y, N b The modifications on ' are different from those on Y'; and The justice chain is conjugated with at least one ligand.

[0063] In one aspect, the present invention provides a double-stranded RNA (dsRNA) agent for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells. The dsRNA agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand contains a region complementary to a portion of the mRNA encoding AASS, wherein each strand is about 14 to about 30 nucleotides in length, and wherein the dsRNA agent is represented by formula (Ij): Justice: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -Na -n q 3′ Antonym: 3′n p ′-N a ′-(X′X′X′) k -N b ′-Y′Y′Y′-N b ′-(Z′Z′Z′) l -N a ′-n q ′5′ (Ij) in: i, j, k, and l are each independently 0 or 1; Each n p n q and n q Each of the '' symbols may or may not be present, and each independently represents a nucleotide at the overhang. p, q, and q′ are each independently 0-6; n p ′>0 and at least one n p It is linked to an adjacent nucleotide via a phosphate thioester bond; Each N a and N a ′ Independently represents an oligonucleotide sequence comprising 0-25 nucleotides, wherein the nucleotides are modified, unmodified, or a combination thereof, and each sequence comprises at least two differently modified nucleotides; Each N b and N b ′ Independently represents an oligonucleotide sequence comprising 0-10 nucleotides, wherein the nucleotides are modified, unmodified, or a combination thereof; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent a motif having three identical modifications on three consecutive nucleotides, wherein the modification is a 2'-O-methyl or 2'-fluoro modification; N b The modifiers on N are different from those on Y, N b The modifications on ' are different from those on Y'; and The positive chain is conjugated with at least one ligand, wherein the ligand is one or more GalNAc derivatives linked by a monovalent, divalent, or trivalent branching linker.

[0064] In one aspect, the present invention provides a double-stranded RNA (dsRNA) agent for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells. The dsRNA agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand contains a region complementary to a portion of the mRNA encoding AASS, wherein each strand is about 14 to about 30 nucleotides in length, and wherein the dsRNA agent is represented by formula (Ij): Justice: 5'n p -N a -(XXX) l -N b -YYY-N b -(ZZZ) j -N a -n q 3′ Antonym: 3′n p ′-N a ′-(X′X′X′) k -N b ′-Y′Y′Y′-N b ′-(Z′Z′Z′) l -N a ′-n q ′5′ (Ij) in: i, j, k, and l are each independently 0 or 1; Each n p n q and n q Each of the '' symbols may or may not be present, and each independently represents a nucleotide at the overhang. p, q, and q′ are each independently 0-6; n p ′>0 and at least one n p It is linked to an adjacent nucleotide via a phosphate thioester bond; Each N a and N a ′ Independently represents an oligonucleotide sequence comprising 0-25 nucleotides, wherein the nucleotides are modified, unmodified, or a combination thereof, and each sequence comprises at least two differently modified nucleotides; Each N b and N b ′ Independently represents an oligonucleotide sequence comprising 0-10 nucleotides, wherein the nucleotides are modified, unmodified, or a combination thereof; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent a motif having three identical modifications on three consecutive nucleotides, wherein the modification is a 2'-O-methyl or 2'-fluoro modification; N b The modifiers on N are different from those on Y, N b The modifications on ' are different from those on Y'; The positive chain contains at least one thiophosphate bond; and The positive chain is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives linked by a monovalent, divalent, or trivalent branching connector.

[0065] In one aspect, the present invention provides a double-stranded RNA (dsRNA) agent for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells. The dsRNA agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand contains a region complementary to a portion of the mRNA encoding AASS, wherein each strand is about 14 to about 30 nucleotides in length, and wherein the dsRNA agent is represented by formula (Ij): Justice: 5'n p -N a -YYY-N a -n q 3′ Antonym: 3′n p ′-N a ′-Y′Y′Y′-N a ′-n q ′5′ (Ik) in: Each n p n q and n q Each of the '' symbols may or may not be present, and each independently represents a nucleotide at the overhang. p, q, and q′ are each independently 0-6; n p ′>0 and at least one n p It is linked to an adjacent nucleotide via a phosphate thioester bond; Each N a and N a ′ Independently represents an oligonucleotide sequence comprising 0-25 nucleotides, wherein the nucleotides are modified, unmodified, or a combination thereof, and each sequence comprises at least two differently modified nucleotides; YYY and Y′Y′Y′ each independently represent a motif having three identical modifications on three consecutive nucleotides, wherein the modifications are 2'-O-methyl and / or 2'-fluoro modifications; The positive chain comprises at least one thiophosphate bond; and the positive chain is conjugated with at least one ligand, wherein the ligand is one or more GalNAc derivatives linked by a monovalent, divalent or trivalent branching linker.

[0066] In one aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells. The dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand contains at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 1, 2, or 3 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by no more than 1, 2, or 3 nucleotides, wherein substantially all or all nucleotides of the sense strand contain modifications selected from 2'-O-methyl and 2'-fluoro, wherein the sense strand contains two thiophosphate nucleotides linked at the 5' end, wherein substantially all or all nucleotides of the antisense strand contain modifications selected from 2'-O-methyl and 2'-fluoro, wherein the antisense strand contains two thiophosphate nucleotides linked at the 5' end and two thiophosphate nucleotides linked at the 3' end, and wherein the sense strand is conjugated at the 3' end to one or more GalNAc derivatives linked by a monovalent, divalent, or trivalent branching adapter. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence of SEQ ID NO: 2, wherein substantially all or all nucleotides of the sense strand comprise a modification selected from 2'-O-methyl and 2'-fluoro, wherein the sense strand comprises two thiophosphate nucleotides linked at the 5' end, wherein substantially all or all nucleotides of the antisense strand comprise a modification selected from 2'-O-methyl and 2'-fluoro, wherein the antisense strand comprises two thiophosphate nucleotides linked at the 5' end and two thiophosphate nucleotides linked at the 3' end, and wherein the sense strand is conjugated at the 3' end to one or more GalNAc derivatives linked by a monovalent, divalent, or trivalent branching adapter.

[0067] In one implementation, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

[0068] In one implementation, the complementary region includes any of the antisense sequences listed in Tables 3-6.

[0069] In one implementation, the sense strand and the antisense strand comprise nucleotide sequences selected from any of the agents listed in Tables 3-6.

[0070] In the various embodiments of the aforementioned dsRNA agent, the dsRNA agent targets hotspot regions of the mRNA encoding AASS.

[0071] In another aspect, the present invention provides a dsRNA agent that targets hotspot regions of aminoadiazine semialdehyde synthase (AASS) mRNA.

[0072] The present invention also provides cell, vector, and pharmaceutical compositions comprising any of the dsRNA agents of the present invention. The dsRNA agents can be formulated in a non-buffered solution, such as saline or water, or in a buffered solution, such as a solution comprising acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS).

[0073] In one aspect, the present invention provides a method for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells. The method comprises contacting cells with the dsRNA agent or pharmaceutical composition of the present invention, thereby inhibiting the expression of AASS in the cells.

[0074] Cells can be present in a subject (such as a human subject).

[0075] In one implementation, AASS expression is suppressed by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or suppressed to below the detection limit of AASS expression.

[0076] In one implementation, the human subject suffers from a disease, condition, or illness associated with AASS. In one implementation, the disease, condition, or illness associated with AASS is a lysine catabolism disorder. In one implementation, the lysine catabolism disorder is type 1 glutaric aciduria (GA1). In one implementation, the lysine catabolism disorder is pyridoxine-dependent epilepsy (PDE).

[0077] In one aspect, the present invention provides a method for inhibiting the expression of AASS in a subject. The method comprises administering a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present invention to the subject, thereby inhibiting the expression of AASS in the subject.

[0078] In another aspect, the present invention provides a method for treating a subject suffering from an AASS-related disease, condition, or illness. The method comprises administering to the subject a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present invention, thereby treating the subject suffering from an AASS-related disease, condition, or illness.

[0079] In another aspect, the present invention provides a method for preventing at least one symptom in a subject suffering from a disease, condition, or ailment that would benefit from a reduction in AASS gene expression. The method comprises administering to the subject a preventatively effective amount of the dsRNA agent or pharmaceutical composition of the present invention, thereby preventing at least one symptom in a subject suffering from a disease, condition, or ailment that would benefit from a reduction in AASS gene expression.

[0080] In one embodiment, administration of a dsRNA agent or pharmaceutical composition to a subject results in a decrease in AASS protein activity, for example, a reduction in the production of yeast amino acids and / or 2-aminoadipic acid-4-halfaldehyde; a reduction in AASS protein accumulation, AASS enzyme activity, glutaric acid and / or 3-hydroxyglutaric acid accumulation, and / or a reduction in α-aminoadipic acid-4-halfaldehyde (α-AASA) in the subject.

[0081] In one implementation, the disease, condition, or symptom associated with AASS is a lysine catabolism disorder.

[0082] In one implementation, the lysine catabolism disorder is glutaric aciduria type 1 (GA1).

[0083] In one implementation, lysine catabolism disorder is pyridoxine-dependent epilepsy (PDE).

[0084] In one embodiment, the method and use of the present invention further include administering additional therapeutic agents to the subject.

[0085] In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg.

[0086] The agent may be administered to the subject intravenously, intramuscularly, or subcutaneously. In one embodiment, the agent is administered to the subject subcutaneously.

[0087] In one embodiment, the method and use of the present invention further include measuring the level of AASS in a subject.

[0088] In one aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any one of any agents in any of Tables 3-6, and the antisense strand comprises a nucleotide sequence of any one of any agents in any of Tables 3-6, wherein substantially all or all nucleotides of the sense strand and substantially all or all nucleotides of the antisense strand are modified nucleotides, and wherein the dsRNA agent is conjugated to a ligand. Detailed Implementation

[0089] This invention provides an iRNA composition that achieves cleavage of the AASS gene RNA transcript mediated by an RNA-induced silencing complex (RISC). The AASS gene is intracellular, for example, within the cells of a subject (such as a human). This invention also provides methods for inhibiting AASS gene expression and treating subjects who would benefit from inhibiting or reducing AASS gene expression using the iRNA composition of this invention, such as subjects who have or are susceptible to AASS-related diseases, conditions, or ailments, such as subjects who have or are susceptible to lysine catabolism disorders, for example, subjects with type 1 glutaric aciduria (GA1) or pyridoxine-dependent epilepsy (PDE).

[0090] The yeast lysine pathway, primarily located in liver mitochondria, is the main lysine catabolism pathway in humans. Aminoadipic acid hemialdehyde synthase (AASS) is a bifunctional enzyme involved in the first two steps of lysine catabolism in this pathway. AASS's lysine-ketoglutarate reductase function breaks down lysine into yeast amino acid, and in the second step, yeast amino acid dehydrogenase function converts yeast amino acid to α-aminoadipic acid hemialdehyde. The next reaction in this pathway is the conversion of α-aminoadipic acid hemialdehyde to α-aminoadipic acid by α-aminoadipic acid hemialdehyde dehydrogenase (ALDH7A1). This subsequently produces 2-ketoadipic acid, which is then converted to glutaryl-CoA. Glutaryl-CoA is then converted to crotonyl-CoA by glutaryl-CoA dehydrogenase (GCDH). Inhibition of the conversion of glutaryl-CoA to crotonyl-CoA may lead to the production and accumulation of toxic catabolites such as glutaric acid and 3-hydroxyglutaric acid. Disruptions to lysine catabolism pathways, such as the deficiency of any enzyme, can lead to various lysine catabolism disorders, including type 1 glutaric aciduria (GA1) and pyridoxine-dependent epilepsy (PDE). GA1 is caused by a deficiency of GCDH, which leads to the accumulation of the toxic byproduct glutaryl-CoA. A deficiency of ALDH7A1 results in the accumulation of α-aminohexanoic acid semialdehyde and L-piperidinic acid, as well as the occurrence of PDE.

[0091] In the lysine catabolism pathway, AASS is upstream of ALDH7A1 and GCDH. Inhibition or reduction of AASS expression leads to a decrease in metabolite accumulation. GCDH knockout mice possess the GA1 phenotype and have been used as a model for studying GA1. GCDH / AASS double knockout mice have improved survival rates, reduced levels of the toxic metabolite glutaric acid, and also reduced levels of the GA1 phenotype (Barzi et al. 2023, Sci. Transl. Med. 15(692): eadf4086; and Leandro et al. 2020, J. Inherited Metabolic Disease, 43: 1154-64, which are incorporated herein by reference). - / - / AASS + / + Hepatocyte transplantation into GCDH / AASS double knockout mice resulted in the restoration of the GA1 phenotype (Barzi et al. 2023, Sci. Transl. Med. 15(692): eadf4086). Therefore, AASS is a target for treating lysine catabolism disorders using the dsRNA agents presented in this paper.

[0092] The iRNA targeting AASS of the present invention may include an RNA strand (antisense strand) having a region of about 30 nucleotides or shorter, such as 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30 nucleotides. A region of 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides, which is substantially complementary to at least a portion of the mRNA transcript of the AASS gene.

[0093] In some embodiments, one or both strands of the double-stranded RNAi agent of the present invention are up to 66 nucleotides in length, for example, 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides in length, and have a region of at least 19 consecutive nucleotides substantially complementary to at least a portion of the mRNA transcript of the AASS gene. In some embodiments, such iRNA agents having a longer antisense strand may include a second RNA strand (sense strand) of 20-60 nucleotides in length, wherein the sense and antisense strands form a doublet of 18-30 consecutive nucleotides.

[0094] Using the iRNA agents described herein allows for targeted degradation of the mRNA of the AASS gene in mammals.

[0095] In particular, very low doses of iRNA can specifically and effectively mediate RNA interference (RNAi), leading to a significant inhibition of AASS gene expression. Therefore, methods and compositions incorporating these iRNAs can be used to treat subjects who would benefit from inhibition or reduction of AASS gene expression, such as subjects with or susceptible to AASS-related diseases, conditions, or illnesses, such as subjects with or susceptible to lysine catabolism disorders, for example, subjects with type 1 glutaric aciduria (GA1) or pyridoxine-dependent epilepsy (PDE).

[0096] The following detailed description discloses how to prepare and use compositions containing iRNA to inhibit the expression of the AASS gene, as well as compositions and methods for treating subjects suffering from diseases and conditions that would benefit from inhibiting and / or reducing the expression of the gene.

[0097] I. Definition To facilitate a clearer understanding of the invention, certain terms are first defined. Additionally, it should be noted that whenever a value or range of a parameter is mentioned, it is intended that values ​​and ranges intermediate in the range are also intended to be part of the invention.

[0098] The articles “a” and “an” are used in this text to refer to one or more (i.e., at least one) grammatical object of the article. For example, “an element” refers to one or more elements, such as multiple elements.

[0099] The term “including” is used in this document to mean “including but not limited to” and is used interchangeably with that expression.

[0100] The term “or” is used herein to refer to the term “and / or” and is used interchangeably with that term unless the context clearly specifies otherwise.

[0101] The term “about” is used herein to refer to a range of tolerances typical in the art. For example, “about” can be understood as approximately 2 standard deviations from the mean. In some embodiments, “about” refers to ±10%. In some embodiments, “about” refers to ±5%. When “about” precedes a series of numbers or ranges, it should be understood that “about” may modify each number in the series or range.

[0102] The term “AASS”, also known as “aminoadipic acid hemialdehyde synthase,” “α-aminoadipic acid hemialdehyde synthase,” “aminoadipic acid hemialdehyde synthase,” “α-aminoadipic acid hemialdehyde dehydrogenase (AASADH),” “lysine-2-oxoglutarate reductase,” or “lysine-ketoglutarate reductase / yeast lysine dehydrogenase,” unless otherwise specified, refers to the well-known gene encoding an AASS protein from any vertebrate or mammalian source, including but not limited to humans, bovines, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys, and guinea pigs.

[0103] The term also refers to fragments and variants of natural AASS that retain at least one in vivo or in vitro activity of natural AASS.

[0104] For Homo sapiens, exemplary nucleotide and amino acid sequences of AASS can be found, for example, in GenBank accession number NM_005763.4 (SEQ ID NO: 1; reverse complementary sequence SEQ ID NO: 2).

[0105] For house mice, exemplary nucleotide and amino acid sequences of AASS can be found, for example, in GenBank accession number NM_013930.4 (SEQ ID NO: 3; reverse complementary sequence SEQ ID NO: 4).

[0106] For brown rats, exemplary nucleotide and amino acid sequences of AASS can be found, for example, in GenBank accession number NM_001100963.1 (SEQ ID NO: 5; reverse complementary sequence SEQ ID NO: 6).

[0107] For cynomolgus monkeys, exemplary nucleotide and amino acid sequences of AASS can be found, for example, in GenBank accession number XM_005550630.2 (SEQ ID NO: 9311; reverse complementary sequence SEQ ID NO: 9312).

[0108] Other instances of AASS mRNA sequences are readily available from public databases such as GenBank, UniProt, and OMIM.

[0109] For example, more information about AASS is available in the NCBI Gene Database (http: / / www.ncbi.nlm.nih.gov / gene / 10157).

[0110] In some implementations, iRNAs that are substantially complementary to regions of mouse or rat AASS mRNA will cross-react with human AASS mRNA and are potential candidates for human targeting.

[0111] As used herein, the term “AASS” also refers to a specific polypeptide expressed in cells by naturally occurring DNA sequence variations of the AASS gene, such as single nucleotide polymorphisms (SNPs) within the AASS gene. Many single nucleotide polymorphisms (SNPs) within the AASS gene have been identified and can be found, for example, in NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp).

[0112] As used herein, a “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the AASS gene, including mRNA that is a product of RNA processing of the primary transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-guided cleavage at or near that nucleotide sequence portion of the mRNA molecule formed during the transcription of the AASS gene.

[0113] The target sequence of the AASS gene can be approximately 9-36 nucleotides in length, for example, approximately 15-30 nucleotides in length. Examples of target sequence lengths include 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, and 19... -29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides. Ranges and lengths falling between those described above are also included as part of this invention.

[0114] As used herein, the term "chain containing the sequence..." refers to an oligonucleotide that contains a nucleotide chain described by a sequence referred to using standard nucleotide nomenclature.

[0115] “G,” “C,” “A,” “T,” and “U” typically represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively. However, it should be understood that the term “ribonucleotide” or “nucleotide” can also refer to modified nucleotides or alternative substitutions as further detailed below (see, for example, Table 2). Those skilled in the art will appreciate that guanine, cytosine, adenine, and uracil can be substituted with other portions without substantially altering the base-pairing properties of oligonucleotides containing nucleotides with such substitutions. For example, but not limited to, nucleotides containing inosine as a base can pair with nucleotides containing adenine, cytosine, or uracil. Therefore, nucleotides containing uracil, guanine, or adenine in the nucleotide sequence of the dsRNA of this invention can be substituted with nucleotides containing, for example, inosine. In another example, adenine and cytosine anywhere in an oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU Wobble base pairing with the target mRNA. Sequences containing such substitutional portions are suitable for the compositions and methods involved in this invention.

[0116] As may be used interchangeably herein, the terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interference agent” refer to an agent containing RNA as defined herein and which mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process called RNA interference (RNAi). iRNA regulates (e.g., inhibits) the expression of the AASS gene in cells (e.g., cells in a subject, such as a mammalian subject).

[0117] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence (e.g., an AASS target mRNA sequence) to guide the cleavage of the target RNA. While not wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is cleaved into siRNA by a type III endonuclease called Dicer (Sharp et al. (2001) Genes Dev. 15: 485). Dicer is a ribonuclease III-like enzyme that processes dsRNA into short interfering RNA of 19-23 base pairs in length and with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409: 363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC), in which one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107: 309). Upon binding to a suitable target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) GenesDev. 15: 188). Therefore, in one aspect, the present invention relates to a single-stranded RNA (sssiRNA) generated within a cell that promotes the formation of the RISC complex to achieve silencing of a target gene (i.e., the AASS gene). Thus, the term "siRNA" is also used herein to refer to RNAi as described above.

[0118] In another embodiment, the RNAi agent may be a single-stranded RNAi agent, which is introduced into a cell or organism to inhibit the target mRNA. The single-stranded RNAi agent (ssRNAi) binds to the RISC endonuclease Argonaute 2, which subsequently cleaves the target mRNA. Single-stranded siRNAs are typically 15-30 nucleotides long and chemically modified. The design and testing of single-stranded RNAi agents are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are hereby incorporated herein by reference. Any antisense nucleotide sequence described herein may be used as a single-stranded siRNA as described herein, or chemically modified using the methods described in Lima et al., (2012) Cell 150:883-894.

[0119] In another embodiment, the "iRNA" used in the compositions and methods of the present invention is double-stranded RNA, and is referred to herein as a "double-stranded RNAi agent," a "double-stranded RNA (dsRNA) molecule," a "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands with "sense" and "antisense" orientations relative to the target RNA (i.e., the AASS gene). In some embodiments of the present invention, the double-stranded RNA (dsRNA) triggers the degradation of the target RNA (e.g., mRNA) through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).

[0120] Typically, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Additionally, as used herein, an "RNAi agent" may include chemically modified ribonucleotides; an RNAi agent may include substantial modifications at multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide having a modified sugar moiety, modified internucleotide linkages, and / or modified nucleobases. Therefore, the term "modified nucleotide" encompasses substitutions, additions, or removals of functional groups or atoms in internucleotide linkages, sugar moieties, or nucleobases. Modifications suitable for use in agents of this invention include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, "RNAi agent" encompasses any such modifications used in siRNA-type molecules.

[0121] The double-stranded region can have any length that allows the desired target RNA to be specifically degraded via the RISC pathway, and the length can be in the range of about 9 to 36 base pairs, for example, about 15-30 base pairs, such as about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 base pairs, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15- 17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19- 21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Ranges and lengths falling between those described above are also included as part of this invention.

[0122] The two strands forming a double-stranded structure can be different parts of a larger RNA molecule, or they can be separate RNA molecules. When the two strands are parts of a larger molecule and thus form a double-stranded structure through a continuous nucleotide chain connecting the 3' end of one strand to the 5' end of the corresponding other strand, this connecting RNA strand is called a "hairpin loop." A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, the hairpin loop may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides.

[0123] When the two substantially complementary strands of dsRNA consist of separate RNA molecules, these molecules do not necessarily need to be covalently linked, but they can be. In cases where the two strands are covalently linked by means other than a continuous nucleotide chain between the 3' end of one strand and the 5' end of the corresponding strand to form a duplex structure, this linking structure is called a "connector." The RNA strands can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, RNAi may also contain one or more nucleotide overhangs.

[0124] In one embodiment, the RNAi agent of the present invention is dsRNA, each strand of which contains fewer than 30 nucleotides, such as 17-27, 19-27, 17-25, 19-25, or 19-23 nucleotides. This agent interacts with a target RNA sequence (e.g., an AASS target mRNA sequence) to guide the cleavage of the target RNA. In another embodiment, the RNAi agent of the present invention is dsRNA, each strand of which contains 19-23 nucleotides. This agent interacts with a target RNA sequence (e.g., an AASS target mRNA sequence) to guide the cleavage of the target RNA. In one embodiment, the sense strand is 21 nucleotides long. In another embodiment, the antisense strand is 23 nucleotides long.

[0125] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of an iRNA (e.g., dsRNA). For example, a nucleotide overhang is present when the 3' end of one strand of dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide. Alternatively, an overhang may contain at least two, three, four, five, or more nucleotides. A nucleotide overhang may contain or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). An overhang may be on the sense strand, antisense strand, or any combination thereof. Furthermore, the nucleotide overhang may be present at the 5' end, 3' end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0126] In one embodiment, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at the 3' and / or 5' ends, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhangs. In another embodiment, the sense strand of the dsRNA has a 1-10 nucleotide overhang at the 3' and / or 5' ends, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhangs. In another embodiment, one or more nucleotides in the overhangs are replaced with nucleoside phosphate thioesters.

[0127] In some embodiments, the overhang on the sense strand or antisense strand, or both, may include an extension length longer than 10 nucleotides, such as 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides. In some embodiments, the extended overhang is on the sense strand of the duplex. In some embodiments, the extended overhang is located at the 3' end of the sense strand of the duplex. In some embodiments, the extended overhang is located at the 5' end of the sense strand of the duplex. In some embodiments, the extended overhang is on the antisense strand of the duplex. In some embodiments, the extended overhang is located at the 3' end of the antisense strand of the duplex. In some embodiments, the extended overhang is located at the 5' end of the antisense strand of the duplex. In some embodiments, one or more nucleotides in the extended overhang are replaced with nucleoside phosphate thioesters.

[0128] As used in this article regarding dsRNA, the term "flat" or "flat-ended" refers to the absence of unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be flat. When both ends of a dsRNA are flat, it is called a flat-ended dsRNA. It is important to clarify that a "flat-ended" dsRNA is a dsRNA with both ends flat, meaning there are no nucleotide overhangs at either end of the molecule. In most cases, such molecules are double-stranded along their entire length.

[0129] The term "antisense strand" or "guide strand" refers to a strand of iRNA (e.g., dsRNA) that contains a region substantially complementary to the target sequence (e.g., AASSmRNA).

[0130] As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., a target sequence, such as an AASS nucleotide sequence as defined herein). In cases where the complementary region is not perfectly complementary to the target sequence, mismatches can occur in internal or terminal regions of the molecule. Typically, the most tolerable mismatches are in terminal regions, such as within 5, 4, 3, or 2 nucleotides of the 5'-terminus and / or 3'-terminus of iRNA.

[0131] As used herein, the term "sense strand" or "passenger strand" refers to a strand of iRNA that includes regions substantially complementary to the regions of the antisense strand as defined herein.

[0132] As used herein, the term "cleavage region" refers to the region immediately adjacent to a cleavage site. A cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases immediately adjacent to the cleavage site at either end. In some embodiments, the cleavage region comprises two bases immediately adjacent to the cleavage site at either end. In some embodiments, the cleavage site specifically occurs at the site where nucleotides 10 and 11 of the antisense strand bind, and the cleavage region comprises nucleotides 11, 12, and 13.

[0133] As used herein, unless otherwise stated, the term "complementarity" when describing a first nucleotide sequence relative to a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions and form a double-stranded structure, as will be understood by those skilled in the art. Such conditions can be, for example, stringent conditions, which may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50 or 70 °C for 12–16 hours, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press"). Other conditions, such as physiologically relevant conditions that may be encountered in a living organism, may also apply. 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.

[0134] Complementary sequences within iRNA (e.g., within dsRNA as described herein) comprise base pairings of an oligonucleotide or polynucleotide containing a first nucleotide sequence with an oligonucleotide or polynucleotide containing a second nucleotide sequence over the entire length of one or both nucleotide sequences. In this document, such sequences may be referred to as “perfectly complementary” relative to each other. However, when referred to herein as “substantially complementary” relative to a second sequence, the two sequences may be perfectly complementary, or they may form one or more, but typically no more than 5, 4, 3, or 2 mismatched base pairs when hybridizing to form a duplex of up to 30 base pairs, while maintaining their ability to hybridize under conditions most relevant to their final application (e.g., repressing gene expression via a RISC pathway). However, in cases where the two oligonucleotides are engineered to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches when determining complementarity. For example, for the purposes described herein, a dsRNA comprising an oligonucleotide of length 21 and another oligonucleotide of length 23, wherein the longer oligonucleotide contains a sequence of 21 nucleotides that are perfectly complementary to the shorter oligonucleotide, may still be referred to as “perfectly complementary”.

[0135] As used herein, “complementary” sequences may also include non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, or may be formed entirely from them, provided that the above requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairs.

[0136] In this article, the terms “complementary,” “fully complementary,” and “substantially complementary” may be used to describe the base match between the sense and antisense strands of dsRNA, or the base match between the antisense strand and the target sequence of iRNA, as understood in the context in which they are used.

[0137] As used herein, a polynucleotide that is substantially complementary to at least a portion of messenger RNA (mRNA) is a polynucleotide that is substantially complementary to a continuous portion of a target mRNA (e.g., the mRNA encoding AASS). For example, if a polynucleotide sequence is substantially complementary to an uninterrupted portion of the mRNA encoding AASS, then the polynucleotide is complementary to at least a portion of the AASS mRNA.

[0138] Therefore, in some embodiments, the antisense polynucleotide disclosed herein is completely complementary to the target AASS sequence. In other embodiments, the antisense polynucleotide disclosed herein is substantially complementary to the target AASS sequence and comprises a continuous nucleotide sequence that is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 1 or the fragment of SEQ ID NO: 1, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0139] In one embodiment, the RNAi agent of the present invention comprises a sense strand substantially complementary to an antisense polynucleotide, which in turn is complementary to a target AASS sequence, and wherein the sense strand polynucleotide comprises a continuous nucleotide sequence that is at least about 80% complementary over its entire length to an equivalent region of the nucleotide sequence of SEQ ID NO: 2 or a fragment of any of SEQ ID NO: 2, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0140] In some embodiments, the iRNA of the present invention comprises an antisense strand that is substantially complementary to the target AASS sequence and comprises a continuous nucleotide sequence that is at least about 80% complementary over its entire length to an equivalent region of the nucleotide sequence of any of the positive strands in Tables 3-6 or a fragment of any of the positive strands in Tables 3-6, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, or 100% complementary.

[0141] As used herein, the term “inhibition” is used interchangeably with “reduction,” “silence,” “downregulation,” “suppression,” and other similar terms, and includes any level of inhibition.

[0142] As used herein, the expression “inhibit AASS gene expression” includes inhibiting the expression of any AASS gene (such as, for example, mouse AASS gene, rat AASS gene, monkey AASS gene, or human AASS gene) as well as inhibiting the expression of variants or mutants of the AASS gene that encodes the AASS protein.

[0143] "Inhibition of AASS gene expression" includes inhibition of the AASS gene at any level, such as at least partial inhibition of AASS gene expression, like at least about 20%. In some embodiments, the inhibition is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0144] AASS gene expression can be assessed based on the level of any variable associated with AASS gene expression, such as AASS mRNA or AASS protein levels. AASS gene expression can also be assessed indirectly based on, for example, a decrease in AASS protein activity, such as a reduction in AASS biological activity, such as a decrease in the production of yeast glutamic acid and / or 2-aminohexanoic acid-4-halfalaldehyde; a decrease in the accumulation of glutaric acid and / or 3-hydroxyglutaric acid in the subject; and / or a decrease in α-aminohexanoic acid-halfalaldehyde (α-AASA) in urine and / or plasma. Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level. A control level can be any type of control level used in the art, such as a baseline level before administration, or a level determined from similar subjects, cells, or untreated or treated samples with a control (such as, for example, a buffer-only control or a non-active agent control).

[0145] In one implementation, at least partial inhibition of AASS gene expression is assessed by the reduction in the amount of AASS mRNA isolated or detected from a first cell or cell group in which the AASS gene has been transcribed and which has been treated to inhibit AASS gene expression, compared to a second cell or cell group (control cells) that is substantially the same as the first cell or cell group but has not been treated in this way.

[0146] The degree of inhibition can be expressed by the following formula: [(mRNA in control cells) - (mRNA in treated cells)] / (mRNA in control cells)] * 100% As used herein, the expression "contacting cells with an RNAi agent (such as dsRNA)" includes contacting cells by any possible means. Contacting cells with an RNAi agent includes contacting cells with iRNA in vitro or in vivo. Contact can be direct or indirect. Thus, for example, the RNAi agent can be physically contacted with cells by a person performing the method, or alternatively, the RNAi agent can be placed in an environment that will allow or result in its subsequent contact with cells.

[0147] In vitro cell contact can be performed, for example, by incubating the cells with an RNAi agent. In vivo cell contact can be performed, for example, by injecting the RNAi agent into or near the tissue where the cells are located, or by injecting the RNAi agent into another area, such as the bloodstream or subcutaneous space, such that the agent subsequently reaches the tissue where the cells to be contacted are located. For example, the RNAi agent may contain a ligand and / or be coupled to a ligand (e.g., GalNAc3) that directs the RNAi agent to a target site (e.g., the liver). Combinations of in vitro and in vivo contact methods are also possible. For example, cells can be contacted with an RNAi agent in vitro and subsequently transplanted into a subject.

[0148] In one embodiment, contacting cells with iRNA includes “introducing” or “delivering” iRNA into cells by promoting or achieving uptake or absorption into the cells. The uptake or absorption of iRNA can be performed by non-assisted diffusion or active cellular processes, or by an adjuvant or device. Introducing iRNA into cells can be performed in vitro and / or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be achieved using 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, the entire contents of which are hereby incorporated herein by reference. In vitro introduction into cells includes methods known in the art such as electroporation and lipid transfection. Other methods will be described below and / or are known in the art.

[0149] The term "lipid nanoparticle" or "LNP" refers to a vesicle containing a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., iRNA or a plasmid from which iRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

[0150] As used in this article, “subject” refers to animals such as mammals, including primates (e.g., humans, non-human primates such as monkeys and chimpanzees), non-primates (e.g., cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, and whales) or birds (e.g., ducks or geese).

[0151] In one implementation, the subject is a person, such as a person receiving treatment or evaluation for a disease, condition, or symptom that would benefit from reduced AASS expression; a person at risk of having a disease, condition, or symptom that would benefit from reduced AASS expression; a person with a disease, condition, or symptom that would benefit from reduced AASS expression; and / or a person receiving treatment as described herein for a disease, condition, or symptom that would benefit from reduced AASS expression.

[0152] As used herein, the terms “treating” or “treatment” refer to a beneficial or desired outcome, including but not limited to the relief or improvement of one or more symptoms associated with AASS gene expression and / or AASS protein production, such as AASS-related diseases, such as lysine catabolism disorders. In one embodiment, the lysine catabolism disorder is type 1 glutaric aciduria (GA1) or pyridoxine-dependent epilepsy (PDE). “Treatment” can also refer to, for example, prolonged survival compared to expected survival without treatment.

[0153] In the context of AASS-related disorders, the term "reduction" refers to a statistically significant reduction in such a level. For example, this reduction could be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more. In some implementations, the reduction is at least 20%. In the context of AASS levels in subjects, "reduction" preferably refers to a reduction to a level generally recognized as being within the normal range for individuals without such a condition.

[0154] As used herein, when used in conjunction with a disease, condition, or illness, “prevention” or “preventing” means reducing the likelihood of a subject developing symptoms associated with such disease, condition, or illness, such as symptoms of AASS gene expression, such as encephalopathy crisis, bilateral striatal brain injury, complex motor disorders, accumulation of glutaryl-CoA byproducts (e.g., glutaric acid (GA) and 3-hydroxyglutaric acid (3-OH-GA)), seizures, neurological abnormalities, elevated concentrations of α-aminohexanoic acid semialdehyde (α-AASA) or piperidine acid in urine and / or plasma, and intellectual disability. The absence of a disease, symptom, or condition, or a reduction in the occurrence of symptoms associated with such a disease, symptom, or condition (e.g., a reduction of at least about 10% according to a clinically recognized scale for that disease or condition), or the manifestation of delayed symptoms (e.g., days, weeks, months, or years) such as encephalopathy crisis, bilateral striatal brain injury, complex motor disorder, accumulation of glutaryl-CoA byproducts (e.g., glutaric acid (GA) and 3-hydroxyglutaric acid (3-OH-GA)), seizures, neurological abnormalities, reduction in the concentration of α-aminohexanoic acid semialdehyde (α-AASA) in urine and / or plasma, and intellectual disability are considered effective prevention.

[0155] As used herein, the term "AASS-related disease" refers to a disease or condition caused by or associated with AASS gene expression or AASS protein production. The term "AASS-related disease" includes diseases, conditions, or illnesses that would benefit from reduced AASS gene expression or protein activity.

[0156] In one implementation, “AASS-related disease” is a lysine catabolism disorder. “Lysine catabolism disorder” is any disease, condition, or ailment associated with the lysine catabolism pathway. Non-limiting examples of lysine catabolism disorders include, for example, type 1 glutaric aciduria (GA1) and pyridoxine-dependent epilepsy (PDE).

[0157] As used herein, “therapeutic effective amount” is intended to include the amount of RNAi agent that is sufficient to effectively treat a disease (e.g., by reducing, improving, or maintaining the existing disease or one or more symptoms of the disease) when administered to a subject with an AASS-related disease, condition, or symptom. “Therapeutic effective amount” may vary depending on the RNAi agent, how it is administered, the disease and its severity and history, age, weight, family history, genetic makeup, type of previous or concurrent treatment (if any), and other individual characteristics of the subject to be treated.

[0158] As used herein, “preventive effective dose” refers to the amount of iRNA sufficient to prevent or improve the disease or one or more symptoms of the disease when administered to a subject with an AASS-related disease, condition, or symptom. Improving the disease includes slowing its progression or reducing the severity of subsequent disease development. “Preventive effective dose” may vary depending on the iRNA, how it is administered, the risk level and history of the disease, age, weight, family history, genetic makeup, type of previous or concurrent treatment (if any), and other individual characteristics of the patient to be treated.

[0159] "Therapeutic effective amount" or "preventive effective amount" also includes the amount of RNAi agent that produces a desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The iRNA used in the method of the present invention can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0160] The term "pharmaceutically acceptable" is used in this document to refer to compounds, materials, compositions, and / or dosage forms that are suitable for tissue contact with human and animal subjects within the limits of reasonable medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications, and in proportion to a reasonable benefit / risk ratio.

[0161] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or medium for carrying or delivering the subject compound from one organ or body part to another, such as liquid or solid fillers, diluents, excipients, manufacturing aids (e.g., lubricants, talc, magnesium stearate, calcium stearate, or zinc stearate, or stearic acid), or solvent encapsulation materials. Each carrier must be "acceptable," meaning it is compatible with the other components of the formulation without causing harm to the subject receiving treatment. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) tragacanth gum powder; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) 1) Polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) Esters, such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, such as magnesium hydroxide and aluminum hydroxide; (15) Alginate; (16) Atherless water; (17) Isotonic saline; (18) Ringer's solution; (19) Ethanol; (20) pH buffer solution; (21) Polyesters, polycarbonates and / or polyanhydrides; (22) Fillers, such as peptides and amino acids; (23) Serum components, such as serum albumin, HDL and LDL; and (24) Other non-toxic and compatible substances used in pharmaceutical preparations.

[0162] As used herein, the term "sample" includes a collection of similar bodily fluids, cells, or tissues isolated from a subject, as well as bodily fluids, cells, or tissues present within a subject. Examples of biological bodily fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, eye fluid, lymph, urine, saliva, etc. Tissue samples may include samples from tissues, organs, or localized areas. For example, a sample may be derived from a specific organ, a portion of an organ, or bodily fluids or cells within those organs. In some embodiments, a sample may be derived from the liver (e.g., the entire liver or certain segments of the liver or certain types of cells in the liver, such as, for example, hepatocytes). In some embodiments, "sample derived from a subject" means blood or plasma drawn from a subject.

[0163] II. The iRNA of the present invention This article describes an iRNA that inhibits the expression of a target gene. In one embodiment, the iRNA inhibits the expression of the AASS gene. In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the AASS gene in cells, such as hepatocytes, such as hepatocytes in a subject (e.g., a mammal, such as a person with a lysine catabolism disorder, condition, or disease).

[0164] The dsRNA includes an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during the expression of the AASS gene. The complementary region is about 30 nucleotides long or shorter (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides long or shorter). When contacted with a cell expressing the target gene, the iRNA inhibits the expression of the target gene (e.g., a human, primate, non-primate, or rodent target gene) by at least about 10%, as determined by methods such as PCR or branched DNA (bDNA) based methods, or by protein-based methods, such as by immunofluorescence analysis using techniques such as Western blotting or flow cytometry.

[0165] dsRNA comprises two complementary RNA strands that hybridize under the conditions of use to form a double-stranded structure. One strand of the dsRNA (antisense strand) contains a complementary region that is substantially complementary (usually perfectly complementary) to the target sequence. The target sequence can be derived from the mRNA sequence formed during the expression of the AASS gene. The other strand (sense strand) contains a region complementary to the antisense strand, such that the two strands hybridize to form a double-stranded structure when combined under appropriate conditions. As described elsewhere herein and as is known in the art, the complementary sequence of the dsRNA can also exist as a self-complementary region of a single nucleic acid molecule, rather than on a separate oligonucleotide.

[0166] Typically, the length of a double-stranded structure is between 15 and 30 base pairs, for example, lengths of 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30. Between 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Ranges and lengths falling between the ranges and lengths mentioned above are also included as part of this invention.

[0167] Similarly, the length of the region complementary to the target sequence is between 15 and 30 nucleotides, for example, lengths of 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30. Between 0, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides. Ranges and lengths falling between the ranges and lengths mentioned above are also included as part of this invention.

[0168] In some implementations, the lengths of the sense and antisense strands of the dsRNA are each independently about 15 to about 30 nucleotides, or about 25 to about 30 nucleotides, for example, the lengths of each strand are independently between 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18- The length of the dsRNA is between 22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides. In some embodiments, the length of the dsRNA is between about 15 and about 23 nucleotides, or between about 25 and about 30 nucleotides. Typically, dsRNAs are long enough to serve as substrates for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than approximately 21-23 nucleotides can serve as substrates for Dicer. Those skilled in the art will also recognize that the RNA region targeted for cleavage is usually a portion of a larger RNA molecule (typically an mRNA molecule). In relevant cases, a “portion” of the mRNA target is a contiguous sequence of length sufficient to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0169] Those skilled in the art will also recognize that the double-stranded regions are the major functional parts of dsRNA, for example, double-stranded regions of approximately 9 to 36 base pairs, such as approximately 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13 -34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 1 5-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19 -26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Therefore, in one embodiment, if an RNA molecule or RNA complex having a double-stranded region of more than 30 base pairs is processed into, for example, a functional double-stranded region of 15-30 base pairs that targets the desired RNA for cleavage, then the RNA molecule or RNA complex is dsRNA. Therefore, those skilled in the art will recognize that, in one embodiment, the microRNA (miRNA) is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In yet another embodiment, the iRNA agent that can be used to target AASS expression is not generated in target cells by cleaving larger dsRNAs.

[0170] As described herein, dsRNA may also contain one or more single-stranded nucleotide overhangs, for example, 1, 2, 3, or 4 nucleotides. dsRNAs with at least one nucleotide overhang can exhibit unexpectedly superior repressive properties compared to their blunt-ended counterparts. The nucleotide overhangs may contain, or be composed of, nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). The overhangs may be on the sense strand, antisense strand, or any combination thereof. Furthermore, the overhanging nucleotides may be present at the 5' end, 3' end, or both ends of either the antisense or sense strand of the dsRNA.

[0171] dsRNA can be synthesized using standard methods known in the art, as discussed further below, for example, by using automated DNA synthesizers, such as those commercially available from, for example, Biosearch, Applied Biosystems, Inc.

[0172] The iRNA compounds of the present invention can be prepared using a two-step procedure. First, each strand of a double-stranded RNA molecule is prepared separately. Then, the component strands are annealed. The strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis, or both. Organic synthesis has the advantage of readily preparing oligonucleotide chains containing non-natural or modified nucleotides. The single-stranded oligonucleotides of the present invention can be prepared using solution-phase or solid-phase organic synthesis, or both.

[0173] In one aspect, the dsRNA of the present invention comprises at least two nucleotide sequences, namely a sense strand sequence and an antisense strand sequence. The sense strand sequence is selected from the sequence sets provided in Tables 3-6, and the corresponding nucleotide sequence of the antisense strand of the sense strand is selected from the sequence sets in Tables 3-6. In this aspect, one of the two sequences is complementary to the other of the two sequences, and one sequence is substantially complementary to the sequence of the mRNA generated in the expression of the AASS gene. Therefore, in this aspect, the dsRNA will comprise two oligonucleotides, one of which is described in Tables 3-6 as the sense strand (passenger strand), and the second oligonucleotide is described in Tables 3-6 as the corresponding antisense strand (guide strand) of the sense strand. In one embodiment, the substantially complementary sequence of the dsRNA is contained on a single oligonucleotide. In another embodiment, the substantially complementary sequence of the dsRNA is contained on a single oligonucleotide.

[0174] It should be understood that although the sequences in Tables 3-6 are described as modified, unmodified, unconjugated, and / or conjugated sequences, the RNA of the iRNA of the present invention (e.g., the dsRNA of the present invention) may contain any of the sequences listed in Tables 3-6 that are unmodified, unconjugated, and / or modified and / or conjugated in a manner different from that described herein.

[0175] Those skilled in the art are well aware that dsRNAs with a double-stranded structure of approximately 20 to 23 base pairs (e.g., 21 base pairs) have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20: 6877-6888). However, other studies have found that shorter or longer RNA double-stranded structures may also be effective (Chu and Rana (2007) RNA14: 1714-1719; Kim et al. (2005) Nat Biotech23: 222-226). In the embodiments described above, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein can comprise at least one chain of at least 21 nucleotides in length. It is reasonable to expect that shorter double-stranded structures, with only a few nucleotides reduced at one or both ends, could be similarly effective compared to the dsRNAs described above. Therefore, any dsRNA having a sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides derived from one of the sequences provided herein, and whose ability to inhibit AASS gene expression differs from that of a dsRNA containing the complete sequence by no more than about 5%, 10%, 15%, 20%, 25% or 30%, is considered to be within the scope of this invention.

[0176] Additionally, the RNAs described in Tables 3-6 identify one or more sites in the AASS transcript that are susceptible to RISC-mediated cleavage. Therefore, the present invention also includes iRNAs that target these one or more sites. As used herein, if an iRNA promotes cleavage of an RNA transcript at any location within a specific site, then the iRNA is said to target that specific site of the transcript. Such iRNAs typically comprise at least about 15 consecutive nucleotides from one of the sequences provided herein and are coupled to an additional nucleotide sequence taken from a region of the gene adjacent to the selected sequence.

[0177] While target sequences are typically 15–30 nucleotides in length, the applicability of a particular sequence within this range for guiding the cleavage of any given target RNA varies considerably. The various software packages and guidelines listed herein provide guidelines for identifying the optimal target sequence for any given gene target, but an empirical approach can also be taken, where a “window” or “mask” of a given size (21 nucleotides as a non-limiting example) is placed literally or symbolically (including, for example, computer simulations) on the target RNA sequence to identify sequences within that size range that could serve as target sequences. By progressively moving this sequence “window” one nucleotide upstream or downstream of the initial target sequence location, the next potential target sequence can be identified until the full suite of possible sequences for any chosen given target size is identified. This process, coupled with the systematic synthesis and testing (using assays as described herein or known in the art) of the identified sequences to identify those that perform best, can identify those RNA sequences that will mediate optimal repression of target gene expression when targeted with an iRNA agent. Therefore, although the sequences identified in this paper represent effective target sequences, it is conceivable that further optimization of inhibition efficiency could be achieved by progressively “walking the window” one nucleotide upstream or downstream of a given sequence to identify sequences with equivalent or better inhibitory characteristics.

[0178] Furthermore, for any sequence identified herein, it is conceivable that further optimization could be achieved by systematically adding or removing nucleotides to produce longer or shorter sequences and testing those sequences by making a window of longer or shorter size walk above or below the target RNA from that point. Similarly, combining this method of generating new candidate targets with testing the effectiveness of iRNAs based on these target sequences using inhibition assays known in the art and / or as described herein can further improve inhibition efficiency. Additionally, such optimized sequences can be modulated to further optimize the molecule (e.g., increase serum stability or circulating half-life, increase thermal stability, enhance transmembrane delivery, target specific locations or cell types, increase interaction with silencing pathway enzymes, increase release from endosomes) as an expression inhibitor, for example, by introducing modified nucleotides as described herein or known in the art, adding or altering protrusions, or other modifications known in the art and / or discussed herein.

[0179] iRNA agents as described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNA as described herein contains no more than three mismatches. If the antisense strand of the iRNA contains a mismatch with the target sequence, the mismatched region is preferably not located within the center of the complementary region. If the antisense strand of the iRNA contains a mismatch with the target sequence, the mismatch is preferably confined to the last 5 nucleotides from the 5' or 3' end of the complementary region. For example, for a 23-nucleotide iRNA agent, the strand complementary to a region of the AASS gene typically contains no mismatches within the central 13 nucleotides. The effectiveness of an iRNA containing a mismatch with the target sequence in inhibiting AASS gene expression can be determined using the methods described herein or methods known in the art. It is important to consider the efficacy of mismatched iRNAs in inhibiting AASS gene expression, especially when specific complementary regions in the AASS gene are known to have polymorphic sequence variations within a population.

[0180] RNA targets may possess regions or nucleotide sequence spans of target RNA that are relatively easier or more suitable for mediating RNA interference induced by the binding of RNAi agents to these regions compared to other regions of the RNA target. This increased sensitivity to RNA interference within such "hotspot regions" (or simply "hotspots") implies that iRNAi agents targeting these regions may be more effective at inducing iRNA interference than those targeting other regions of the target RNA. For example, without being bound by theory, the accessibility of the target region of the target RNA may affect the efficacy of iRNAi agents targeting that region, and some hotspot regions exhibit increased accessibility. For instance, secondary structures formed within the RNA target (e.g., within or near hotspot regions) may influence the ability of iRNAi agents to bind to the target region and induce RNA interference.

[0181] According to certain aspects of the invention, an iRNA agent can be designed to target hotspot regions of any target RNA described herein, including any identified portion of the target RNA (e.g., a specific exon). As used herein, a hotspot region may refer to a region of approximately 19-200, 19-150, 19-100, 19-75, 19-50, 21-200, 21-150, 21-100, 21-75, 21-50, 50-200, 50-150, 50-100, 50-75, 75-200, 75-150, 75-100, 100-200, or 100-150 nucleotides in the target RNA sequence, where targeting with an RNAi agent provides an observably higher probability of effective silencing compared to targeting other regions of the same target RNA. According to certain aspects of the invention, hotspot regions may constitute a limited region of the target RNA, and in some cases, constitute a substantially restricted region of the target, for example, less than half the length of the target RNA, such as about 5%, 10%, 15%, 20%, 25%, or 30% of the target RNA length. Conversely, other regions compared to the hotspots may cumulatively constitute at least a majority of the target RNA length. For example, other regions may cumulatively constitute at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% of the target RNA length.

[0182] Hotspots can be identified by empirically evaluating comparative regions of target RNAs using efficacy data obtained from in vitro or in vivo screening assays. For example, RNAi agents targeting different regions of the target RNA can be compared to assess the frequency with which effective iRNAs bind to each region (e.g., the amount of target gene expression suppressed, as measured by mRNA or protein expression). Typically, hotspots can be identified by observing aggregations of multiple effective RNAi agents that bind to a limited region of the RNA target. Hotspots are adequately characterized by observing the efficacy of iRNAs that collectively cover at least about 60%, such as about 70%, about 80%, about 90%, or about 95% or more of the length of the target region identified as a hotspot, including both ends of the region (i.e., the iRNA targets at least about 60%, 70%, 80%, 90%, or 95% or more of the nucleotides within the region, including the nucleotides at each end of the region). According to some aspects of the invention, iRNA agents exhibiting at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% inhibition in this region (e.g., residual mRNA not exceeding about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) can be identified as effective.

[0183] The suitability of targeting RNA regions can also be assessed using quantitative comparisons of inhibition measurements on different regions of defined sizes (e.g., 25, 30, 40, 50, 60, 70, 80, 90 or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 nt). For example, the mean inhibition level of each region can be determined and the mean of each region can be compared. The mean inhibition level in hotspot regions may be significantly higher than the mean of all evaluated regions. According to some aspects, the mean inhibition level in hotspot regions may be at least about 10%, 20%, 30%, 40%, or 50% higher than the mean of said mean. According to some aspects, the mean inhibition level in hotspot regions may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 standard deviations higher than the mean of said mean. The average inhibition level may be significantly higher than statistically significant (e.g., p < 0.05). According to some aspects, each inhibition measurement within a hotspot region may be higher than a threshold amount (e.g., equal to or lower than a threshold amount of residual mRNA). According to some aspects, each inhibition measurement within this region may be significantly higher than the mean of all inhibition measurements across all measurement regions. For example, each inhibition measurement within a hotspot region may be at least about 10%, 20%, 30%, 40%, or 50% higher than the mean of all inhibition measurements. According to some aspects, each inhibition measurement may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 standard deviations higher than the mean of all inhibition measurements. Each inhibition measurement may be significantly higher than the mean of all inhibition measurements (e.g., p < 0.05). The criteria for evaluating hotspots may include various combinations of the above criteria under compatible conditions (e.g., the average inhibition level is at least about a first amount, and there is no inhibition measurement below a threshold level of a second amount, which is less than the first amount).

[0184] Therefore, it is explicitly envisioned that any iRNA agent targeting a hotspot region of the target RNA, including the specific exemplary iRNA agents described herein, may be preferred to induce RNA interference in the target mRNA, because targeting such hotspot regions may exhibit a more robust inhibitory response than targeting regions that are not hotspot regions. RNAi agents targeting target sequences that substantially overlap with a hotspot region (e.g., overlap of at least about 70%, 75%, 80%, 85%, 90%, or 95% of the target sequence length) or preferably are located entirely within the hotspot region can be considered to be targeting such a hotspot region. The hotspot regions of the RNA targets of this invention may include any regions (including regions confirmed by any criteria described elsewhere herein) that the data disclosed herein indicate a high frequency of targeting by effective RNAi agents, regardless of whether the extent of such hotspot regions is explicitly specified.

[0185] In various embodiments, the dsRNA agent of the present invention targets hotspot regions of mRNA encoding AASS.

[0186] III. The modified iRNA of the present invention In one embodiment, the RNA of the iRNA (e.g., dsRNA) of the present invention is unmodified and does not contain, for example, chemical modifications and / or conjugations known in the art and described herein. In another embodiment, the RNA of the iRNA (e.g., dsRNA) of the present invention is chemically modified to enhance stability or other beneficial characteristics. In some embodiments of the present invention, substantially all nucleotides of the iRNA of the present invention are modified. In other embodiments of the present invention, all nucleotides of the iRNA of the present invention are modified. The iRNA of the present invention wherein "substantially all nucleotides are modified" is largely but not entirely modified and may contain no more than 5, 4, 3, 2, or 1 unmodified nucleotide.

[0187] In some aspects of the invention, substantially all nucleotides of the iRNA of the invention are modified, and the iRNA agent comprises no more than 10 nucleotides containing a 2'-fluorine modification (e.g., no more than 9, 8, 7, 6, 5, 4, 5, 4, 3, or 2 2'-fluorine modifications). For example, in some embodiments, the positive strand comprises no more than 4 nucleotides containing a 2'-fluorine modification (e.g., no more than 3 or 2 2'-fluorine modifications). In other embodiments, the antisense strand comprises no more than six nucleotides containing 2'-fluorine modifications (e.g., no more than five 2'-fluorine modifications, no more than four 2'-fluorine modifications, no more than four 2'-fluorine modifications, or no more than two 2'-fluorine modifications).

[0188] In other aspects of the invention, all nucleotides of the iRNA of the invention are modified, and the iRNA agent comprises no more than 10 nucleotides containing 2'-fluoro modifications (e.g., no more than 9 2'-fluoro modifications, no more than 8 2'-fluoro modifications, no more than 7 2'-fluoro modifications, no more than 6 2'-fluoro modifications, no more than 5 2'-fluoro modifications, no more than 4 2'-fluoro modifications, no more than 5 2'-fluoro modifications, no more than 4 2'-fluoro modifications, no more than 3 2'-fluoro modifications, or no more than 2 2'-fluoro modifications).

[0189] In one embodiment, the double-stranded RNAi agent of the present invention further comprises a 5'-phosphate or a 5'-phosphate mimic at the 5' nucleotide of the antisense strand. In another embodiment, the double-stranded RNAi agent further comprises a 5'-phosphate mimic at the 5' nucleotide of the antisense strand. In a specific embodiment, the 5'-phosphate mimic is 5'-vinylphosphonic acid (5'-VP). In one embodiment, the phosphate mimic is 5'-cyclopropylphosphonic acid. In some embodiments, the 5' end of the antisense strand of the double-stranded iRNA agent does not contain 5'-vinylphosphonic acid (VP).

[0190] In one embodiment, at least one of the modified nucleotides is selected from deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, diol modified nucleotides (GNA) (e.g., Ggn, Cgn, Tgn, or Agn), nucleotides having a 2' phosphate (e.g., G2p, C2p, A2p, or U2p), and vinylphosphonic acid nucleotides; and combinations thereof. In other embodiments, each of the duplexes in Table 4 or 6 may be specifically modified to provide another duplex iRNA agent of this disclosure. In one example, the 3' end of each positive-sense duplex may be modified by removing the 3'-terminal L96 ligand and exchanging the two phosphodiester nucleotide links between the three 3'-terminal nucleotides with the thiophosphate nucleotide links. That is, formula: 5'-N1-…-N n-2 N n-1 N n L96 3' The three 3'-terminal nucleotides (N) of the positive sequence can be replaced with 5'-N1-…-N n-2 s N n-1 s N n 3', The antisense sequence remains unchanged to provide another double-stranded iRNA agent of this disclosure.

[0191] The nucleic acids involved in this invention can be synthesized and / or modified using methods recognized in the art, such as those described in “Currentprotocols in nucleic acid chemistry,” Beaucage, S. Let al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example: terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inversion linking) or 3'-end modifications (conjugation, DNA nucleotides, inversion linking, etc.); base modifications, such as substitutions for stabilizing bases, destabilizing bases, or bases paired with extended partner libraries, base removal (debase nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and / or backbone modifications, including modifications or substitutions of phosphodiester bonds. Specific examples of iRNA compounds that can be used in the embodiments described herein include, but are not limited to, RNA containing a modified backbone or without natural nucleoside inter-linking. RNAs with a modified backbone include, among others, those that do not have a phosphorus atom in their backbone. For the purposes of this specification and as sometimes discussed in the art, modified RNAs that do not have a phosphorus atom in their internucleotide backbone may also be considered oligonucleotides. In some embodiments, the modified iRNA will have a phosphorus atom in its internucleotide backbone.

[0192] Modified RNA backbones include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates (including 3′-alkylene phosphonates and chiral phosphonates), hypophosphonates, aminophosphates (including 3′-aminoaminophosphates and aminoalkylaminophosphates), thioaminophosphates, thioalkylphosphonates, thioalkyl phosphate triesters, and boroalkyl phosphates having normal 3′-5′ linkages, their 2′-5′ linker analogs, and those having opposite polarities, wherein adjacent nucleoside unit pairs are 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′ linked. Various salts, mixed salts, and free acid forms are also included. In some embodiments of the invention, the dsRNA agent of the invention is in free acid form. In other embodiments of the invention, the dsRNA agent of the invention is in salt form. In one embodiment, the dsRNA agent of the invention is in sodium salt form. In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ions are present in the agent as counterions to substantially all phosphodiester and / or thiophosphate groups present in the agent. The agent wherein substantially all phosphodiester and / or thiophosphate bonds have sodium counterions comprises no more than 5, 4, 3, 2, or 1 phosphodiester and / or thiophosphate bonds without sodium counterions. In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ions are present in the dsRNA agent as counterions to all phosphodiester and / or thiophosphate groups present in the dsRNA agent.

[0193] Representative U.S. patents teaching the preparation of the above phosphorus-containing bonds include, but are not limited to: U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; and 5,286,717. 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5 587,361;5,625,050;6,028,188;6,124,445;6,160,109;6,169,170;6,172,209;6,239,265;6,277,603;6,326,199;6,346,614;6,444,423;6,531,590;6, U.S. Patent RE39464, the entire contents of each of these patents are hereby incorporated herein by reference.

[0194] Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short-chain alkyl or cycloalkyl nucleosides, mixed heteroatoms and alkyl or cycloalkyl nucleosides, or one or more short-chain heteroatoms or heterocyclic nucleosides. These include those with the following structures: morpholino linkages (formed from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formylacetyl and thioformylacetyl backbones; methyleneformylacetyl and thioformylacetyl backbones; alkene-containing backbones; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S, and CH2 component moieties.

[0195] Representative U.S. patents teaching the preparation of the above oligonucleotides include, but are not limited to: U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; and 5,470,967. 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

[0196] In other embodiments, suitable RNA mimics for iRNA are conceived in which the sugar-nucleoside linkage (i.e., backbone) of the nucleotide unit is replaced with a new group. The base unit is retained for hybridization with a suitable nucleic acid target compound. One such oligomer (an RNA mimic that has shown excellent hybridization properties) is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, specifically an aminoethylglycine backbone. The nucleobase is retained and directly or indirectly bound to the aza-nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Further PNA compounds suitable for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0197] Some embodiments of the present invention include RNA having a thiophosphate backbone and oligonucleotides having a heteroatom backbone, particularly the --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [referred to as methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- [wherein the native phosphodiester backbone is represented as --O--P--O--CH2--] of the aforementioned U.S. Patent No. 5,602,240, and the amide backbone of the aforementioned U.S. Patent No. 5,602,240. In some embodiments, the RNA described herein has the morpholino backbone structure of the aforementioned U.S. Patent No. 5,034,506.

[0198] Modified RNA may also contain one or more substituted sugar moieties. The iRNA (e.g., dsRNA) described herein may include 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. 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 includes one of the following at the 2' position: C1 to C 10Lower 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 gene groups, intercalating agents, groups for improving the pharmacokinetic properties of iRNA or groups for improving the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2′-O--CH2CH2OCH3, also known as 2′-O-(2-methoxyethoxy) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78: 486-504), i.e., alkoxy-alkoxy groups. Another exemplary modification is 2'-dimethylaminoethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described below in the 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. Further exemplary modifications include: 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (all three series contain R and S isomers); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0199] Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2), and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the iRNA, particularly at the 3′ terminal nucleotide or at the 3′ position of the sugar and the 5′ position of the 5′ terminal nucleotide in 2′-5′ linked dsRNAs. iRNAs can also have sugar mimics, such as replacing the cyclobutyl moiety of the pentofuranosyl sugar. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to: U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, some of which belong to the same applicant as this application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

[0200] The iRNA of the present invention may also include nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as: 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl derivatives and other alkyl derivatives of adenine and guanine; 2-propyl derivatives and other alkyl derivatives of adenine and guanine; 2-thiouracil; 2-thiothymine and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, 6-azo Cytosine and 6-azothymidine; 5-uracil (pseudouracil); 4-thiouracil; 8-halogenated, 8-amino, 8-mercapto, 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-deazoguanine and 7-deazoadenine, and 3-deazoguanine and 3-deazoadenine. Further nucleobases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in *Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P.ed. Wiley-VCH, 2008; those disclosed in *The Concise Encyclopedia Of Polymer Science and Engineering,* pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990; those disclosed in *Angewandte Chemie,* (1991), International Edition, 30:613; and those disclosed in *Sanghvi, Y. S., Chapter 15, dsRNA Research and Applications,* pages 289-302, Crooke, ST. and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomers involved in this invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.It has been shown that 5-methylcytosine substitution increases the stability of nucleic acid duplexes by 0.6–1.2 °C (Sanghvi, YS, Crooke, STand Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276–278) and is an exemplary base substitution, even more so when combined with 2'-O-methoxyethyl sugar modification.

[0201] Representative U.S. patents teaching the preparation of certain modified nucleobases and other modified nucleobases described above include, but are not limited to, the aforementioned U.S. patent numbers 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; and 5,587,469. 5,594,121; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

[0202] The iRNA of the present invention can also be modified to include one or more locking nucleic acids (LNAs). Locking nucleic acids are nucleotides having a modified ribose moiety, wherein the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endonucleotide conformation. Adding locking nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1): 439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3): 833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12): 3185-3193).

[0203] The iRNA of the present invention can also be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanyl ring modified by a two-atom bridging. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety containing a bridge connecting the two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In some embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Therefore, in some embodiments, the agent of the present invention may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety containing an additional bridge connecting the 2'-carbon and 4'-carbon. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively “locks” the ribose in a 3'-endonucleotide conformation. Adding locking nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1): 439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3): 833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12): 3185-3193). Examples of bicyclic nucleosides used in the polynucleotides of the present invention include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In some embodiments, the antisense polynucleotides of the present invention comprise one or more bicyclic nucleosides containing a 4' to 2' bridge.Examples of such 4′ to 2′ bridged bicyclic nucleotides include, but are not limited to: 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′; 4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also known as “restricted ethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and their analogues; see, for example, U.S. Patent No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and their analogues; see, for example, U.S. Patent No. 7,399,845); 8,278,283); 4′-CH2-N(OCH3)-2′ (and its analogues; see, for example, U.S. Patent No. 8,278,425); 4′-CH2-ON(CH3)-2′ (see, for example, U.S. Patent Publication No. 2004 / 0171570); 4′-CH2-N(R)-O-2′, wherein R is H, C1-C12 alkyl or protecting group (see, for example, U.S. Patent No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, for example, Chattopadhyaya et al., J.Org.Chem., 2009, 74, 118-134); and 4′-CH2-C(-CH2)-2′ (and its analogues; see, for example, U.S. Patent No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.

[0204] Other representative U.S. patents and U.S. patent disclosures teaching the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008 / 0039618; and US 2009 / 0012281, the entire contents of each of which are hereby incorporated herein by reference.

[0205] Any of the aforementioned bicyclic nucleotides can be prepared to have one or more stereochemical sugar configurations, including, for example, α-L-ribofuranosyl and β-D-ribofuranosyl (see WO 99 / 14226).

[0206] The iRNA of the present invention can also be modified to include one or more restriction ethyl nucleotides. As used herein, "restriction ethyl nucleotide" or "cEt" refers to a locked nucleic acid containing a bicyclic sugar moiety comprising a 4'-CH(CH3)-O-2' bridge. In one embodiment, the restriction ethyl nucleotide is in the S conformation and is referred to herein as "S-cEt".

[0207] The iRNA of the present invention may also comprise one or more "conformation-restricted nucleotides" ("CRNs"). A CRN is a nucleotide analog having a linker connecting the C2' and C4' carbons of the ribose or the C3 and -C5' carbons of the ribose. The CRN locks the ribose ring into a stable conformation and increases hybridization affinity to the mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity, resulting in less ribose ring puckering.

[0208] Representative disclosures teaching the preparation of certain CRNs described above include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383; and PCT Publication WO 2013 / 036868, the entire contents of each of which are hereby incorporated herein by reference.

[0209] In some embodiments, the iRNA of the present invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid in which any bond of the sugar has been removed, thereby forming an unlocked “sugar” residue. In one instance, UNA also encompasses monomers in which the bond between C1′ and C4′ (i.e., the covalent carbon-oxygen-carbon bond between C1′ and C4′ carbons) has been removed. In another instance, the C2′-C3′ bond of the sugar (i.e., the covalent carbon-carbon bond between C2′ and C3′ carbons) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol Biosyst., 2009, 10, 1039, which are hereby incorporated by reference).

[0210] Representative U.S. publications teaching the preparation of UNA include, but are not limited to: U.S. Patent No. 8,314,227; and U.S. Patent Publications Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of each of which are hereby incorporated herein by reference.

[0211] The RNAi agent disclosed herein may also comprise one or more “cyclohexene nucleic acids” or (“CeNA”). CeNA is a nucleotide analog in which the furanose portion of DNA is replaced by a cyclohexene ring. Binding a cyclohexene nucleotide to the DNA strand increases the stability of the DNA / RNA hybrid. CeNA is stable against degradation in serum, and the CeNA / RNA hybrid is able to activate E. coli RNase H, leading to cleavage of the RNA strand. (See Wang et al., Am. Chem. Soc. 2000, 122, 36, 8595-8602, which is incorporated herein by reference).

[0212] Potential stabilizing modifications to the ends of RNA molecules may include N-(acetylaminohexanoyl)-4-hydroxyproline (Hyp-C6-NHAc), N-(hexanoyl-4-hydroxyproline (Hyp-C6), N-(acetyl-4-hydroxyproline (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminohexanoyl)-4-hydroxyproline (Hyp-C6-amino), 2-docosyl-uridine-3”-phosphate, reverse base dT (idT), and others. Disclosure of such modifications can be found in PCT Publication No. WO2011 / 005861.

[0213] Other modifications to the iRNA of the present invention include 5' phosphate or 5' phosphate mimics, such as the 5'-terminal phosphate or phosphate mimics on the antisense strand of an RNAi agent. Suitable phosphate mimics are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0214] In some specific embodiments, the RNAi agent of the present invention is an RNAi agent that inhibits AASS gene expression, selected from the group of RNAi agents listed in Tables 3-6. Any of these RNAi agents may further contain a ligand.

[0215] A. Modified iRNA containing the motif of this invention In some aspects of the invention, the double-stranded RNAi agents of the invention include chemically modified agents, such as those disclosed, for example, in WO 2013 / 075035, filed November 16, 2012, the entire contents of which are incorporated herein by reference.

[0216] Therefore, the present invention provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene (i.e., the AASS gene) in vivo. This RNAi agent comprises a sense strand and an antisense strand. The length of each strand of the RNAi agent can be in the range of 12-30 nucleotides. For example, the length of each strand can be between 14-30 nucleotides, between 17-30 nucleotides, between 25-30 nucleotides, between 27-30 nucleotides, between 17-23 nucleotides, between 17-21 nucleotides, between 17-19 nucleotides, between 19-25 nucleotides, between 19-23 nucleotides, between 19-21 nucleotides, between 21-25 nucleotides, or between 21-23 nucleotides. In one embodiment, the length of the sense strand is 21 nucleotides. In one embodiment, the length of the antisense strand is 23 nucleotides.

[0217] The sense and antisense strands typically form a double-stranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” The length of the double-stranded region in an RNAi agent can range from 12 to 30 nucleotide pairs. For example, the length of the double-stranded region can be between 14 and 30 nucleotide pairs, between 17 and 30 nucleotide pairs, between 27 and 30 nucleotide pairs, between 17 and 23 nucleotide pairs, between 17 and 21 nucleotide pairs, between 17 and 19 nucleotide pairs, between 19 and 25 nucleotide pairs, between 19 and 23 nucleotide pairs, between 19 and 21 nucleotide pairs, between 21 and 25 nucleotide pairs, or between 21 and 23 nucleotide pairs. In another example, the length of the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides.

[0218] In one embodiment, the RNAi agent may contain one or more overhanging regions and / or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs may be 1-6 nucleotides long, for example, 2-6 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, or 1-2 nucleotides. These overhangs may result from one strand being longer than the other, or from two strands of the same length being interleaved. The overhangs may form a mismatch with the target mRNA, or may be complementary to the targeted gene sequence or another sequence. The first and second strands may also be joined, for example, by additional bases to form a hairpin, or by other non-base linkers.

[0219] In one embodiment, the nucleotides in the overhang region of the RNAi agent can each be independently modified or unmodified nucleotides, including but not limited to those modified with 2'-sugars, such as 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT can be an overhang sequence at any end of any strand. The overhang may form a mismatch with the target mRNA, or it may be complementary to the targeted gene sequence or it may be another sequence.

[0220] The 5' or 3' overhang of the sense strand, antisense strand, or both strands of the RNAi agent may be phosphorylated. In some embodiments, the one or more overhang regions contain two nucleotides with a phosphate thioester between them, wherein the two nucleotides may be the same or different. In one embodiment, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, this 3' overhang is located in the antisense strand. In one embodiment, this 3' overhang is located in the sense strand.

[0221] RNAi agents may contain only a single overhang, which can enhance the interfering activity of RNAi without affecting its overall stability. For example, the single-stranded overhang may be located at the 3' end of the sense strand, or alternatively, at the 3' end of the antisense strand. RNAi may also have a blunt end at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. Generally, the antisense strand of RNAi has a nucleotide overhang at the 3' end and a blunt 5' end. While it is undesirable to be bound by theory, the asymmetric blunt end at the 5' end and the overhang at the 3' end of the antisense strand are beneficial for guiding strand loading into the RISC process.

[0222] In one embodiment, the RNAi agent is a double-ended blunt-ended derivative of 19 nucleotides in length, wherein the sense strand contains at least one motif with three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 starting from the 5' end. The antisense strand contains at least one motif with three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 starting from the 5' end.

[0223] In another embodiment, the RNAi agent is a 20-nucleotide-long, double-ended compound, wherein the sense strand contains at least one motif with three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 starting from the 5' end. The antisense strand contains at least one motif with three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 starting from the 5' end.

[0224] In another embodiment, the RNAi agent is a 21-nucleotide-long, double-ended compound, wherein the sense strand contains at least one motif with three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 starting from the 5' end. The antisense strand contains at least one motif with three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 starting from the 5' end.

[0225] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif with three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 starting from the 5' end; and the antisense strand contains at least one motif with three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 starting from the 5' end, wherein one end of the RNAi agent is flat, and the other end contains a 2-nucleotide overhang. Preferably, the 2-nucleotide overhang is located at the 3' end of the antisense strand.

[0226] When the overhangs of the two nucleotides are at the 3' end of the antisense strand, two phosphate thioester nucleotides may be present between the three terminal nucleotides, wherein two of the three nucleotides are overhang nucleotides and the third nucleotide is a pairing nucleotide immediately adjacent to the overhang nucleotides. In one embodiment, the RNAi agent further has two phosphate thioester nucleotides between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, each nucleotide (including nucleotides that are part of a motif) in the sense and antisense strands of the RNAi agent is a modified nucleotide. In one embodiment, each residue is independently modified, for example, by a 2'-O-methyl or 3'-fluoro modification in an alternating motif. Optionally, the RNAi agent also comprises a ligand (preferably GalNAc3).

[0227] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25-30 nucleotide residues long, wherein positions 1 to 23 of the first strand, starting from the 5' terminal nucleotide (position 1), contain at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues long, and, starting from the 3' terminal nucleotide, contains at least 8 ribonucleotides at positions 1-23 of the sense strand to form a double strand; wherein at least the 3' terminal nucleotide of the antisense strand is unpaired with the sense strand, and at most 6 consecutive 3' terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; wherein the 5' end of the antisense strand contains 10-3 The positive strand contains 0 consecutive unpaired nucleotides on the sense strand, forming a single-stranded 5' overhang of 10-30 nucleotides; wherein, when aligned with the sense and antisense strands for maximum complementarity, at least the 5' and 3' terminal nucleotides of the sense strand pair with the nucleotide bases of the antisense strand, thereby forming a substantially double-stranded region between the sense and antisense strands; and along the length of the antisense strand, for at least 19 ribonucleotides, the antisense strand is sufficiently complementary to the target RNA to reduce target gene expression when the double-stranded nucleic acid is introduced into mammalian cells; and wherein the sense strand contains at least one motif having three 2'-F modifications on three consecutive nucleotides, wherein at least one of the motifs occurs at or near a cleavage site. The antisense strand contains at least one motif having three 2'-O-methyl modifications on three consecutive nucleotides at or near a cleavage site.

[0228] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the RNAi agent comprises a first strand and a second strand, the first strand having a length of at least 25 and at most 29 nucleotides, and the second strand having a length of at most 30 nucleotides and having at least one motif with three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 starting from the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand form a blunt end, and the second strand is 1-4 nucleotides longer than the first strand at its 3' end, wherein the length of the double-stranded region is at least 25 nucleotides, and for at least 19 nucleotides along the length of the second strand, the second strand is sufficiently complementary to the target mRNA to reduce target gene expression when the RNAi agent is introduced into mammalian cells, and wherein dicer cleavage of the RNAi agent preferably produces siRNA comprising the 3' end of the second strand, thereby reducing target gene expression in mammals. Optionally, the RNAi agent also comprises a ligand.

[0229] In one embodiment, the sense strand of the RNAi agent contains at least one motif having three identical modifications on three consecutive nucleotides, wherein one of these motifs occurs at a cleavage site in the sense strand.

[0230] In one embodiment, the antisense strand of the RNAi agent may also contain at least one motif having three identical modifications on three consecutive nucleotides, wherein one of these motifs occurs at or near a cleavage site in the antisense strand.

[0231] For RNAi agents with a double-stranded region of 17–23 nucleotides in length, the cleavage site on the antisense strand is typically around positions 10, 11, and 12, starting from the 5' end. Therefore, these motifs with three identical modifications can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 on the antisense strand, counted from the 5' end of the first nucleotide, or counted from the 5' end of the antisense strand starting from the first paired nucleotide within the double-stranded region. The cleavage site in the antisense strand can also vary depending on the length of the double-stranded region of the RNAi starting from the 5' end.

[0232] The sense strand of an RNAi agent may contain at least one motif at a cleavage site on the strand, having three identical modifications on three consecutive nucleotides; and the antisense strand may contain at least one motif at or near a cleavage site on the strand, having three identical modifications on three consecutive nucleotides. When the sense and antisense strands form a dsRNA duplex, the sense and antisense strands may be aligned such that a motif having three nucleotides on the sense strand and a motif having three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

[0233] In one embodiment, the positive strand of the RNAi agent may contain more than one motif having three identical modifications on three consecutive nucleotides. The first motif may occur at or near a cleavage site on the strand, and the other motifs may be wing modifications. The term "wing modification" herein refers to a motif occurring at a portion of the strand that is separate from the motif at or near a cleavage site on the same strand. Wing modifications are either adjacent to the first motif or separated by at least one or more nucleotides. When these motifs are closely adjacent to each other, their chemical properties differ from each other, and when these motifs are separated by one or more nucleotides, their chemical properties may be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may be present at one end relative to the first motif at or near a cleavage site or on either side of the leading motif.

[0234] Similar to the sense strand, the antisense strand of an RNAi agent may contain more than one motif with three identical modifications on three consecutive nucleotides, at least one of which occurs at or near the cleavage site of the strand. The antisense strand may also contain one or more wing modifications similar to those that can be present on the sense strand.

[0235] In one implementation, the wing modifications on the sense or antisense strand of the RNAi agent typically exclude the first or two terminal nucleotides at the 3'-end, 5'-end, or both ends of the strand.

[0236] In another embodiment, the wing modifications on the sense or antisense strand of the RNAi agent typically do not include the preceding one or two paired nucleotides in the double-stranded region at the 3'-end, 5'-end, or both ends of the strand.

[0237] When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modification may fall on the same end of the duplex region and have an overlap of one, two or three nucleotides.

[0238] When the sense and antisense strands of an RNAi agent each contain at least two wing modifications, the sense and antisense strands can be aligned such that the two modifications, each from one strand, fall at one end of the double-stranded region with an overlap of one, two, or three nucleotides; the two modifications, each from one strand, fall at the other end of the double-stranded region with an overlap of one, two, or three nucleotides; and the two modifications, each from one strand, fall on each side of the leader motif with an overlap of one, two, or three nucleotides in the double-stranded region.

[0239] In one implementation, each nucleotide (including nucleotides that are part of a motif) in the sense and antisense strands of the RNAi agent may be modified. Each nucleotide may be modified with the same or different modifications, which may include: one or more changes to one or two non-linked phosphate groups and / or one or more linked phosphate groups; changes to the components of the ribose ring, such as changes to the 2' hydroxyl group on the ribose ring; complete replacement of the phosphate ester portion with a "dephosphorylation" linker; modification or replacement of naturally occurring bases; and replacement or modification of the ribose-phosphate backbone.

[0240] Because nucleic acids are polymers of subunits, many modifications occur at repetitive sites within the nucleic acid, such as modifications to bases or phosphate moieties, or to non-linked O sites on phosphate moieties. In some cases, modifications will occur at all target sites in the nucleic acid, but in many cases, this is not the case. For example, modifications may occur only at the 3' or 5' ends, or only in terminal regions, such as on the terminal nucleotides of the strand or in the last 2, 3, 4, 5, or 10 nucleotides. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in double-stranded regions of RNA or only in single-stranded regions of RNA. For example, phosphate thioester modifications at non-linked O sites may be present only at one or both ends, or only in terminal regions, such as on the terminal nucleotides of the strand or in the last 2, 3, 4, 5, or 10 nucleotides, or may be present in both double-stranded and single-stranded regions, particularly at the ends. One or more 5' ends may be phosphorylated.

[0241] To enhance stability, it may be possible, for example, to include specific bases at the overhang or to include modified nucleotides or nucleotide substitutes in the single-stranded overhang (e.g., at the 5' or 3' overhang or both). For example, the inclusion of purine nucleotides at the overhang may be desirable. In some embodiments, all or some bases in the 3' or 5' overhang may be modified with modifications such as those described herein. Modifications may include, for example, modifications at the 2' position of the ribose ring with modifications known in the art, such as replacing the ribose of the nucleoside with a 2'-deoxy-2'-fluoro(2'-F) or 2'-O-methyl modified deoxyribonucleotide, and modifications in the phosphate ester group, such as thiophosphate modifications. The overhang need not be homologous to the target sequence.

[0242] In one embodiment, each residue of the sense and antisense strands is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxy, or 2'-fluoro. These strands may contain more than one modification. In one embodiment, each residue of the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluoro. The term "HNA" refers to hexitol or hexone nucleic acid.

[0243] At least two distinct modifications are typically present on both the sense and antisense strands. These two modifications can be 2'-O-methyl or 2'-fluoro, or other modifications.

[0244] In one implementation, N a and / or N bModifications containing alternating patterns. As used herein, the term "alternating motif" refers to a motif having one or more modifications, each occurring on alternating nucleotides on one strand. Alternating nucleotides can refer to one every other nucleotide, one every two nucleotides, or similar patterns. For example, if A, B, and C each represent a type of modification to the nucleotide, then the alternating motif could be "ABABABABABAB…", "AABBAABBAABB…", "AABAABAABAAB…", "AAABAAABAAAB…", "AAABBBAAABBB…", or "ABCABCABCABC…", etc.

[0245] The types of modifications contained in an alternating motif can be the same or different. For example, if A, B, C, and D each represent a type of modification on a nucleotide, the alternation pattern (i.e., the modification on every other nucleotide) can be the same, but each of the sense or antisense strands can be selected from several possible modifications within an alternating motif such as “ABABAB…”, “ACACAC…”, “BDBDBD…”, or “CDCDCD…”.

[0246] In one embodiment, the RNAi agent of the present invention comprises a modification pattern for an alternating motif on the sense strand that is shifted relative to a modification pattern for an alternating motif on the antisense strand. This shift allows a modified nucleotide set on the sense strand to correspond to a differently modified nucleotide set on the antisense strand, and vice versa. For example, when the sense strand pairs with the antisense strand in a dsRNA duplex, the alternating motif on the sense strand may begin with “ABABAB” from the 5'-3' of that strand, and the alternating motif on the antisense strand may begin with “BABABA” from the 5'-3' of that strand within the duplex region. As another example, the alternating motif on the sense strand may begin with “AABBAABB” from the 5'-3' of that strand, and the alternating motif on the antisense strand may begin with “BBAABBAA” from the 5'-3' of that strand within the duplex region, resulting in a complete or partial shift of the modification pattern between the sense and antisense strands.

[0247] In one embodiment, the RNAi agent comprises an alternating motif pattern initially having 2'-O-methyl and 2'-F modifications on the sense strand, shifted relative to an alternating motif pattern initially having 2'-O-methyl and 2'-F modifications on the antisense strand; that is, the 2'-O-methyl modified nucleotide on the sense strand pairs with the 2'-F modified nucleotide on the antisense strand, and vice versa. The 1 position on the sense strand may begin with a 2'-F modification, while the 1 position on the antisense strand may begin with a 2'-O-methyl modification.

[0248] Introducing one or more motifs with three identical modifications on three consecutive nucleotides into the sense and / or antisense strands disrupts the initial modification patterns present in the sense and / or antisense strands. This disruption of the modification patterns in the sense and / or antisense strands by introducing one or more motifs with three identical modifications on three consecutive nucleotides into the sense and / or antisense strands unexpectedly enhances gene silencing activity against the target gene.

[0249] In one implementation, when a motif having three identical modifications on three consecutive nucleotides is introduced into any of these chains, the modification of the nucleotide immediately adjacent to the motif is different from the modification of the motif. For example, a portion of the sequence containing the motif is "...N". a YYYN b ……, where “Y” represents a modification with three identical motifs on three consecutive nucleotides, “N” a " and "N b ' represents a modification of the nucleotide immediately preceding the motif "YYY" that is different from the modification of Y, and where N a and N b These can be the same or different modifiers. Alternatively, when a wing modifier is present, N a and / or N b It may exist or it may not exist.

[0250] The RNAi agent may also contain at least one phosphate-thioester or methylphosphonate nucleotide inter-link. The phosphate-thioester or methylphosphonate nucleotide inter-link modification may occur on any nucleotide at any position on the sense strand or antisense strand, or on either strand. For example, the nucleotide inter-link modification may occur on each nucleotide on the sense strand and / or antisense strand; each nucleotide inter-link modification may occur in an alternating pattern on the sense strand and / or antisense strand; or the sense strand or antisense strand may contain two nucleotide inter-link modifications in an alternating pattern. The alternating pattern of the nucleotide inter-link modification on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of the nucleotide inter-link modification on the sense strand may have a shift relative to the alternating pattern of the nucleotide inter-link modification on the antisense strand. In one embodiment, the double-stranded RNAi agent contains 6-8 phosphate-thioester nucleotide inter-links. In one embodiment, the antisense strand contains two phosphate thioester nucleotides linked at the 5' end and two phosphate thioester nucleotides linked at the 3' end, and the sense strand contains at least two phosphate thioester nucleotides linked at either the 5' end or the 3' end.

[0251] In one embodiment, RNAi includes an internucleotide-linking modification of phosphate thioester or methylphosphonate nucleotides in the overhang region. For example, the overhang region may contain two nucleotides with an internucleotide-linking of phosphate thioester or methylphosphonate nucleotides between them. Internucleotide-linking modifications may also be made to link the overhang nucleotides to terminally paired nucleotides within the double-stranded region. For example, at least two, three, four, or all of the overhang nucleotides may be linked by internucleotide-linking of phosphate thioester or methylphosphonate nucleotides, and optionally, additional internucleotide-linking of phosphate thioester or methylphosphonate nucleotides may be present to link the overhang nucleotides to their adjacent paired nucleotides. For example, at least two internucleotide-linking of phosphate thioester nucleotides may be present between three terminal nucleotides, wherein two of these three nucleotides are overhang nucleotides, and the third is an adjacent paired nucleotide. These terminal three nucleotides may be located at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and / or the 5' end of the antisense strand.

[0252] In one embodiment, the two nucleotides have a 3'-end on the antisense strand, and there are two phosphate thioester nucleotides linked between the three terminal nucleotides, wherein two of these three nucleotides are the 3'-end nucleotides, and the third nucleotide is the pairing nucleotide immediately adjacent to the 3'-end nucleotide. Optionally, the RNAi agent may additionally have two phosphate thioester nucleotides linked between the three terminal nucleotides at the 5'-end of both the sense and antisense strands.

[0253] In one embodiment, the RNAi agent contains one or more mismatches, or combinations thereof, with the target within the duplex. Mismatches can occur in overhang regions or duplex regions. Base pairs can be graded based on their tendency to promote dissociation or melting (e.g., the association or dissociation free energy for a particular pair; the simplest method is to examine these pairs individually, but adjacent pairs or similar analyses can also be used). Regarding promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I = inosine). Mismatches, such as non-canonical pairings or pairings other than canonical (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings; and pairings including universal bases are preferred over canonical pairings.

[0254] In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs in the duplex region starting from the 5' end of the antisense strand, these base pairs being independently selected from: A:U, G:U, I:C, and mismatched pairs, such as non-canonical pairings or pairings other than canonical, or pairings including universal bases, in order to promote dissociation of the antisense strand at the 5' end of the duplex.

[0255] In one embodiment, the nucleotide at position 1 from the 5' end of the antisense strand within the double-stranded region is selected from A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs from the 5' end of the antisense strand within the double-stranded region is an AU base pair. For example, the first base pair from the 5' end of the antisense strand within the double-stranded region is an AU base pair.

[0256] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxy-thymine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxy-thymine (dT). In one embodiment, a short sequence of deoxy-thymine nucleotides, such as two dT nucleotides, is present at the 3' end of the sense strand and / or antisense strand.

[0257] In one implementation, the justice chain sequence can be represented by equation (I): 5′n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3′(I) in: i and j are each independently 0 or 1; p and q are each independently 0-6; Each N a Independently represent oligonucleotide sequences containing 0-25 modified nucleotides, each sequence containing at least two differently modified nucleotides; Each N b Independently represent oligonucleotide sequences containing 0-10 modified nucleotides; Each n p and n q Independently represent the overhanging nucleotide; Nb and Y do not have the same modification; and XXX, YYY, and ZZZ each independently represent a motif with three identical modifications on three consecutive nucleotides. Preferably, YYY consists entirely of nucleotides modified with 2'-F.

[0258] In one implementation, N a and / or N b Includes alternation pattern modification.

[0259] In one implementation, the YYY motif occurs at or near the cleavage site on the positive strand. For example, when the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the YYY motif may occur at or near the cleavage site on the positive strand (e.g., at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), with counting starting from the 5' end at the first nucleotide; or optionally, counting may start from the 5' end at the first paired nucleotide within the double-stranded region.

[0260] In one implementation, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The justice chain can therefore be represented by the following equation: 5′n p -N a -YYY-N b -ZZZ-N a -n q 3′(Ib); 5′n p -N a -XXX-N b -YYY-N a -n q 3′(Ic); or 5′n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3′(Id).

[0261] When the justice chain is represented by equation (Ib), N b This indicates an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N a It can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0262] When the justice chain is represented by equation (Ic), N b This indicates an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N a It can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0263] When the justice chain is represented as equation (Id), each N bIndependently represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Preferably, N b It can be 0, 1, 2, 3, 4, 5, or 6. Each N a It can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0264] Each of X, Y, and Z can be the same as or different from each other.

[0265] In other implementations, i is 0 and j is 0, and the justice chain can be represented by the following formula: 5′n p -N a -YYY-N a -n q 3′(Ia).

[0266] When the justice chain is represented by equation (Ia), each N a It can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0267] In one implementation, the antisense strand sequence of RNAi can be represented by formula (Ie): 5′n q '-N a ′-(Z'Z′Z′) k -N b ′-Y′Y′Y′-N b ′-(X′X′X′) l -N′ a -n p ′3′(Ie) in: k and l are each independently 0 or 1; p' and q' are each independently 0-6; Each N a ′ Independently represents an oligonucleotide sequence containing 0-25 modified nucleotides, each sequence containing at least two differently modified nucleotides; Each N b ′ Independently represents an oligonucleotide sequence containing 0-10 modified nucleotides; Each n p ′ and n q ′ Independently represents the overhanging nucleotide; Where N b 'and Y' do not have the same modifiers; and X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent a motif with three identical modifications on three consecutive nucleotides.

[0268] In one implementation, N a 'and / or N b 'Includes alternating pattern modifications.'

[0269] The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 on the antisense strand, with counting starting from the 5′ end from the first nucleotide; or optionally, counting starts from the 5′ end at the first paired nucleotide within the double-stranded region. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, and 13.

[0270] In one implementation, the Y'Y'Y' motif consists entirely of nucleotides modified with 2'-OMe.

[0271] In one implementation, k is 1 and l is 0, or k is 0 and l is 1, or both k and 1 are 1.

[0272] Therefore, the antisense chain can be represented by the following formula: 5′n q '-N a ′-Z′Z′Z′-N b ′-Y′Y′Y′-N a ′-n p '3'(Ig); 5′n q '-N a ′-Y′Y′Y′-N b ′-X′X′X′-n p’ 3′(Ih); or 5′n q’ -N a ′-Z′Z′Z′-N b ′-Y′Y′Y′-N b ′-X′X′X′-N a ′-n p’ 3′(Ii).

[0273] When the antisense chain is represented by equation (Ig), N b ' indicates an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N a 'Independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0274] When the antisense chain is represented as (Ih), N b ' indicates an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N a 'Independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0275] When the antisense chain is represented as equation (Ii), each N b 'Independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N a 'Independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N b It can be 0, 1, 2, 3, 4, 5 or 6.

[0276] In other implementations, k is 0 and l is 0, and the antisense chain can be represented by the following formula: 5′n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3′(If).

[0277] When the antisense chain is represented as (If), each N a 'Independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0278] Each of X′, Y′, and Z′ can be the same as or different from each other.

[0279] Each nucleotide in the sense and antisense strands may be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxy, or 2'-fluoro. For example, each nucleotide in the sense and antisense strands may be independently modified with 2'-O-methyl or 2'-fluoro. In particular, each X, Y, Z, X', Y', and Z' may represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0280] In one embodiment, the positive strand of the RNAi agent may contain a YYY motif, which occurs at positions 9, 10, and 11 of the strand when the double-stranded region is 21 nt, and is counted starting from the first nucleotide at the 5' end, or optionally, starting from the first paired nucleotide at the 5' end within the double-stranded region; and Y represents a 2'-F modification. The positive strand may additionally contain an XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region; and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0281] In one embodiment, the antisense strand may contain a Y'Y'Y' motif occurring at positions 11, 12, or 13 of the strand, counted starting from the first nucleotide at the 5' end, or optionally, counted starting from the first paired nucleotide at the 5' end within the double-stranded region; and Y' represents a 2'-O-methyl modification. The antisense strand may additionally contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at opposite ends of the double-stranded region; and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0282] The justice chain represented by any one of the above equations (Ia), (Ib), (Ic), and (Id) forms a double chain with the antisense chain represented by any one of the equations (If), (Ig), (Ih), and (Ii).

[0283] Therefore, the RNAi agent used in the method of the present invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi duplex is represented by formula (Ij): Justice: 5′np-N a -(XXX)i-Nb-Y Y Y-Nb-(ZZZ)j-Na-nq 3′ Antonym: 3′np'-Na'-(X'X′X′)k-Nb'-Y′Y′Y′-Nb'-(Z′Z′Z′)l-Na'-nq'5′(Ij) in: i, j, k, and l are each independently 0 or 1; p, p′, q, and q′ are each independently 0-6; Each Na and Na' independently represents an oligonucleotide sequence containing 0-25 modified nucleotides, each sequence containing at least two differently modified nucleotides; Each Nb and Nb' independently represents an oligonucleotide sequence containing 0-10 modified nucleotides; Each of np', np, nq', and nq, which may or may not be present, independently represents a terminal nucleotide; and XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent a motif with three identical modifications on three consecutive nucleotides.

[0284] In one implementation, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another implementation, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0285] Exemplary combinations of the sense and antisense strands that form an RNAi duplex include the following: 5′np-Na-Y Y Y-Na-nq 3′ 3′np'-Na'-Y′Y′Y′-Na'nq'5′(Ik) 5′np-Na-Y Y Y-Nb-Z Z Z-Na-nq 3′ 3′np'-Na'-Y′ Y′ Y′-Nb'-Z′ Z′ Z′-Na' nq' 5′(Il) 5′np-Na-X X X-Nb-Y Y Y-Na-nq 3′ 3′np'-Na'-X′ X′ X′-Nb'-Y′ Y′ Y′-Na'-nq' 5′(Im) 5′np-Na-X X X-Nb-Y Y Y-Nb-Z Z Z-Na-nq 3′ 3′np'-Na'-X′ X′ X′-Nb'-Y′ Y′ Y′-Nb'-Z′ Z′ Z′-Na-nq' 5′(In) When the RNAi agent is represented by formula (Ik), each Na independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0286] When the RNAi agent is represented by formula (II), each Nb independently represents an oligonucleotide sequence containing 1-10, 1-7, 1-5, or 1-4 modified nucleotides. Each Na independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0287] When the RNAi agent is represented by formula (Im), each Nb and Nb' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0288] When the RNAi agent is represented by formula (In), each Nb and Nb' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na and Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Each of Na, Na', Nb, and Nb' independently contains an alternating pattern of modifications.

[0289] Each of X, Y, and Z in equations (Ij), (Ik), (Il), (Im), and (In) may be the same as or different from each other.

[0290] When the RNAi agent is represented by formulas (Ij), (Ik), (Il), (Im), and (In), at least one of the Y nucleotides may form a base pair with one of the Y' nucleotides. Alternatively, at least two of the Y nucleotides may form a base pair with the corresponding Y' nucleotide; or all three of the Y nucleotides may form a base pair with the corresponding Y' nucleotide.

[0291] When the RNAi agent is represented by formula (Il) or (In), at least one of the Z nucleotides may form a base pair with one of the Z′ nucleotides. Alternatively, at least two of the Z nucleotides may form a base pair with the corresponding Z′ nucleotide; or all three of the Z nucleotides may form a base pair with the corresponding Z′ nucleotide.

[0292] When the RNAi agent is represented by formula (Im) or (In), at least one of the X nucleotides may form a base pair with one of the X′ nucleotides. Alternatively, at least two of the X nucleotides may form a base pair with the corresponding X′ nucleotide; or all three of the X nucleotides may form a base pair with the corresponding X′ nucleotide.

[0293] In one embodiment, the modification on the Y nucleotide is different from the modification on the Y' nucleotide, the modification on the Z nucleotide is different from the modification on the Z' nucleotide, and / or the modification on the X nucleotide is different from the modification on the X' nucleotide.

[0294] In one embodiment, when the RNAi agent is represented by formula (In), the Na modification is 2'-O-methyl or 2'-fluoro. In another embodiment, when the RNAi agent is represented by formula (In), the Na modification is 2'-O-methyl or 2'-fluoro and np' > 0, and at least one np' is linked to an adjacent nucleotide via a thiophosphate bond. In yet another embodiment, when the RNAi agent is represented by formula (In), the Na modification is 2'-O-methyl or 2'-fluoro, np' > 0, and at least one np' is linked to an adjacent nucleotide via a thiophosphate bond, and the positive strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branching adapter (described below). In another embodiment, when the RNAi agent is represented by formula (In), the Na modification is 2'-O-methyl or 2'-fluoro, np' > 0 and at least one np' is linked to an adjacent nucleotide via a thiophosphate bond, the sense strand contains at least one thiophosphate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked by a divalent or trivalent branching linker.

[0295] In one embodiment, when the RNAi agent is represented by formula (Ik), the Na modification is 2'-O-methyl or 2'-fluoro, np' > 0 and at least one np' is linked to an adjacent nucleotide via a thiophosphate bond, the sense strand contains at least one thiophosphate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked by a divalent or trivalent branched linker.

[0296] In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formulas (Ij), (Ik), (Il), (Im), and (In), wherein the duplexes are linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer also contains a ligand. Each of the duplexes may target the same gene or two different genes; or each of the duplexes may target the same gene at two different target sites.

[0297] In one embodiment, the RNAi agent is a multimer containing three, four, five, six, or more duplexes represented by formulas (Ij), (Ik), (Il), (Im), and (In), wherein the duplexes are linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer also includes a ligand. Each of the duplexes may target the same gene or two different genes; or each of the duplexes may target the same gene at two different target sites.

[0298] In one embodiment, two RNAi agents represented by formulas (Ij), (Ik), (Il), (Im), and (In) are linked to each other at one or both of their 5' and 3' ends, and optionally conjugated to a ligand. Each of the agents may target the same gene or two different genes; or each of the agents may target the same gene at two different target sites.

[0299] In some embodiments, the RNAi agent of the present invention may contain a small number of nucleotides with 2'-fluorination modification, such as 10 or fewer nucleotides with 2'-fluorination modification. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides with 2'-fluorination modification. In one specific embodiment, the RNAi agent of the present invention contains 10 nucleotides with 2'-fluorination modification, for example, 4 nucleotides with 2'-fluorination modification in the sense strand and 6 nucleotides with 2'-fluorination modification in the antisense strand. In another specific embodiment, the RNAi agent of the present invention contains 6 nucleotides with 2'-fluorination modification, for example, 4 nucleotides with 2'-fluorination modification in the sense strand and 2 nucleotides with 2'-fluorination modification in the antisense strand.

[0300] In other embodiments, the RNAi agent of the present invention may contain an extremely low number of nucleotides with 2'-fluorine modification, such as two or fewer nucleotides with 2'-fluorine modification. For example, the RNAi agent may contain two, one, or zero nucleotides with 2'-fluorine modification. In one specific embodiment, the RNAi agent may contain two nucleotides with 2'-fluorine modification, for example, zero nucleotides with 2'-fluorine modification in the sense strand and two nucleotides with 2'-fluorine modification in the antisense strand.

[0301] Various publications describe polyRNAi agents that can be used in the methods of the present invention. Such publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, the entire contents of each of which are hereby incorporated herein by reference.

[0302] In some embodiments, the compositions and methods of this disclosure include vinylphosphonate (VP) modification of the RNAi agent as described herein. In exemplary embodiments, the vinylphosphonate of this disclosure has the following structure: For example, when the phosphate mimic is 5'-vinylphosphonate (VP), the 5'-terminal nucleotide can have the following structure: The asterisk (*) indicates the position where the nucleotide is bonded to the 5' position of the adjacent nucleotide. R is hydrogen, hydroxyl, methoxy, fluorine (e.g., hydroxyl or methoxy) or another modification described herein; and B is a nucleobase or a modified nucleobase, optionally wherein B is adenine, guanine, cytosine, thymine, or uracil.

[0303] The vinylphosphonate of this disclosure can be linked to either the antisense or sense strand of the dsRNA of this disclosure. In some 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. The dsRNA agent may contain a phosphorus-containing group at the 5' end of either the sense or antisense strand. The 5'-terminal phosphorus-containing group may be a 5'-terminal phosphate (5'-P), a 5'-terminal thiophosphate (5'-PS), a 5'-terminal dithiophosphate (5'-PS2), a 5'-terminal vinylphosphonate (5'-VP), a 5'-terminal methylphosphonate (MePhos), or a 5'-deoxy-5'-C-malonyl group. When the 5'-terminal phosphorus-containing group is a 5'-terminal vinylphosphonate (5'-VP), the 5'-VP may be a 5'-E-VP isomer (i.e., trans-vinylphosphonate). ), 5'-Z-VP isomer (i.e., cis-vinylphosphonate) (or mixtures thereof).

[0304] The compositions and methods disclosed herein also cover vinyl phosphate modification. An exemplary vinyl phosphate structure is as follows: For example, when the phosphate mimic is 5'-vinyl phosphate, the 5'-terminal nucleotide can have the structure shown, wherein the phosphonate group is replaced with a phosphate ester.

[0305] As described in more detail below, RNAi agents containing one or more carbohydrate moieties conjugated to an RNAi agent can optimize one or more properties of that RNAi agent. In many cases, the carbohydrate moieties are linked to modified subunits of the RNAi agent. For example, the ribose ring of one or more ribonucleotide subunits of a dsRNA agent can be replaced by another moieties (e.g., a non-carbohydrate (preferably cyclic) carrier linked to a carbohydrate ligand). The ribonucleotide subunit whose ribose ring has been so replaced is referred herein to as a ribose substitution modified subunit (RRMS). Cyclic carriers can be carbocyclic systems (i.e., all ring atoms are carbon atoms) or heterocyclic systems (i.e., one or more ring atoms may be heteroatoms, e.g., nitrogen, oxygen, sulfur). Cyclic carriers can be monocyclic systems or may contain two or more rings, such as fused rings. Cyclic carriers can be fully saturated cyclic systems or may contain one or more double bonds.

[0306] The ligand can be linked to the polynucleotide via a carrier. The carrier includes (i) at least one “backbone connection point,” preferably two “backbone connection points,” and (ii) at least one “tether connection point.” As used herein, a “backbone connection point” refers to a functional group (e.g., a hydroxyl group), or generally, a bond (e.g., a phosphate ester or a modified phosphate ester) that can be used and is suitable for incorporating the carrier into the backbone (e.g., a sulfur-containing backbone) of the ribonucleic acid. In some embodiments, a “tether connection point” (TAP) refers to a constitutive ring atom of the cyclic carrier that links a selected portion, such as a carbon atom or a heteroatom (different from the atom providing the backbone connection point). This portion can be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, and polysaccharide. Optionally, the selected portion is linked to the cyclic carrier by an intermediate tether. Thus, the cyclic carrier will generally include a functional group (e.g., an amino group), or generally provide a bond suitable for binding or tethering another chemical entity (e.g., a ligand) to the constitutive ring.

[0307] RNAi agents can be conjugated to ligands via a carrier, wherein the carrier can be a cyclic group or an acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, [1,3]dioxacyclopentane, oxazolinyl, isoxazolinyl, morpholinyl, thiazolinyl, isothiazolinyl, quinoxolinyl, pyridazinoneyl, tetrahydrofuranyl, and decahydronaphthalene; preferably, the acyclic group is selected from a serinel skeleton or a diethanolamine skeleton.

[0308] In another embodiment of the invention, the iRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. This RNAi agent can be represented by formula (L): In formula (L), B1, B2, B3, B1', B2', B3', and B4' are each independently a modified nucleotide selected from 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halogenated, ENA, and BNA / LNA. In some embodiments, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification. In some embodiments, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe or 2'-F modification. In some embodiments, at least one of B1, B2, B3, B1', B2', B3', and B4' contains a 2′-ON-methylacetamido (2′-O-NMA) modification.

[0309] C1 is a heat-destabilized nucleotide located at a site opposite the seed region of the antisense strand (i.e., position 2-8 at the 5' end of the antisense strand). For example, C1 is located on the sense strand at positions 2-8 at the 5' end of the antisense strand. In one example, C1 is located at position 15 from the 5' end of the sense strand. The C1 nucleotide has a heat-destabilizing modification, which may include: a base-depleting modification; a nucleotide mismatch with the opposite nucleotide in the duplex; and a sugar modification, such as a 2'-deoxy modification or an acyclic nucleotide, such as a non-locked nucleic acid (UNA) or a glycerol nucleic acid (GNA). In some embodiments, C1 has a heat-destabilizing modification selected from: i) a nucleotide mismatch with the opposite nucleotide in the antisense strand; ii) a base-depleting modification selected from: and iii) sugar modifications selected from the following: Where B is a modified or unmodified nucleobase, and R... 1 and R 2 Independently H, halogen, OR3, or alkyl; and R ３ It is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In some embodiments, the thermal destabilizing modification in C1 is a mismatch selected from the following: G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T; and optionally, at least one nucleobase in the mismatch is a 2'-deoxynucleobase. In one example, the thermal destabilizing modification in C1 is GNA or

[0310] T1, T1', T2', and T3' each independently represent a modified nucleotide that provides steric hindrance less than or equal to that of a 2'-OMe modification. Steric hindrance refers to the sum of the steric hindrance effects of modifications. Methods for determining the steric hindrance effects of nucleotide modifications are known to those skilled in the art. Modifications can be made at the 2' position of the ribose ring of the nucleotide, or at a position in the backbone of a non-ribonucleotide, acyclic nucleotide, or nucleotide that is similar to or equivalent to the 2' position of the ribose ring, and provide steric hindrance less than or equal to that of a 2'-OMe modification. For example, T1, T1', T2', and T3' are each independently selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl. In some embodiments, T1 is DNA. In some embodiments, T1' is DNA, RNA, or LNA. In some embodiments, T2' is DNA or RNA. In some embodiments, T3' is DNA or RNA.

[0311] n 1 n 3 and q 1 The length of each nucleotide independently ranges from 4 to 15 nucleotides.

[0312] n 5 q 3 and q 7 The length of each nucleotide is independently 1-6 nucleotides.

[0313] n 4 q 2 and q 6 The length of n is independently 1-3 nucleotides; alternatively, n 4 It is 0.

[0314] q 5 The length of each nucleotide is independently 0-10 nucleotides.

[0315] n 2 and q 4 The length of each nucleotide is independently 0-3 nucleotides.

[0316] Alternatively, n 4 The length is 0-3 nucleotides.

[0317] In some implementations, n 4 It can be 0. In one instance, n... 4 It is 0, and q 2 and q 6 It is 1. In another instance, n 4 It is 0, and q 2 and q 6=1, wherein: there are two inter-thiophosphate nucleotide linkage modifications at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and there are two inter-thiophosphate nucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two inter-thiophosphate nucleotide linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand).

[0318] In some implementations, n 4 q 2 and q 6 Each is 1.

[0319] In some implementations, n 2 n 4 q 2 q 4 and q 6 Each is 1.

[0320] In some implementations, when the length of the sense strand is 19-22 nucleotides, C1 is located at position 14-17 at the 5' end of the sense strand, and n 4 The value is 1. In some implementations, C1 is located at position 15 at the 5' end of the justice chain.

[0321] In some implementations, T3' starts at position 2, beginning from the 5' end of the antisense chain. In one instance, T3' is at position 2, beginning from the 5' end of the antisense chain, and q 6 It equals 1.

[0322] In some implementations, T1' begins at position 14 starting from the 5' end of the antisense chain. In one example, T1' is at position 14 starting from the 5' end of the antisense chain and q 2 It equals 1.

[0323] In one exemplary implementation, T3' starts at position 2 from the 5' end of the antisense chain, while T1' starts at position 14 from the 5' end of the antisense chain. In one instance, T3' starts at position 2 from the 5' end of the antisense chain and q 6 It equals 1, and T1' starts at position 14 from the 5' end of the antisense chain and q 2 It equals 1.

[0324] In some implementations, T1' and T3' are separated by a length of 11 nucleotides (i.e., T1' and T3' nucleotides are not counted).

[0325] In some implementations, T1' is located at position 14 starting from the 5' end of the antisense chain. In one example, T1' is located at position 14 starting from the 5' end of the antisense chain and q 2It equals 1 and is used at the 2' position or at a non-ribose, acyclic, or backbone position, providing less steric hindrance than 2'-OMe ribose.

[0326] In some implementations, T3' is located at position 2 starting from the 5' end of the antisense chain. In one example, T3' is located at position 2 starting from the 5' end of the antisense chain and q 6 It equals 1 and is used at the 2' position or at a non-ribose, acyclic, or backbone position, providing a spatial steric hindrance less than or equal to the spatial steric hindrance of 2'-OMe ribose.

[0327] In some implementations, T1 is located at the cleavage site of the positive strand. In one example, when the positive strand is 19-22 nucleotides long, T1 is located at position 11 from the 5' end of the positive strand, and n 2 The value is 1. In one exemplary embodiment, when the length of the positive strand is 19-22 nucleotides, T1 is located at the positive strand cleavage site at position 11 starting from the 5' end of the positive strand, and n 2 The value is 1.

[0328] In some implementations, T2' starts at position 6, beginning at the 5' end of the antisense chain. In one example, T2' is located at positions 6-10, beginning at the 5' end of the antisense chain, and q 4 The value is 1.

[0329] In one exemplary embodiment, T1 is located at the cleavage site of the positive strand, for example, at position 11 starting from the 5' end of the positive strand when the length of the positive strand is 19-22 nucleotides, and n 2 =1; T1' is at position 14 starting from the 5' end of the antisense chain, and q 2 The value is equal to 1, and the modification of T1' is at the 2' position of the ribose ring or at a position in the non-ribose, acyclic, or backbone, providing less steric hindrance than that of 2'-OMe ribose; T2' is at positions 6-10 starting from the 5' end of the antisense chain, and q 4 =1; T3' is at position 2 starting from the 5' end of the antisense chain, and q 6 It equals 1, and the modification of T3' at the 2' position or at a position in a non-ribose, acyclic, or backbone location provides a steric hindrance less than or equal to the steric hindrance of 2'-OMe ribose.

[0330] In some implementations, T2' starts at position 8, beginning at the 5' end of the antisense chain. In one example, T2' starts at position 8, beginning at the 5' end of the antisense chain, and q 4 The value is 2.

[0331] In some implementations, T2' begins at position 9, starting from the 5' end of the antisense chain. In one example, T2' is at position 9, starting from the 5' end of the antisense chain, and q 4 The value is 1.

[0332] In some implementations, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 B3 is 1, B3 is 2'-OMe or 2'-F, q 5 =6, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 =1; wherein: there are two inter-thiophosphate nucleotide linkage modifications at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and there are two inter-thiophosphate nucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two inter-thiophosphate nucleotide linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand).

[0333] In some implementations, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 B3' is 1, B3' is 2'-OMe or 2'-F, q 5 =6, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 =1; wherein: there are two inter-thiophosphate nucleotide linkage modifications at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and there are two inter-thiophosphate nucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two inter-thiophosphate nucleotide linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand).

[0334] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'OMe, n 5For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1.

[0335] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 =1; wherein: there are two inter-thiophosphate nucleotide linkage modifications at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and there are two inter-thiophosphate nucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two inter-thiophosphate nucleotide linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand).

[0336] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 6, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =7, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1.

[0337] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 6, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =7, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 =1; wherein: there are two inter-thiophosphate nucleotide linkage modifications at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and there are two inter-thiophosphate nucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two inter-thiophosphate nucleotide linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand).

[0338] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 B3' is 1, B3' is 2'-OMe or 2'-F, q 5 =6, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1.

[0339] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 B3' is 1, B3' is 2'-OMe or 2'-F, q 5 =6, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 =1; wherein: there are two inter-thiophosphate nucleotide linkage modifications at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and there are two inter-thiophosphate nucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two inter-thiophosphate nucleotide linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand).

[0340] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =5, T2' is 2'-F, q 4 B3' is 1, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 =1; optionally, at least 2 additional TTs are present at the 3' end of the antisense chain.

[0341] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1=9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =5, T2' is 2'-F, q 4 B3' is 1, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 1; optionally having at least two additional TTs at the 3' end of the antisense strand; wherein: there are two inter-thiophosphate nucleotide linkage modifications at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and two inter-thiophosphate nucleotide linkage modifications at positions 1 and 2 of the antisense strand and two inter-thiophosphate nucleotide linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand).

[0342] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1.

[0343] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q6 B4' is 1, B4' is 2'-OMe, and q 7 =1; wherein: there are two inter-thiophosphate nucleotide linkage modifications at positions 1-5 on the sense strand (counting from the 5' end), and two inter-thiophosphate nucleotide linkage modifications at positions 1 and 2 on the antisense strand, and two inter-thiophosphate nucleotide linkage modifications at positions 18-23 (counting from the 5' end).

[0344] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1.

[0345] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7=1; wherein: there are two inter-thiophosphate nucleotide linkage modifications at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and there are two inter-thiophosphate nucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two inter-thiophosphate nucleotide linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand).

[0346] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1.

[0347] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 =1; wherein: there are two inter-thiophosphate nucleotide linkage modifications at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), and there are two inter-thiophosphate nucleotide linkage modifications at positions 1 and 2 of the antisense strand, and two inter-thiophosphate nucleotide linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand).

[0348] RNAi agents can contain a phosphorus-containing group at the 5' end of either the sense or antisense strand. This 5'-terminal phosphorus-containing group can be a 5'-terminal phosphate ester (5'-P), a 5'-terminal thiophosphate ester (5'-PS), a 5'-terminal dithiophosphate ester (5'-PS2), a 5'-terminal vinylphosphonate ester (5'-VP), a 5'-terminal methylphosphonate ester (MePhos), or a 5'-deoxy-5'-C-malonyl group. When the 5'-terminal phosphorus-containing group is a 5'-terminal vinylphosphonate (5'-VP), 5'-VP can be a 5'-E-VP isomer (i.e., trans-vinylphosphonate). 5'-Z-VP isomer (i.e., cis-vinylphosphonate, (or mixtures thereof).

[0349] In some embodiments, the RNAi agent contains a phosphorus-containing group at the 5' end of the sense strand. In some embodiments, the RNAi agent contains a phosphorus-containing group at the 5' end of the antisense strand.

[0350] In some embodiments, the RNAi agent contains 5'-P. In some embodiments, the RNAi agent contains 5'-P in the antisense strand.

[0351] In some embodiments, the RNAi agent contains 5'-PS. In some embodiments, the RNAi agent contains 5'-PS in the antisense strand.

[0352] In some embodiments, the RNAi agent contains 5'-VP. In some embodiments, the RNAi agent contains 5'-VP in the antisense strand. In some embodiments, the RNAi agent contains 5'-E-VP in the antisense strand. In some embodiments, the RNAi agent contains 5'-Z-VP in the antisense strand.

[0353] In some embodiments, the RNAi agent contains 5'-PS2. In some embodiments, the RNAi agent contains 5'-PS2 in the antisense strand.

[0354] In some embodiments, the RNAi agent comprises 5'-PS2. In some embodiments, the RNAi agent comprises a 5'-deoxy-5'-C-malonyl group in the antisense strand.

[0355] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1=9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also contain 5'-PS.

[0356] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also contain 5'-P.

[0357] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1. The RNAi agent also contains 5'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0358] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also contain 5'-PS2.

[0359] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also contain a 5'-deoxy-5'-C-malonyl group.

[0360] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkages at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkages at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkages at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-P.

[0361] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-phosphothionucleotide (PS) linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two PS linkage modifications at positions 1 and 2 on the antisense strand, and two PS linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-PS.

[0362] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkages at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkages at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkages at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0363] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-phosphothionucleotide linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-phosphothionucleotide linkage modifications at positions 1 and 2 on the antisense strand, and two inter-phosphothionucleotide linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-PS2.

[0364] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-thiophosphate nucleotide linkages at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-thiophosphate nucleotide linkages at positions 1 and 2 on the antisense strand, and two inter-thiophosphate nucleotide linkages at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group.

[0365] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also contain 5'-P.

[0366] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6B4' is 1, B4' is 2'-OMe, and q 7 The dsRNA agent also contains 5'-PS.

[0367] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1. The RNAi agent also contains 5'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0368] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also contain 5'-PS2.

[0369] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1. RNAi agents also contain a 5'-deoxy-5'-C-malonyl group.

[0370] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkages at positions 1-5 on the sense strand (counting from the 5' end), and two inter-nucleotide thiophosphate linkages at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkages at positions 18-23 (counting from the 5' end). The RNAi agent also contains 5'-P.

[0371] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkages at positions 1-5 on the sense strand (counting from the 5' end), and two inter-nucleotide thiophosphate linkages at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkages at positions 18-23 (counting from the 5' end). The RNAi agent also contains 5'-PS.

[0372] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkages (counting from the 5' end) at positions 1-5 on the sense strand, and two inter-nucleotide thiophosphate linkages (counting from the 5' end) at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkages (counting from the 5' end) at positions 18-23. The RNAi agent also contains 5'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0373] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkages (counting from the 5' end) at positions 1-5 on the sense strand, and two inter-nucleotide thiophosphate linkages (counting from the 5' end) at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkages (counting from the 5' end) at positions 18-23. The RNAi agent also contains 5'-PS2.

[0374] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-thiophosphate nucleotide linkages (counting from the 5' end) at positions 1-5 on the sense strand, and two inter-thiophosphate nucleotide linkages (counting from the 5' end) at positions 1 and 2 on the antisense strand, and two inter-thiophosphate nucleotide linkages (counting from the 5' end) at positions 18-23. The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group.

[0375] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5=5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also contain 5'-P.

[0376] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also contain 5'-PS.

[0377] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1. The RNAi agent also contains 5'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0378] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4B3 is 0, B3 is 2'OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The dsRNA RNA agent also contains 5'-PS2.

[0379] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also contain a 5'-deoxy-5'-C-malonyl group.

[0380] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkages at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkages at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkages at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-P.

[0381] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1; wherein: there are two inter-phosphothionucleotide (PS) linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two PS linkage modifications at positions 1 and 2 on the antisense strand, and two PS linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-PS.

[0382] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkages at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkages at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkages at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0383] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1; wherein: there are two inter-phosphothionucleotide linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-phosphothionucleotide linkage modifications at positions 1 and 2 on the antisense strand, and two inter-phosphothionucleotide linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-PS2.

[0384] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q6 B4' is 1, B4' is 2'-F, and q 7 The value is 1; wherein: there are two inter-thiophosphate nucleotide linkages at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-thiophosphate nucleotide linkages at positions 1 and 2 on the antisense strand, and two inter-thiophosphate nucleotide linkages at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group.

[0385] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also contain 5'-P.

[0386] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also contain 5'-PS.

[0387] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1. The RNAi agent also contains 5'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0388] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also contain 5'-PS2.

[0389] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5=7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1. RNAi agents also contain a 5'-deoxy-5'-C-malonyl group.

[0390] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkages at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkages at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkages at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-P.

[0391] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7The value is 1; wherein: there are two inter-phosphothionucleotide (PS) linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two PS linkage modifications at positions 1 and 2 on the antisense strand, and two PS linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-PS.

[0392] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkages at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkages at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkages at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0393] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q7 The value is 1; wherein: there are two inter-phosphothionucleotide linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-phosphothionucleotide linkage modifications at positions 1 and 2 on the antisense strand, and two inter-phosphothionucleotide linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-PS2.

[0394] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1; wherein: there are two inter-thiophosphate nucleotide linkages at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-thiophosphate nucleotide linkages at positions 1 and 2 on the antisense strand, and two inter-thiophosphate nucleotide linkages at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group.

[0395] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkage modifications at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-P and a targeting ligand. In some embodiments, 5'-P is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0396] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkage modifications at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains a 5'-PS and a targeting ligand. In some embodiments, the 5'-PS is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0397] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-thiophosphate nucleotide linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-thiophosphate nucleotide linkage modifications at positions 1 and 2 on the antisense strand, and two inter-thiophosphate nucleotide linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand.

[0398] In some implementations, the 5'-VP is located at the 5'-end of the antisense strand, and the targeting ligand is located at the 3'-end of the sense strand.

[0399] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkage modifications at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-PS2 and a targeting ligand. In some embodiments, 5'-PS2 is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0400] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkage modifications at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group and a targeting ligand. In some embodiments, the 5'-deoxy-5'-C-malonyl group is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0401] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkage modifications (counting from the 5' end) at positions 1-5 on the sense strand, and two inter-nucleotide thiophosphate linkage modifications (counting from the 5' end) at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkage modifications (counting from the 5' end) at positions 18-23. The RNAi agent also contains 5'-P and a targeting ligand. In some embodiments, 5'-P is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0402] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkage modifications (counting from the 5' end) at positions 1-5 on the sense strand, and two inter-nucleotide thiophosphate linkage modifications (counting from the 5' end) at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkage modifications (counting from the 5' end) at positions 18-23. The RNAi agent also contains a 5'-PS and a targeting ligand. In some embodiments, the 5'-PS is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0403] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7The value is 1; wherein: two inter-thiophosphate nucleotide linkage modifications (counting from the 5' end) are present in positions 1-5 of the sense strand, and two inter-thiophosphate nucleotide linkage modifications (counting from the 5' end) are present in positions 1 and 2 of the antisense strand, and two inter-thiophosphate nucleotide linkage modifications (counting from the 5' end) are present in positions 18-23. The RNAi agent also comprises a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In some embodiments, the 5'-VP is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0404] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkage modifications (counting from the 5' end) at positions 1-5 on the sense strand, and two inter-nucleotide thiophosphate linkage modifications (counting from the 5' end) at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkage modifications (counting from the 5' end) at positions 18-23. The RNAi agent also contains 5'-PS2 and a targeting ligand. In some embodiments, 5'-PS2 is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0405] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-OMe, and q 7 The value is 1; wherein: there are two inter-thiophosphate nucleotide linkage modifications (counting from the 5' end) at positions 1-5 on the sense strand, and two inter-thiophosphate nucleotide linkage modifications (counting from the 5' end) at positions 1 and 2 on the antisense strand, and two inter-thiophosphate nucleotide linkage modifications (counting from the 5' end) at positions 18-23. The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group and a targeting ligand. In some embodiments, the 5'-deoxy-5'-C-malonyl group is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0406] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkage modifications at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-P and a targeting ligand. In some embodiments, 5'-P is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0407] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkage modifications at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains a 5'-PS and a targeting ligand. In some embodiments, the 5'-PS is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0408] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1; wherein: two phosphate-thionucleotide inter-linked modifications are present at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two phosphate-thionucleotide inter-linked modifications are present at positions 1 and 2 on the antisense strand, and two phosphate-thionucleotide inter-linked modifications are present at positions 18-23 on the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also comprises a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In some embodiments, the 5'-VP is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0409] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkage modifications at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-PS2 and a targeting ligand. In some embodiments, 5'-PS2 is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0410] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 =4, T2' is 2'-F, q 4 For 2, B3' is 2'-OMe or 2'-F, q 5 =5, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7The value is 1; wherein: there are two inter-nucleotide thiophosphate linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkage modifications at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group and a targeting ligand. In some embodiments, the 5'-deoxy-5'-C-malonyl group is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0411] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkage modifications at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-P and a targeting ligand. In some embodiments, 5'-P is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0412] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkage modifications at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains a 5'-PS and a targeting ligand. In some embodiments, the 5'-PS is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0413] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1; wherein: two phosphate-thionucleotide inter-linked modifications are present at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two phosphate-thionucleotide inter-linked modifications are present at positions 1 and 2 on the antisense strand, and two phosphate-thionucleotide inter-linked modifications are present at positions 18-23 on the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also comprises a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In some embodiments, the 5'-VP is located at the 5' end of the antisense strand, and the targeting ligand is located at the 3' end of the sense strand.

[0414] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkage modifications at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains 5'-PS2 and a targeting ligand. In some embodiments, 5'-PS2 is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0415] In some implementations, B1 is 2'-OMe or 2'-F, n 1 The value is 8, T1 is 2'F, n 2 B2 is 3, B2 is 2'-OMe, n 3 For 7, n 4 B3 is 0, B3 is 2'-OMe, n 5 For 3, B1' is 2'-OMe or 2'-F, q 1 =9, T1' is 2'-F, q 2 B2' is 1, B2' is 2'-OMe or 2'-F, q 3 4, q 4 B3' is 0, B3' is 2'-OMe or 2'-F, q 5 =7, T3' is 2'-F, q 6 B4' is 1, B4' is 2'-F, and q 7 The value is 1; wherein: there are two inter-nucleotide thiophosphate linkage modifications at positions 1-5 on the sense strand (counting from the 5' end of the sense strand), and two inter-nucleotide thiophosphate linkage modifications at positions 1 and 2 on the antisense strand, and two inter-nucleotide thiophosphate linkage modifications at positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains a 5'-deoxy-5'-C-malonyl group and a targeting ligand. In some embodiments, the 5'-deoxy-5'-C-malonyl group is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0416] In one particular embodiment, the RNAi agent of the present invention comprises: (a) A chain of justice, which has: (i) The length of 21 nucleotides; (ii) an ASGPR ligand attached to the 3'-end, wherein the ASGPR ligand comprises three GalNAc derivatives linked via a trivalent branching linker; and (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19 and 21 and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14 to 16, 18 and 20 (starting from the 5' end); as well as (b) An antisense chain, which has: (i) The length of 23 nucleotides; (ii) the 2'-OMe modifications at positions 1, 3, 5, 9, 11 to 13, 15, 17, 19, 21 and 23, and the 2'F modifications (counting from the 5' end) at positions 2, 4, 6 to 8, 10, 14, 16, 18, 20 and 22; and (iii) Phosphothiophosphate nucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23 (starting from the 5' end); wherein the dsRNA agent has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0417] In another particular embodiment, the RNAi agent of the present invention comprises: (a) A chain of justice, which has: (i) The length of 21 nucleotides; (ii) An ASGPR ligand connected to the 3'-end, wherein the ASGPR ligand comprises three GalNAc derivatives connected by a trivalent branching linker. (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 15, 17, 19 and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18 and 20 (counting from the 5' end); and (iv) Phosphothiophosphate nucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) An antisense chain, which has: (i) The length of 23 nucleotides; (ii) the 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19 and 21 to 23, and the 2'F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18 and 20 (counting from the 5' end); and (iii) Phosphophosphate nucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent described herein has two nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0418] In another particular embodiment, the RNAi agent of the present invention comprises: (a) A chain of justice, which has: (i) The length of 21 nucleotides; (ii) An ASGPR ligand connected to the 3'-end, wherein the ASGPR ligand comprises three GalNAc derivatives connected by a trivalent branching linker. (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21; 2'-F modifications at po...

Claims

1. A double-stranded RNA (dsRNA) agent for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells, wherein 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 mRNA encoding AASS, comprising at least 15 consecutive nucleotides differing from any antisense sequence listed in any of Tables 3-6 by no more than 3 nucleotides.

2. The dsRNA agent according to claim 1, wherein the dsRNA agent comprises at least one modified nucleotide.

3. The dsRNA agent according to claim 1 or 2, wherein substantially all nucleotides of the sense strand contain modifications.

4. The dsRNA agent according to claim 1 or 2, wherein substantially all nucleotides of the antisense strand contain modifications.

5. The dsRNA agent according to claim 1 or 2, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand contain modifications.

6. A double-stranded RNA (dsRNA) agent for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells, wherein the double-stranded RNA agent comprises a sense strand and an antisense strand forming a double-stranded region. The sense strand comprises at least 15 consecutive nucleotides that differ from any sense sequence listed in any of Tables 3-6 by no more than 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from any antisense sequence listed in any of Tables 3-6 by no more than 3 nucleotides. The positive strand and the antisense strand are substantially all modified nucleotides, wherein the positive strand is conjugated to a linked ligand at the 3' end.

7. The dsRNA agent of claim 6, wherein all nucleotides of the sense strand contain modifications.

8. The dsRNA agent according to claim 6, wherein all nucleotides of the antisense strand are modified.

9. The dsRNA agent of claim 6, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand contain modifications.

10. The dsRNA agent according to any one of claims 2-9, wherein at least one of the modified nucleotides is selected from deoxynucleotides, 3′-terminal deoxythymidine (dT) nucleotides, 2′-O-methyl modified nucleotides, 2′-fluoro modified nucleotides, 2′-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, debased nucleotides, 2′-amino modified nucleotides, 2′-O-allyl modified nucleotides, 2′-C-alkyl modified nucleotides, 2′-hydroxy modified nucleotides, 2′-methoxyethyl modified nucleotides, 2′-O-alkyl modified nucleotides, morpholino nucleotides, aminophosphates, nucleotides containing non-natural bases, tetrahydropyran modified nucleotides, 1,5-dehydrated hexadiol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5′-phosphates, nucleotides containing 5′-phosphate mimics, diol modified nucleotides, and 2-O-(N-methylacetamide) modified nucleotides and combinations thereof.

11. The dsRNA agent according to claim 11, wherein the nucleotide modification is a 2'-O-methyl and / or a 2'-fluoro modification.

12. The dsRNA agent according to any one of claims 1-11, wherein the length of the complementary region is at least 17 nucleotides.

13. The dsRNA agent according to any one of claims 1-12, wherein the length of the complementary region is 19 to 30 nucleotides.

14. The dsRNA agent according to claim 13, wherein the complementary region is 19-25 nucleotides in length.

15. The dsRNA agent according to claim 14, wherein the length of the complementary region is 21 to 23 nucleotides.

16. The dsRNA agent according to any one of claims 1-15, wherein the length of each of the sense strand and the antisense strand does not exceed 30 nucleotides.

17. The dsRNA agent according to any one of claims 1-16, wherein the length of each of the sense strand and the antisense strand is independently 19-30 nucleotides.

18. The dsRNA agent of claim 17, wherein each of the sense strand and the antisense strand is independently 19-25 nucleotides in length.

19. The dsRNA agent of claim 17, wherein each of the sense strand and the antisense strand is independently 21-23 nucleotides in length.

20. The dsRNA agent according to any one of claims 1-19, wherein at least one of the sense strand or antisense strand comprises a 3' overhang having at least one nucleotide.

21. The dsRNA agent of claim 20, wherein at least one of the sense strand or antisense strand comprises a 3' overhang having at least 2 nucleotides.

22. The dsRNA agent according to any one of claims 1-5 and 10-21, wherein the dsRNA agent further comprises a ligand.

23. The dsRNA agent of claim 22, wherein the ligand is conjugated to the 3' end of the positive strand of the dsRNA agent.

24. The dsRNA agent according to claim 6 or 23, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

25. The dsRNA agent according to claim 24, wherein the ligand is 26. The dsRNA agent of claim 25, wherein the dsRNA agent is conjugated to the ligand as illustrated below. Furthermore, X is either O or S.

27. The dsRNA agent according to claim 26, wherein X is O.

28. The dsRNA agent of claim 1, wherein the complementary region comprises any one of the antisense sequences of any one of Tables 3-6.

29. A double-stranded RNA (dsRNA) agent for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region. The sense strand comprises at least 15 consecutive nucleotides that differ from any sense sequence listed in any of Tables 3-6 by no more than 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from any sense sequence listed in any of Tables 3-6 by no more than 3 nucleotides. The positive strand of the nucleotide chain contains modifications selected from 2'-O-methyl and 2'-fluoro modifications. The positive strand contains two phosphate thioester nucleotides linked at the 5' end. The antisense strand contains substantially all nucleotides selected from 2'-O-methyl and 2'-fluoro modifications. The antisense strand contains two phosphate-thioester nucleotides linked at the 5' end and two phosphate-thioester nucleotides linked at the 3' end, and The justice chain is fused at the 3'-end with one or more GalNAc derivatives connected by a monovalent, divalent, or trivalent branch connector.

30. The dsRNA agent of claim 29, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

31. The dsRNA agent according to claim 29 or 30, wherein the dsRNA agent comprises any one of the antisense sequences listed in any one of Tables 3-6.

32. The dsRNA agent according to any one of claims 1-31, wherein the sense strand and the antisense strand comprise nucleotide sequences selected from any of the dsRNA agents listed in any one of Tables 3-6.

33. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence of any dsRNA agent of any one of Tables 3-6, and the antisense strand comprises the nucleotide sequence of any dsRNA agent of any one of Tables 3-6. The dsRNA agent is conjugated with a ligand, wherein substantially all nucleotides of the sense strand and substantially all or all nucleotides of the antisense strand are modified nucleotides.

34. The dsRNA agent according to any one of claims 1-33, wherein the dsRNA agent is selected from AD-2320882.1, AD-2320938.1, AD-2321352.1, AD-2321573.1, AD-2321665.1, AD-2321839.1, AD-2321863.1, AD-2322178.1, AD-2322257.1, AD-2322371.1, AD-2322980.1, AD-2323248.1, AD-2323253.1, AD-2323445.1, AD-2323482.1, AD-2323584.1, AD -2323585.1, AD-2323784.1, AD-2323856.1, AD-2323858.1, AD-2323886.1, AD-2323961.1, AD-2323965.1, AD-2323970.1, AD-2323972.1, AD-2323976 .

1. AD-2324043.1, AD-2324044.1, AD-2324046.1, AD-2324047.1, AD-2324276.1, AD-2324670.1, AD-2325059.1, AD-2325148.1, AD-2325356.1, AD-23 25357.1, AD-2325369.1, AD-2325373.1, AD-2325448.1, AD-2325450.1, AD -2325473.1, AD-2325474.1, AD-2325477.1, AD-2325546.1, AD-2325552.1, AD-2325646.1, AD-2325648.1, AD-2325674.1, AD-2325677.1, AD-2325678.1, AD-2325679.1, AD-2325681.1, AD-2325744.1, AD-2325745.1, AD-23257 46.1, AD-2325748.1, AD-2325752.1, AD-2325762.1, AD-2325763.1, AD-2325764.1, AD-2325765.1, AD-2325769.1, AD-2325780.1, AD-2325781.1, AD- 2325782.1, AD-2325784.1, AD-2325785.1, AD-2325786.1, AD-2325837.1, AD-2325838.1, AD-2325839.1, AD-2325840.1, AD-2325847.1, AD-2325848.

1. AD-2325955.1, AD-2325959.1, AD-2325968.1, AD-2325969.1, AD-2325973.1, AD-2325976.1, AD-2325979.1, AD-2325984.1, AD-2326042.1, AD-2326067.1, AD-2326170.1, AD-2328140.1, AD-2328186.1, AD-2328257.1, AD-2328266.1 and AD-2328843.

1.

35. The dsRNA according to any one of claims 1-34, wherein the dsRNA agent targets a hotspot region of the mRNA encoding AASS.

36. A dsRNA agent that targets hotspot regions of aminoadiazine semialdehyde synthase (AASS) mRNA.

37. A cell containing a dsRNA agent according to any one of claims 1-36.

38. A vector encoding at least one of the sense or antisense strands of the dsRNA agent according to any one of claims 1-36.

39. A pharmaceutical composition for inhibiting the expression of the aminoadiazine semialdehyde synthase (AASS) gene, said pharmaceutical composition comprising a dsRNA agent according to any one of claims 1-36.

40. The pharmaceutical composition of claim 39, wherein the dsRNA agent is formulated in a non-buffered solution.

41. The pharmaceutical composition according to claim 40, wherein the non-buffered solution is saline or water.

42. The pharmaceutical composition of claim 39, wherein the dsRNA agent is formulated with a buffer solution.

43. The pharmaceutical composition of claim 42, wherein the buffer solution comprises acetate, citrate, prolyl, carbonate, or phosphate, or any combination thereof.

44. The pharmaceutical composition according to claim 42, wherein the buffer solution is phosphate-buffered saline (PBS).

45. A method for inhibiting the expression of aminoadiazine semialdehyde synthase (AASS) in cells, the method comprising contacting the cells with a dsRNA agent according to any one of claims 1-36 or a pharmaceutical composition according to any one of claims 39-44, thereby inhibiting the expression of AASS in the cells.

46. ​​The method of claim 45, wherein the cells are in the subject.

47. The method of claim 46, wherein the subject is a human being.

48. The method according to any one of claims 45-47, wherein the AASS expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or inhibited to below the detection limit of AASS expression.

49. The method of claim 48, wherein the human subject suffers from a disease, condition, or illness associated with AASS.

50. The method of claim 49, wherein the disease, condition or symptom associated with AASS is a lysine catabolism disorder.

51. The method of claim 50, wherein the lysine catabolism disorder is type 1 glutaric aciduria (GA1).

52. The method of claim 50, wherein the lysine catabolism disorder is pyridoxine-dependent epilepsy (PDE).

53. A method for inhibiting the expression of AASS in a subject, the method comprising administering to the subject a therapeutically effective amount of a dsRNA agent according to any one of claims 1-36 or a pharmaceutical composition according to any one of claims 39-44, thereby inhibiting the expression of AASS in the subject.

54. A method of treating a subject suffering from an AASS-related disease, condition, or illness, the method comprising administering to the subject a therapeutically effective amount of a dsRNA agent according to any one of claims 1-36 or a pharmaceutical composition according to any one of claims 39-44, thereby treating the subject suffering from an AASS-related disease, condition, or illness.

55. A method for preventing at least one symptom in a subject suffering from a disease, condition, or illness that would benefit from a reduction in AASS gene expression, the method comprising administering to the subject a preventatively effective amount of a dsRNA agent according to any one of claims 1-36 or a pharmaceutical composition according to any one of claims 39-44, thereby preventing at least one symptom in a subject suffering from a disease, condition, or illness that would benefit from a reduction in AASS gene expression.

56. The method according to any one of claims 53-55, wherein the disease, condition or symptom associated with AASS is a lysine catabolism disorder.

57. The method of claim 56, wherein the lysine catabolism disorder is type 1 glutaric aciduria (GA1).

58. The method of claim 57, wherein the lysine catabolism disorder is pyridoxine-dependent epilepsy (PDE).

59. The method according to any one of claims 46-58, further comprising administering an additional therapeutic agent to the subject.

60. The method according to any one of claims 46-59, wherein the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg.

61. The method according to any one of claims 46-60, wherein the dsRNA agent is administered intravenously, intramuscularly, or subcutaneously to the subject.

62. The method according to any one of claims 46-61, the method further comprising measuring the level of AASS in the subject.

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