METHODS FOR THE TREATMENT OF TRINUCLEOTIDE REPEAT EXPANSION DISORDERS ASSOCIATED WITH MLH1 ACTIVITY

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The contents of the text file named “4398_005 PC03_Seqlisting_ST25.txt,” which was created on Nov. 24, 2019 and is 694,786 bytes in size, are hereby incorporated by reference in their entireties.

BACKGROUND

Trinucleotide repeat expansion disorders are genetic disorders caused by trinucleotide repeat expansions. Trinucleotide repeat expansions are a type of genetic mutation where nucleotide repeats in certain genes or introns exceed the normal, stable threshold for that gene. The trinucleotide repeats can result in defective or toxic gene products, impair RNA transcription, and/or cause toxic effects by forming toxic mRNA transcripts.

Trinucleotide repeat expansion disorders are generally categorized by the type of repeat expansion. For example, Type 1 disorders such as Huntington's disease are caused by CAG repeats which result in a series of glutamine residues known as a polyglutamine tract, Type 2 disorders are caused by heterogeneous expansions that are generally small in magnitude, and Type 3 disorders such as fragile X syndrome are characterized by large repeat expansions that are generally located outside of the protein coding region of the genes. Trinucleotide repeat expansion disorders are characterized by a wide variety of symptoms such as progressive degeneration of nerve cells that is common in the Type 1 disorders.

Subjects with a trinucleotide repeat expansion disorder or those who are considered at risk for developing a trinucleotide repeat expansion disorder have a constitutive nucleotide expansion in a gene associated with disease (i.e., the trinucleotide repeat expansion is present in the gene during embryogenesis). Constitutive trinucleotide repeat expansions can undergo expansion after embryogenesis (i.e., somatic trinucleotide repeat expansion). Both constitutive trinucleotide repeat expansion and somatic trinucleotide repeat expansion can be associated with presence of disease, age at onset of disease, and/or rate of progression of disease.

SUMMARY OF THE DISCLOSURE

The present disclosure features useful compositions and methods to treat trinucleotide repeat expansion disorders, e.g., in a subject in need thereof. In some aspects, the compositions and methods described herein are useful in the treatment of disorders associated with MLH1 activity. Such compositions include administering an antisense oligonucleotide (“ASO”) or a dsRNA (e.g., siRNA or shRNA).

Antisense Oligonucleotides

In some aspects, a single-stranded antisense oligonucleotide of 10-30 linked nucleosides in length, wherein the antisense oligonucleotide comprises a region of at least 10 contiguous nucleobases having at least 80% complementarity to an MLH1 gene are provided. In some aspects, the antisense oligonucleotide comprises:

(a) a DNA core sequence comprising linked deoxyribonucleosides;

(b) a 5′ flanking sequence comprising linked nucleosides; and

wherein the DNA core comprises a region of at least 10 contiguous nucleobases having at least 80% complementarity to an MLH1 gene and is positioned between the 5′ flanking sequence and the 3′ flanking sequence; wherein the 5′ flanking sequence and the 3′ flanking sequence each comprises at least two linked nucleosides; and wherein at least one nucleoside of each flanking sequence comprises an alternative nucleoside.

In some aspects, a single-stranded antisense oligonucleotide of 10-30 linked nucleosides in length for inhibiting expression of a human MLH1 gene in a cell, wherein the antisense oligonucleotide comprises a region of at least 10 contiguous nucleobases having at least 80% complementarity to an MLH1 gene is provided herein. In some aspects, the antisense oligonucleotide comprises:

(a) a DNA core comprising linked deoxyribonucleosides;

(b) a 5′ flanking sequence comprising linked nucleosides; and

In some aspects, the region of at least 10 nucleobases has at least 90% complementary to an MLH1 gene. In some aspects, the region of at least 10 nucleobases has at least 95% complementary to an MLH1 gene.

In some aspects, the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 193-258, 289-607, 629-734, 757-836, 865-1125, 1177-1206, 1218-1286, 1324-1408, 1433-1747, 1759-1814, 1852-1901, 1959-2029, 2053-2240, 2250-2356, 2382-2479, 2510-2546, or 2573-2598 of the MLH1 gene. In some aspects, the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 193-251, 289-607, 629-734, 757-836, 865-1125, 1177-1206, 1218-286, 1324-1408, 1433-1747, 1759-1814, 1852-1901, 1959-2029, 2053-2240, 2250-2356, 2382-2479, 2510-2546, or 2573-2598 of the MLH1 gene. In some aspects, the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 193-251, 289-607, 629-734, 758-836, 865-1125, 1177-1206, 1218-1286, 1324-1408, 1433-1747, 1759-1814, 1852-1901, 1959-2029, 2053-2240, 2250-2356, 2382-2479, 2510-2546, or 2573-2598 of the MLH1 gene. In some aspects, the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 312-391, 410-508, 522-607, 629-726, 759-1125, 1177-1206, 1221-1286, 1324-1407, 1433-1747, 1764-1814, 1854-1901, 1959-2029, 2053-2113, 2184-2240, 2251-2283, 2303-2351, 2384-2479, or 2510-2546 of the MLH1 gene. In some aspects, the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 575-602, 662-724, 805-830, 891-960, 1002-1027, 1056-1081, 1100-1125, 1342-1384, 1443-1498, 1513-1561, 1600-1625, 1652-1747, 1876-1901, 2001-2026, or 2430-2459 of the MLH1 gene. In some aspects, the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 307-332, 458-500, 571-602, 758-787, 865-890, 892-917, 1045-1084, 1624-1649, 1786-1813, 1871-1901, 2053-2081, 2086-2114, or 2149-2176 of the MLH1 gene.

In some aspects, the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 6-1393. In some aspects, the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 81-84, 86-87, 90, 99-101, 106-107, 111, 113-114, 117, 122-126, 129-131, 137-138, 140, 144, 146-160, 172, 188-191, 211, 215-220, 222-226, 229, 231-239, 242-249, 270-271, 274-279, 286-293, 295-298, 310-320, 322-328, 332-335, 337, 345, 383-388, 397-402, 405-413, 415-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-598, 600-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699, 701-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 1118-1123, 1131-1134, 1136, 1138-1139, 1140-1144, 1151-1152, 1161-1163, 1187-1192, 1194-1195, 1197, 1199-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, or 1343. In some aspects, the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 81-84, 86-87, 99-101, 106, 113-114, 122-126, 129-131, 137-138, 140, 144, 146-159, 172, 188-191, 211, 215-217, 219, 223-226, 229, 232-239, 242-249, 270-271, 274-279, 286-293, 295-298, 310-320, 322-328, 332-335, 337, 345, 384-388, 397-402, 405-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 1118-1123, 1131-1134, 1136, 1138-1144, 1151-1152, 1161-1163, 1187-1191, 1194-1195, 1197, 1199-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, or 1343. In some aspects, the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 81-87, 99-101, 106, 113-114, 122-126, 129-131, 137-138, 140, 144, 146, 147, 148-151, 153-159, 172, 188-191, 211215-217, 219, 223-226, 229, 232-239, 242-245, 248-249, 270-271274-276, 278-279, 286-293, 295-298, 310-320, 322-338, 332-335, 337, 345, 384-386, 387-388, 397-402, 405-413, 415-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-598, 600-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699, 701-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 1118-1123, 1131-1134, 1136, 1138-1144, 1151-1152, 1161-1163, 1187-1191, 1194-1195, 1197, 1199, 1200-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, or 1343. In some aspects, the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 122, 123, 125-126, 129-130, 131, 137, 146-147, 153-156, 158, 188-191, 211, 216, 223, 226, 235, 237, 245, 248, 270-271, 276, 278-279, 286, 289-293, 297-298, 310, 312-320, 323-328, 332, 334-335, 337, 385-386, 397-402, 407, 410-412, 415-417, 419-420, 476, 478, 482-487, 490-491, 493-494, 497-501, 522-523, 525-526, 528-530, 546-548, 557, 563-565, 586, 603, 605-610, 613, 619-622, 631-632, 634, 639-641, 645-646, 650-651, 655, 699, 701, 704-709, 714, 729-731, 733-734, 736-740, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 847, 850, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 928-931, 936-939, 977, 981-986, 1019, 1024-1026, 1034, 1036-1041, 1083-1085, 1087, 1111, 1132-1134, 1136, 1138-1140, 1142, 1161-1163, 1188-1190, 1194-1195, 1207, 1218, 1241, 1244, 1247, 1257-1259, 1262-1269, 1277-1278, or 1314-1315. In some aspects, the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 291-293, 313, 316-317, 325-326, 334-335, 415, 483, 485-486, 499-500, 523, 525, 564, 607, 622, 704-705, 707-709, 739, 752, 768-769, 788, 827, 861, 871-872, 877-879, 882, 885, 886, 901, 986, 1038, 1263, or 1265-1267. In some aspects, the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 117, 215-226, 229-232, 287-293, 384-385, 387-388, 458, 484, 596-610, 850, 936-938, 981-986, 1083-1086, 1109-1112, or 1121-1123.

In some aspects, the nucleobase sequence of the antisense oligonucleotide consists of any one of SEQ ID NOs: 6-1393. In some aspects, the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 81-87, 90, 99-101, 106-107, 111, 113-114, 117, 122-126, 129-131, 137-138, 140, 144, 146-160, 172, 188-191, 211, 215-220, 22-226, 229, 231-239, 242-249, 270-271, 274-279, 286-293, 295-297, 298, 310-320, 322-328, 332-335, 337, 345, 383-388, 397-402, 405-413, 415-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-598, 600-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699, 701-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 118-1123, 1131-1134, 1136, 1138-1144, 1151-1152, 1161-1163, 1187-1192, 1194-1195, 1197, 1199-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1222, 1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, or 1343. In some aspects, the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 81-84, 86-87, 99-101, 106, 113-114, 122-126, 129-131, 137-138, 140, 144, 146-159, 172, 188-191, 211, 215-217, 219, 223-226, 229, 232-239, 242-249, 270-271, 274-279, 286-293, 295-298, 310-320, 322-328, 332-335, 337, 345, 384-388, 397-402, 405-413, 415-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-598, 600-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699, 701-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 1118-1123, 1131-1134, 1136, 1138-1144, 1151-1152, 1161-1163, 1187-1191, 1194-1195, 1197, 1199-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, or 1343. In some aspects, the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 81-84, 86-87, 99-101, 106, 113-114, 122-126, 129-131, 137-138, 140, 144, 146-151, 153-159, 172, 188-191, 211, 215-217, 219223-226, 229, 232-239, 242-245, 248-249, 270-271, 274-276, 278-279, 286-293, 295-298, 310-320, 322-328, 332-335, 337, 345, 384-388, 397-402, 405-413, 415-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-525, 526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-598, 600-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699, 701-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 1118-1123, 1131-1134, 1136, 1138-1144, 1151-1152, 1161-1163, 1187-1191, 1194-1195, 1197, 1199-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, or 1343. In some aspects, the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 122-123, 125-126, 129-131, 137, 146-147, 153-156, 158, 188-191, 211, 216, 223, 226, 235, 237, 245, 248, 270-271, 276, 278-279, 286, 289-293, 297-298, 310, 312-320, 323-328, 332, 334-335, 337, 385-386, 397-402, 407, 410-412, 415-417, 419-420, 476, 478, 482-487, 490-491, 493-494, 497-501, 522-523, 525-526, 528-530, 546-548, 557, 563-565, 586, 603, 605-610, 613, 619-622, 631-632, 634, 639-641, 645-646, 650-651, 655, 699, 701, 704-709, 714, 729-731, 733-734, 736-740, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 847, 850, 859, 861, 866-867, 868-872, 877-882, 884-887, 889-890, 893-894, 901, 928-931, 936-939, 977, 981-986, 1019, 1024-1026, 1034, 1036-1041, 1083-1085, 1087, 1111, 1132-1134, 1136, 1138-1140, 1142, 1161-1163, 1188-1190, 1194-1195, 1207, 1218, 1241, 1244, 1247, 1257-1259, 1262-1269, 1277-1278, or 1314-1315. In some aspects, the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 291-293, 313, 316-317, 325-326, 334-335, 415, 483, 485-486, 499-500, 523, 525, 564, 607, 622, 704-705, 707-709, 739, 752, 768-769, 788, 827, 861, 871-872, 877-879, 882, 885, 886, 901, 986, 1038, 1263, or 1265-1267. In some aspects, the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 117, 215-226, 229-232, 287-293, 384-385, 387-388, 458-484, 596-610, 850, 936-938, 981-986, 1083-1086, 1109-1112 or 1121-1123.

In some aspects, the antisense oligonucleotide exhibits at least 50% mRNA inhibition at a 20 nM oligonucleotide concentration when determined using a cell assay when compared with a control cell. In some aspects, the antisense oligonucleotide exhibits at least 60% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell. In some aspects, the antisense oligonucleotide exhibits at least 70% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell. In some aspects, the antisense oligonucleotide exhibits at least 85% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell. In some aspects, the antisense oligonucleotide exhibits at least 50% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell. In some aspects, the antisense oligonucleotide exhibits at least 60% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell. In some aspects, the antisense oligonucleotide exhibits at least 70% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell. In some aspects, the antisense oligonucleotide exhibits at least 85% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell. The cell assay can comprise transfecting a mammalian cell, such as HEK293, NIH3T3, or HeLa, with oligonucleotides using Lipofectamine 2000 (Invitrogen) and measuring mRNA levels compared to a mammalian cell transfected with a mock oligonucleotide.

In some aspects, he antisense oligonucleotide comprises at least one alternative internucleoside linkage. In some aspects, the at least one alternative internucleoside linkage is a phosphorothioate internucleoside linkage. In some aspects, the at least one alternative internucleoside linkage is a 2′-alkoxy internucleoside linkage. In some aspects, the at least one alternative internucleoside linkage is an alkyl phosphate internucleoside linkage.

In some aspects, the antisense oligonucleotide comprises at least one alternative nucleobase. In some aspects, the alternative nucleobase is 5′-methylcytosine, pseudouridine, or 5-methoxyuridine.

In some aspects, the antisense oligonucleotide comprises at least one alternative sugar moiety. In some aspects, the alternative sugar moiety is 2′-OMe or a bicyclic nucleic acid.

In some aspects, the antisense oligonucleotide further comprises a ligand conjugated to the 5′ end or the 3′ end of the antisense oligonucleotide through a monovalent or branched bivalent or trivalent linker.

In some aspects, the antisense oligonucleotide comprises a region complementary to at least 17 contiguous nucleotides of a MLH1 gene. In some aspects, the antisense oligonucleotide comprises a region complementary to at least 19 contiguous nucleotides of a MLH1 gene. In some aspects, the antisense oligonucleotide comprises a region complementary to 19 to 23 contiguous nucleotides of a MLH1 gene. In some aspects, the antisense oligonucleotide comprises a region complementary to 19 contiguous nucleotides of a MLH1 gene. In some aspects, the antisense oligonucleotide comprises a region complementary to 20 contiguous nucleotides of a MLH1 gene. In some aspects, the antisense oligonucleotide is from about 15 to 25 nucleosides in length. In some aspects, the antisense oligonucleotide is 20 nucleosides in length.

Pharmaceutical Compositions and Methods of Treatment Using Antisense Oligonucleotides

In some aspects, the application is directed to a pharmaceutical composition comprising one or more of the antisense oligonucleotides described herein and a pharmaceutically acceptable carrier or excipient. In some aspects, the application is directed to a composition comprising one or more of the antisense oligonucleotides described herein and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome.

In some aspects, the application is directed to a method of inhibiting transcription of MLH1 in a cell, the method comprising contacting the cell with: one or more of the oligonucleotides described herein; a pharmaceutical composition of one or more of the oligonucleotides described herein and a pharmaceutically acceptable carrier or excipient; or the composition of one or more of the oligonucleotides described herein and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle or a liposome; for a time sufficient to obtain degradation of an mRNA transcript of a MLH1 gene, inhibiting expression of the MLH1 gene in the cell.

In some aspects, the application is directed to a method of treating, preventing, or delaying the progression a trinucleotide repeat expansion disorder in a subject in need thereof, the method comprising administering to the subject: one or more of the oligonucleotides described herein; the pharmaceutical composition of one or more oligonucleotides described herein and a pharmaceutically acceptable carrier or excipient; or the composition of one or more of the oligonucleotides described herein and a lipid nanoparticle, polyplex nanoparticle, a lipoplex nanoparticle, or a liposome.

In some aspects, the application is directed to a method of reducing the level and/or activity of MLH1 in a cell of a subject identified as having a trinucleotide repeat expansion disorder, the method comprising contacting the cell with: one or more of the oligonucleotides described herein; the pharmaceutical composition of one or more oligonucleotides described herein and a pharmaceutically acceptable carrier or excipient; or the composition of one or more of the oligonucleotides described herein and a lipid nanoparticle, polyplex nanoparticle, a lipoplex nanoparticle, or a liposome.

In some aspects, the application is directed to a method for inhibiting expression of an MLH1 gene in a cell comprising contacting the cell with: one or more of the oligonucleotides described herein; the pharmaceutical composition of one or more oligonucleotides described herein and a pharmaceutically acceptable carrier or excipient; or the composition of one or more of the oligonucleotides described herein and a lipid nanoparticle, polyplex nanoparticle, a lipoplex nanoparticle, or a liposome, and maintaining the cell for a time sufficient to obtain degradation of a mRNA transcript of an MLH1 gene, thereby inhibiting expression of the MLH1 gene in the cell.

In some aspects, the application is directed to a method of decreasing trinucleotide repeat expansion in a cell, the method comprising contacting the cell with: one or more of the oligonucleotides described herein; the pharmaceutical composition of one or more oligonucleotides described herein and a pharmaceutically acceptable carrier or excipient; or the composition of one or more of the oligonucleotides described herein and a lipid nanoparticle, polyplex nanoparticle, a lipoplex nanoparticle, or a liposome.

In some aspects, a method of treating, preventing, or delaying the progression a disorder in a subject in need thereof wherein the subject is suffering from trinucleotide repeat expansion disorder, comprising administering to said subject the oligonucleotide described herein. In some aspects, the method further comprises administering a second therapeutic agent. In some aspects, the second therapeutic agent is a second oligonucleotide that hybridizes to an mRNA encoding the Huntingtin gene.

In some aspects, a method of preventing or delaying the progression of a trinucleotide repeat expansion disorder in a subject, the method comprising administering to the subject an oligonucleotide in an amount effective to delay progression of a trinucleotide repeat expansion disorder of the subject.

In some aspects, the cell is in a subject. In some aspects, the subject is a human. In some aspects, the cell is a cell of the central nervous system or a muscle cell.

In some aspects, the subject is identified as having a trinucleotide repeat expansion disorder. In some aspects, the trinucleotide repeat expansion disorder is a polyglutamine disease. In some aspects the polyglutamine disease is selected from the group consisting of dentatorubropallidoluysian atrophy, Huntington's disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 17, or Huntington's disease-like 2. In some aspects, the trinucleotide repeat expansion disorder is Huntington's disease.

In some aspects, the trinucleotide repeat expansion disorder is a non-polyglutamine disease. IN some aspects, the non-polyglutamine disease is selected from the group consisting of fragile X syndrome, fragile X-associated tremor/ataxia syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy type 1, spinocerebellar ataxia type 8, spinocerebellar ataxia type 12, oculopharyngeal muscular dystrophy, Fragile X-associated premature ovarian failure, FRA2A syndrome, FRA7A syndrome, or early infantile epileptic encephalopathy. In some aspects, the trinucleotide repeat expansion disorder is Friedreich's ataxia. In some aspects, the trinucleotide repeat expansion disorder is myotonic dystrophy type 1.

In some aspects, the antisense oligonucleotide, pharmaceutical composition, or composition is administered intrathecally. In some aspects, the antisense oligonucleotide, pharmaceutical composition, or composition is administered intraventricularly. In some aspects, the antisense oligonucleotide, pharmaceutical composition, or composition is administered intramuscularly.

In some aspects, the progression of the trinucleotide repeat expansion disorder is delayed by at least 120 days, for example, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years or more, when compared with the predicted progression.

dsRNAs

In some aspects, a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand is complementary to at least 15 contiguous nucleobases of an MLH1 gene, and wherein the dsRNA comprises a duplex structure of between 15 and 30 linked nucleosides in length is provided herein.

In some aspects, a dsRNA for reducing expression of MLH1 in a cell, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand is complementary to at least 15 contiguous nucleobases of an MLH1 gene, and wherein the dsRNA comprises a duplex structure of between 15 and 30 linked nucleosides in length is provided herein.

In some aspects, the dsRNA comprises a duplex structure of between 19 and 23 linked nucleosides in length.

In some aspects, the dsRNA further comprises a loop region joining the sense strand and antisense strand, wherein the loop region is characterized by a lack of base pairing between nucleobases within the loop region.

In some aspects, the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MLH1 gene corresponding to reference mRNA NM_000249.3 at one or more of positions 153-176, 267-388, 417-545, 792-995, 1639-1727, 1849-1900, 2105-2207, 2337-2387, 2426-2479 and 2508-2600 of the MLH1 gene. IN some aspects, the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MLH1 gene corresponding to reference mRNA NM_000249.3 at one or more of positions 326-388, 459-511, 805-878, 903-926, 1639-1720, and 2141-2192 of the MLH1 gene. In some aspects, the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MLH1 gene corresponding to reference mRNA NM_000249.3 at one or more of positions 267-388, 417-545, 805-878, 903-995, 1639-1727, 1849-1900, 2141-2207, 2337-2387, and 2426-2479 of the MLH1 gene. In some aspects, the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MLH1 gene corresponding to reference mRNA NM_000249.3 at one or more of positions 153-176, 267-388, 417-545, 792-995, 1639-1727, 1849-1900, 2105-2207, 2337-2387, and 2426-2479 of the MLH1 gene. In some aspects, the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MLH1 gene corresponding to reference mRNA NM_000249.3 at one or more of positions 332-355, 459-545, 836-859, 1849-1900, 2141-2164, and 2426-2449 of the MLH1 gene. In some aspects, the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MLH1 gene corresponding to reference mRNA NM_000249.3 at one or more of positions 267-388, 417-545, 805-995, 1639-1722, 1849-1900, 2105-2207, 2337-2387, 2426-2479, and 2508-2600 of the MLH1 gene.

In some aspects, the antisense strand comprises an antisense nucleobase sequence selected from a list in Table 4, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T), and the sense strand comprises a sense nucleobase sequence complementary to the antisense nucleobase sequence. In some aspects, the antisense nucleobase sequence consists of an antisense strand in Table 4, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T), and the sense nucleobase sequence consists of a sequence complementary to the antisense nucleobase sequence. In some aspects, the sense strand comprises a sense nucleobase sequence selected from a list in Table 4, and the antisense strand comprises an antisense nucleobase sequence complementary to the sense nucleobase sequence. In some aspects, the sense nucleobase sequence consists of a sense sequence in Table 4, and the antisense nucleobase sequence consists of a sequence complementary to the sense nucleobase sequence.

In some aspects, the sense strand comprises a sense nucleobase sequence selected from any one of the lists in Tables 5-11, and the antisense strand comprises an antisense nucleobase sequence complementary to the sense nucleobase sequence. In some aspects, the sense nucleobase sequence consists of a sense sequence in any one of Tables 5-11, and the antisense nucleobase sequence consists of a sequence complementary to the sense nucleobase sequence.

In some aspects, the antisense strand comprises an antisense nucleobase sequence selected from a list in Table 13, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T), and the sense strand comprises a sense nucleobase sequence complementary to the antisense nucleobase sequence. In some aspects, the antisense nucleobase sequence consists of an antisense sequence in Table 13, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T), and the sense nucleobase sequence consists of a sequence complementary to the antisense nucleobase sequence. In some aspects, the sense strand comprises a sense nucleobase sequence selected from a list in Table 13, and the antisense strand comprises an antisense nucleobase sequence complementary to the sense nucleobase sequence. In some aspects, the sense nucleobase sequence consists of a sense sequence in Table 13, and the antisense nucleobase sequence consists of a sequence complementary to the sense nucleobase sequence.

In some aspects, the dsRNA comprises at least one alternative nucleobase, at least one alternative internucleoside linkage, and/or at least one alternative sugar moiety. In some aspects, the at least one alternative internucleoside linkage is a phosphorothioate internucleoside linkage. In some aspects, the at least one alternative internucleoside linkage is a 2′-alkoxy internucleoside linkage. In some aspects, the at least one alternative internucleoside linkage is an alkyl phosphate internucleoside linkage.

In some aspects, the at least one alternative nucleobase is 5′-methylcytosine, pseudouridine, or 5-methoxyuridine. In some aspects, the alternative sugar moiety is 2′-OMe or a bicyclic nucleic acid. In some aspects, the dsRNA comprises at least one 2′-OMe sugar moiety and at least one phosphorothioate internucleoside linkage.

In some aspects, the dsRNA further comprises a ligand conjugated to the 3′ end of the sense strand through a monovalent or branched bivalent or trivalent linker.

In some aspects, the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1486, 1492, 1494, 1496, 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, 3164, 3210, 3212, 3216, 3248, 3272, 3274, 3276, 3278, and 3288. In some aspects, the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, and 2966. In some aspects, the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, and 3164. In some aspects, the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1486, 1492, 1494, 1496, 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, and 3164. In some aspects, the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1584, 1598, 1604, 1608, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1998, 2008, 2012, 2024, 2720, 2744, 2746, 2748, 2752, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 3120, 3122, 3124, 3130, 3134, 3136, and 3148. In some aspects, the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, 3164, 3210, 3212, 3216, 3248, 3272, 3274, 3276, 3278, and 3288.

In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1487, 1493, 1495, 1497, 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, 3165, 3211, 3213, 3217, 3249, 3273, 3275, 3277, 3279, and 3289, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, and 2967, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, and 3165, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1487, 1493, 1495, 1497, 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, and 3165, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1585, 1599, 1605, 1609, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1999, 2009, 2013, 2025, 2721, 2745, 2747, 2749, 2753, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 3121, 3123, 3125, 3131, 3135, 3137, and 3149, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, 3165, 3211, 3213, 3217, 3249, 3273, 3275, 3277, 3279, and 3289, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1486, 1492, 1494, 1496, 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, 3164, 3210, 3212, 3216, 3248, 3272, 3274, 3276, 3278, and 3288. In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, and 2966. In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, and 3164. In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1486, 1492, 1494, 1496, 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, and 3164. In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1584, 1598, 1604, 1608, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1998, 2008, 2012, 2024, 2720, 2744, 2746, 2748, 2752, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 3120, 3122, 3124, 3130, 3134, 3136, and 3148. In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, 3164, 3210, 3212, 3216, 3248, 3272, 3274, 3276, 3278, and 3288.

In some aspects, the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1487, 1493, 1495, 1497, 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, 3165, 3211, 3213, 3217, 3249, 3273, 3275, 3277, 3279, and 3289, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, and 2967, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, and 3165, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1487, 1493, 1495, 1497, 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, and 3165, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1585, 1599, 1605, 1609, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1999, 2009, 2013, 2025, 2721, 2745, 2747, 2749, 2753, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 3121, 3123, 3125, 3131, 3135, 3137, and 3149, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, 3165, 3211, 3213, 3217, 3249, 3273, 3275, 3277, 3279, and 3289, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

In some aspects, the dsRNA exhibits at least 50% mRNA inhibition at a 0.5 nM dsRNA concentration when determined using a cell assay when compared with a control cell. In some aspects, the dsRNA exhibits at least 40% mRNA inhibition at a 0.5 nM dsRNA concentration when determined using a cell assay when compared with a control cell. In some aspects, the dsRNA exhibits at least 30% mRNA inhibition at a 0.5 nM dsRNA concentration when determined using a cell assay when compared with a control cell. In some aspects, the dsRNA exhibits at least 60% mRNA inhibition at a 10 nM dsRNA concentration when determined using a cell assay when compared with a control cell. In some aspects, the dsRNA exhibits at least 50% mRNA inhibition at a 10 nM dsRNA concentration when determined using a cell assay when compared with a control cell.

In some aspects, the antisense strand is complementary to at least 17 contiguous nucleotides of an MLH1 gene. In some aspects, the antisense strand is complementary to at least 19 contiguous nucleotides of an MLH1 gene. In some aspects, the antisense strand is complementary to 19 contiguous nucleotides of an MLH1 gene.

In some aspects, the antisense strand and/or the sense strand comprises a 3′ overhang of at least 1 linked nucleoside; or a 3′ overhang of at least 2 linked nucleosides.

Pharmaceutical Compositions and Methods of Treatment Using dsRNAs

In some aspects, the application is directed to a pharmaceutical composition comprising one or more of the dsRNAs described herein and a pharmaceutically acceptable carrier or excipient. In some aspects, the application is directed to a composition comprising one or more of the antisense oligonucleotides described herein and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome. In some aspects, the application is directed to a vector encoding at least one strand of the dsRNAs described herein. In some aspects, the application is directed to a cell comprising the vector that encodes at least one strand of the dsRNAs described herein.

In some aspects, the application is directed to a method of inhibiting transcription of MLH1 in a cell, the method comprising contacting the cell with: one or more of the dsRNAs described herein; a pharmaceutical composition of one or more of the dsRNAs described herein and a pharmaceutically acceptable carrier or excipient; or the composition of one or more of the dsRNAs described herein; a vector encoding at least one strand of the dsRNAs; or a cell comprising a vector that encodes at least one strand of the dsRNAs for a time sufficient to obtain degradation of an mRNA transcript of a MLH1 gene, thereby reducing expression of the MLH1 gene in the cell.

In some aspects, the application is directed to a method of treating, preventing, or delaying progression of a trinucleotide repeat expansion disorder in a subject in need thereof, the method comprising administering to the subject one or more of the dsRNAs described herein; a pharmaceutical composition of one or more of the dsRNAs described herein and a pharmaceutically acceptable carrier or excipient; or the composition of one or more of the dsRNAs described herein; a vector encoding at least one strand of the dsRNAs; or a cell comprising a vector that encodes at least one strand of the dsRNAs.

In some aspects, the application is directed to a method of reducing the level and/or activity of MLH1 in a cell of a subject identified as having a trinucleotide repeat expansion disorder, the method comprising contacting the cell with one or more of the dsRNAs described herein; a pharmaceutical composition of one or more of the dsRNAs described herein and a pharmaceutically acceptable carrier or excipient; or the composition of one or more of the dsRNAs described herein; a vector encoding at least one strand of the dsRNAs; or a cell comprising a vector that encodes at least one strand of the dsRNAs.

In some aspects, the application is directed to a method for reducing expression of MLH1 in a cell comprising contacting the cell with one or more of the dsRNAs described herein; a pharmaceutical composition of one or more of the dsRNAs described herein and a pharmaceutically acceptable carrier or excipient; or the composition of one or more of the dsRNAs described herein; a vector encoding at least one strand of the dsRNAs; or a cell comprising a vector that encodes at least one strand of the dsRNAs and maintaining the cell for a time sufficient to obtain degradation of an mRNA transcript of MLH1, thereby reducing expression of MLH1 in the cell.

In some aspects, the application is directed to a method of decreasing trinucleotide repeat expansion in a cell, the method comprising contacting the cell with one or more of the dsRNAs described herein; a pharmaceutical composition of one or more of the dsRNAs described herein and a pharmaceutically acceptable carrier or excipient; or the composition of one or more of the dsRNAs described herein; a vector encoding at least one strand of the dsRNAs; or a cell comprising a vector that encodes at least one strand of the dsRNAs.

In some aspects, a method of treating, preventing, or delaying the progression a disorder in a subject in need thereof wherein the subject is suffering from trinucleotide repeat expansion disorder, comprising administering to said subject the dsRNA described herein. In some aspects, the method further comprises administering a second therapeutic agent. In some aspects, the second therapeutic agent is an oligonucleotide that hybridizes to an mRNA encoding the Huntingtin gene.

In some aspects, a method of preventing or delaying the progression of a trinucleotide repeat expansion disorder in a subject, the method comprising administering to the subject a dsRNA in an amount effective to delay progression of a trinucleotide repeat expansion disorder of the subject.

In some aspects, the cell is in a subject. In some aspects, the subject is a human. In some aspects, the cell is a cell of the central nervous system or a muscle cell.

In some aspects, the dsRNA, pharmaceutical composition, composition, cell, or vector is administered intrathecally. In some aspects, the dsRNA, pharmaceutical composition, composition, cell, or vector is administered intraventricularly. In some aspects, the dsRNA, pharmaceutical composition, composition, cell, or vector is administered intramuscularly.

Definitions

For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular aspects, and are not intended to limit the claimed technology, because the scope of the technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

In this application, unless otherwise clear from context, (i) the term “a” can be understood to mean “at least one”; (ii) the term “or” can be understood to mean “and/or”; and (iii) the terms “including” and “comprising” can be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps.

As used herein, the terms “about” and “approximately” refer to a value that is within 10% above or below the value being described. For example, the term “about 5 nM” indicates a range of from 4.5 to 5.5 nM.

The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 18 nucleotides of a 21-nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range. “At least” is also not limited to integers (e.g., “at least 5%” includes 5.0%, 5.1%, 5.18% without consideration of the number of significant figures.

As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, an antisense oligonucleotide with “no more than 3 mismatches to a target sequence” has 3, 2, 1, or 0 mismatches to a target sequence or a duplex with an overhang of “no more than two linked nucleosides” has a 2, 1, or 0 linked nucleoside overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.

As used herein, the term “administration” refers to the administration of a composition (e.g., a compound or a preparation that includes a compound as described herein) to a subject or system. Administration to an animal subject (e.g., to a human) can be by any appropriate route, such as one described herein.

As used herein, a “combination therapy” or “administered in combination” means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. The treatment regimen defines the doses and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some aspects, the delivery of the two or more agents is simultaneous or concurrent and the agents can be co-formulated. In some aspects, the two or more agents are not co-formulated and are administered in a sequential manner as part of a prescribed regimen. In some aspects, administration of two or more agents or treatments in combination is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, one therapeutic agent of the combination can be administered by intravenous injection while another therapeutic agent of the combination can be administered orally.

As used herein, the term “MLH1” refers to MutL Homolog 1, a DNA mismatch repair protein, having an amino acid sequence from any vertebrate or mammalian source, including, but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig, unless specified otherwise. The term also refers to fragments and variants of native MLH1 that maintain at least one in vivo or in vitro activity of a native MLH1. The term encompasses full-length unprocessed precursor forms of MLH1 as well as mature forms resulting from post-translational cleavage of the signal peptide. MLH1 is encoded by the MLH1 gene. The nucleic acid sequence of an exemplary Homo sapiens (human) MLH1 gene is set forth in NCBI Reference No. NM_000249.3 or in SEQ ID NO: 1. The term “MLH1” also refers to natural variants of the wild-type MLH1 protein, such as proteins having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% identity, or more) to the amino acid sequence of wild-type human MLH1, which is set forth in NCBI Reference No. NP_000240.1 or in SEQ ID NO: 2. The nucleic acid sequence of an exemplary Mus musculus (mouse) MLH1 gene is set forth in NCBI Reference No. NM_026810.2 or in SEQ ID NO: 3. The nucleic acid sequence of an exemplary Rattus norvegicus (rat) MLH1 gene is set forth in NCBI Reference No. NM_031053.1 or in SEQ ID NO: 4. The nucleic acid sequence of an exemplary Macaca fascicularis (cyno) MLH1 gene is set forth in NCBI Reference No. XM_005546623.2 or in SEQ ID NO: 5.

The term “MLH1” as used herein also refers to a particular polypeptide expressed in a cell by naturally occurring DNA sequence variations of the MLH1 gene, such as a single nucleotide polymorphism in the MLH1 gene. Numerous SNPs within the MLH1 gene have been identified and can be found at, for example, NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov/snp). Non-limiting examples of SNPs within the MLH1 gene can be found at, NCBI dbSNP Accession Nos.: rs748766, rs1540354, rs1558528, rs1799977, rs1800146, rs1800149, rs1800734, rs2020873, rs2241031, rs2286939, rs3774332, rs3774338, rs4234259, rs4647256, rs4647269, rs9876116, rs11129748, rs11541859, rs28930073, rs34213726, rs35001569, rs35045067, rs35502531, rs35831931, rs41295280, rs41295282, rs41295284, rs41562513, rs56198082, rs63749792, rs63750114, rs63750447, rs63750549, rs63751592, rs63751684, rs63751597, rs1803985, rs2286940, rs3774339, rs4647225, rs4647277, rs9852810, rs9857293, rs13320360, and rs34285587.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an MLH1 gene, including mRNA that is a product of RNA processing of a primary transcription product. In one aspect, the target portion of the sequence will be at least long enough to serve as a substrate for antisense-oligonucleotide-directed or dsRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a MLH1 gene. The target sequence can be, for example, from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or 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 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated.

“G,” “C,” “A,” “T,” and “U” each generally stand for a naturally-occurring nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively. However, it will be understood that the term “nucleotide” can also refer to an alternative nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of a nucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of antisense oligonucleotides or dsRNAs by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the antisense oligonucleotide or dsRNA can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods described herein.

The terms “nucleobase” and “base” include the purine (e.g. adenine and guanine) and pyrimidine (e.g. uracil, thymine, and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization. The term nucleobase also encompasses alternative nucleobases which can differ from naturally-occurring nucleobases, but are functional during nucleic acid hybridization. In this context “nucleobase” refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as alternative nucleobases. Such variants are for example described in Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.

The term “nucleoside” refers to a monomeric unit of an oligonucleotide or a polynucleotide having a nucleobase and a sugar moiety. A nucleoside can include those that are naturally-occurring as well as alternative nucleosides, such as those described herein. The nucleobase of a nucleoside can be a naturally-occurring nucleobase or an alternative nucleobase. Similarly, the sugar moiety of a nucleoside can be a naturally-occurring sugar or an alternative sugar.

The term “alternative nucleoside” refers to a nucleoside having an alternative sugar or an alternative nucleobase, such as those described herein.

In some aspects, the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as an “alternative nucleobase” selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uridine, 5-bromouridine 5-thiazolo-uridine, 2-thio-uridine, pseudouridine, 1-methylpseudouridine, 5-methoxyuridine, 2′-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

The nucleobase moieties can be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C, or U, wherein each letter can include alternative nucleobases of equivalent function. In some aspects, e.g., for gapmers, 5-methyl cytosine LNA nucleosides can be used.

A “sugar” or “sugar moiety,” includes naturally occurring sugars having a furanose ring. A sugar also includes an “alternative sugar,” defined as a structure that is capable of replacing the furanose ring of a nucleoside. In some aspects, alternative sugars are non-furanose (or 4′-substituted furanose) rings or ring systems or open systems. Such structures include simple changes relative to the natural furanose ring, such as a six-membered ring, or can be more complicated as is the case with the non-ring system used in peptide nucleic acid. Alternative sugars can include sugar surrogates wherein the furanose ring has been replaced with another ring system such as, for example, a morpholino or hexitol ring system. Sugar moieties useful in the preparation of oligonucleotides (e.g., ASO or dsRNA) having motifs include, without limitation, β-D-ribose, β-D-2′-deoxyribose, substituted sugars (such as 2′, 5′ and bis substituted sugars), 4′-S-sugars (such as 4′-S-ribose, 4′-S-2′-deoxyribose and 4′-S-2′-substituted ribose), bicyclic alternative sugars (such as the 2′-O—CH₂-4′ or 2′-O—(CH₂)₂-4′ bridged ribose derived bicyclic sugars) and sugar surrogates (such as when the ribose ring has been replaced with a morpholino or a hexitol ring system). The type of heterocyclic base and internucleoside linkage used at each position is variable and is not a factor in determining the motif. In most nucleosides having an alternative sugar moiety, the heterocyclic nucleobase is generally maintained to permit hybridization.

A “nucleotide,” as used herein, refers to a monomeric unit of an oligonucleotide or polynucleotide that comprises a nucleoside and an internucleosidic linkage. The internucleosidic linkage can include a phosphate linkage. Similarly, “linked nucleosides” can be linked by phosphate linkages. Many “alternative internucleosidic linkages” are known in the art, including, but not limited to, phosphate, phosphorothioate, and boronophosphate linkages. Alternative nucleosides include bicyclic nucleosides (BNAs) (e.g., locked nucleosides (LNAs) and constrained ethyl (cEt) nucleosides), peptide nucleosides (PNAs), phosphotriesters, phosphorothionates, phosphoramidates, and other variants of the phosphate backbone of native nucleoside, including those described herein. An “alternative nucleotide,” as used herein, refers to a nucleotide having an alternative nucleoside or an alternative sugar, and an internucleoside linkage, which can include alternative nucleoside linkages.

The terms “oligonucleotide” and “polynucleotide” as used herein are defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides can be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The oligonucleotide can be man-made. For example, the oligonucleotide can be chemically synthesized and be purified or isolated. Oligonucleotide is also intended to include (i) compounds that have one or more furanose moieties that are replaced by furanose derivatives or by any structure, cyclic or acyclic, that can be used as a point of covalent attachment for the base moiety, (ii) compounds that have one or more phosphodiester linkages that are either modified, as in the case of phosphoramidate or phosphorothioate linkages, or completely replaced by a suitable linking moiety as in the case of formacetal or riboacetal linkages, and/or (iii) compounds that have one or more linked furanose-phosphodiester linkage moieties replaced by any structure, cyclic or acyclic, that can be used as a point of covalent attachment for the base moiety. The oligonucleotides (e.g., ASOs or dsRNA) can comprise one or more alternative nucleosides or nucleotides (e.g., including those described herein). It is also understood that oligonucleotide includes compositions lacking a sugar moiety or nucleobase but is still capable of forming a pairing with or hybridizing to a target sequence.

“Oligonucleotide” refers to a short polynucleotide (e.g., of 100 or fewer linked nucleosides). As used herein, the term “strand” refers to an oligonucleotide comprising a chain of linked nucleosides. A “strand comprising a nucleobase sequence” refers to an oligonucleotide comprising a chain of linked nucleosides that is described by the sequence referred to using the standard nucleobase nomenclature.

The term “antisense,” as used herein, refers to a nucleic acid comprising an oligonucleotide or polynucleotide that is sufficiently complementary to all or a portion of a gene, primary transcript, or processed mRNA, so as to interfere with expression of the endogenous gene (e.g., MLH1). “Complementary” polynucleotides are those that are capable of base pairing according to the standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. It is understood that two polynucleotides can hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other.

The terms “antisense strand” and “guide strand” refer to the strand of a dsRNA that includes a region that is substantially complementary to a target sequence, e.g., an MLH1 mRNA.

The terms “sense strand” and “passenger strand,” as used herein, refer to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

The term “dsRNA” refers to an agent that includes a sense strand and antisense strand that contains linked nucleosides as that term is defined herein. dsRNA includes, for example, siRNAs and shRNAs, which mediate the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The dsRNA reduces the expression of MLH1 in a cell, e.g., a cell within a subject, such as a mammalian subject. In general, the majority of linked nucleosides of each strand of a dsRNA are ribonucleosides, but as described in detail herein, each or both strands can include one or more non-ribonucleosides, e.g., deoxyribonucleosides and/or alternative nucleosides.

The terms “siRNA” and “short interfering RNA” (also known as “small interfering RNA”) refer to an RNA agent, such as a double-stranded agent, of about 10-50 nucleotides in length, the strands optionally having overhanging ends comprising, for example 1, 2, or 3 overhanging linked nucleosides, which is capable of directing or mediating RNA interference. Naturally-occurring siRNAs are generated from longer dsRNA molecules (e.g., >25 linked nucleosides in length) by a cell's RNAi machinery (e.g., Dicer or a homolog thereof).

The terms “shRNA” and “short hairpin RNA,” as used herein, refer to an RNA agent having a stem-loop structure, comprising at least two regions of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, at least two of the regions being joined by a loop region which results from a lack of base pairing between nucleobases within the loop region.

“Chimeric antisense oligonucleotides” are antisense oligonucleotides which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide or nucleoside in the case of an oligonucleotide. Chimeric antisense oligonucleotides also include “gapmers.”

“Chimeric dsRNA” is dsRNA which contains two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleoside or nucleotide in the case of a dsRNA.

The antisense oligonucleotide can be of any length that permits specific degradation of a desired target RNA through an RNase H-mediated pathway, and can range from about 10-30 nucleosides in length, e.g., about 15-30 nucleosides in length or about 18-20 nucleosides in length, for example, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides in length, 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 nucleosides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated.

As used herein, the term “antisense oligonucleotide comprising a nucleobase sequence” refers to an antisense oligonucleotide comprising a chain of nucleotides or nucleosides that is described by the sequence referred to using the standard nucleotide nomenclature.

The term “contiguous nucleobase region” refers to the region of the antisense oligonucleotide or dsRNA (e.g., the antisense strand of the dsRNA) which is complementary to the target nucleic acid. The term can be used interchangeably herein with the term “contiguous nucleotide sequence” or “contiguous nucleobase sequence.” In some aspects, all of the nucleotides of the antisense oligonucleotide or dsRNA are present in the contiguous nucleotide or nucleoside region. In some aspects the antisense oligonucleotide or dsRNA comprises the contiguous nucleotide region and can comprise further nucleotide(s) or nucleoside(s), for example a nucleotide linker region which can be used to attach a functional group to the contiguous nucleotide sequence. The nucleotide linker region can be complementary to the target nucleic acid. In some aspects the internucleoside linkages present between the nucleotides of the contiguous nucleotide region are all phosphorothioate internucleoside linkages. In some aspects, the contiguous nucleotide region comprises one or more sugar-modified nucleosides.

The term “gapmer,” as used herein, refers to an antisense oligonucleotide which comprises a region of RNase H recruiting oligonucleotides (gap or DNA core) which is flanked 5′ and 3′ by regions which comprise one or more affinity enhancing alternative nucleosides (wings or flanking sequence). Various gapmer designs are described herein. Headmers and tailmers are oligonucleotides capable of recruiting RNase H where one of the flanks is missing, i.e. only one of the ends of the oligonucleotide comprises affinity enhancing alternative nucleosides. For headmers the 3′ flanking sequence is missing (i.e. the 5′ flanking sequence comprises affinity enhancing alternative nucleosides) and for tailmers the 5′ flanking sequence is missing (i.e. the 3′ flanking sequence comprises affinity enhancing alternative nucleosides). A “mixed flanking sequence gapmer” refers to a gapmer wherein the flanking sequences comprise at least one alternative nucleoside, such as at least one DNA nucleoside or at least one 2′ substituted alternative nucleoside, such as, for example, 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-Fluoro-RNA, 2′-F-ANA nucleoside(s), or bicyclic nucleosides (e.g., locked nucleosides or constrained ethyl (cEt) nucleosides). In some aspects the mixed flanking sequence gapmer has one flanking sequence which comprises alternative nucleosides (e.g. 5′ or 3′) and the other flanking sequence (3′ or 5′ respectfully) comprises 2′ substituted alternative nucleoside(s).

The duplex region of the dsRNA can be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and can range from about 9 to 36 base pairs in length, e.g., about 10-30 base pairs in length, e.g., about 15-30 base pairs in length or about 18-20 base pairs in length, for example, 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 in length, 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 in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated.

The two strands forming the duplex structure can be different portions of one longer oligonucleotide molecule, or they can be separate oligonucleotide molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of linked nucleosides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleobase. In some aspects, the hairpin loop can comprise 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 nucleobases. In some aspects, the hairpin loop can be 10 or fewer linked nucleosides. In some aspects, the hairpin loop can be 8 or fewer unpaired nucleobases. In some aspects, the hairpin loop can be 4-10 unpaired nucleobases. In some aspects, the hairpin loop can be 4-8 linked nucleosides.

Multiple dsRNAs can be joined together by a linker. The linker can be cleavable or non-cleavable. The dsRNAs can be the same or different.

In one aspect, each strand of the dsRNA includes 19-23 linked nucleosides that interacts with a target RNA sequence, e.g., an MLH1 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-Ill-like enzyme, processes the RNA into 19-23 base pair short interfering RNAs with characteristic two-base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The dsRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the dsRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Where the two substantially complementary strands of a dsRNA are comprised of separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.”

“Linker” or “linking group,” as applied to a dsRNA, means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. The RNA strands can have the same or a different number of linked nucleosides. The maximum number of base pairs is the number of linked nucleosides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA can comprise one or more nucleoside overhangs. In one aspect of the dsRNA, at least one strand comprises a 3′ overhang of at least 1 nucleoside. In another aspect, at least one strand comprises a 3′ overhang of at least 2 linked nucleosides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 linked nucleosides. In other aspects, at least one strand of the dsRNA comprises a 5′ overhang of at least 1 nucleoside. In some aspects, at least one strand comprises a 5′ overhang of at least 2 linked nucleosides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 linked nucleosides. In still other aspects, both the 3′ and the 5′ end of one strand of the dsRNA comprise an overhang of at least 1 nucleoside.

A linker or linking group is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. Conjugate moieties can be attached to the antisense oligonucleotide or dsRNA directly or through a linking moiety (e.g. linker or tether). Linkers serve to covalently connect a third region, e.g. a conjugate moiety to an antisense oligonucleotide or dsRNA (e.g. the termini of region A or C). In some aspects, the conjugate, antisense oligonucleotide conjugate, or dsRNA comprises a linker region which is positioned between the antisense oligonucleotide or dsRNA and the conjugate moiety. In some aspects, the linker between the antisense conjugate and oligonucleotide or dsRNA is biocleavable. Phosphodiester containing biocleavable linkers are described in more detail in WO 2014/076195 (herein incorporated by reference).

As used herein, the term “nucleoside overhang” refers to at least one unpaired nucleobase that protrudes from the duplex structure of a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleoside overhang. A dsRNA can comprise an overhang of at least one nucleoside; alternatively, the overhang can comprise at least two nucleosides, at least three nucleosides, at least four nucleosides, at least five nucleosides or more. A nucleoside overhang can comprise or consist of an alternative nucleoside, including a deoxynucleotide/nucleoside. A nucleoside overhang can comprise or consist of one or more phosphorothioates bonds. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleoside(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA. In some aspects, the overhang includes a self-complementary portion such that the overhang is capable of forming a hairpin structure that is stable under physiological conditions.

The terms “blunt” and “blunt ended,” as used herein in reference to a dsRNA, mean that there are no unpaired nucleobases at a given terminal end of a dsRNA, i.e., no nucleoside overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleoside overhang at either end of the molecule. Most often such a molecule will be double stranded over its entire length. As used herein, the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some aspects, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some aspects, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some aspects, the cleavage site specifically occurs at the site bound by nucleosides 10 and 11 of the antisense strand, and the cleavage region comprises nucleosides 11, 12, and 13.

The term “contiguous nucleobase region” refers to the region of the dsRNA (e.g., the antisense strand of the dsRNA) which is complementary to the target nucleic acid. The term can be used interchangeably herein with the term “contiguous nucleotide sequence” or “contiguous nucleobase sequence.” In some aspects, all the nucleotides of the dsRNA are present in the contiguous nucleotide or nucleoside region. In some aspects, the dsRNA comprises the contiguous nucleotide region and can comprise further nucleotide(s) or nucleoside(s), for example a nucleotide linker region which can be used to attach a functional group to the contiguous nucleotide sequence. The nucleotide linker region can be complementary to the target nucleic acid. In some aspects, the internucleoside linkages present between the nucleotides of the contiguous nucleotide region are all phosphorothioate internucleoside linkages. In some aspects, the contiguous nucleotide region comprises one or more sugar-modified nucleosides.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide or nucleoside sequence in relation to a second nucleotide or nucleoside sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide or nucleoside sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C., or 70° C., for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can be used. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides or nucleosides.

“Complementary” sequences, as used herein, can include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and alternative nucleotides or nucleosides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing. Complementary sequences within a dsRNA or between an antisense oligonucleotide and a target sequence as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide or nucleoside sequence to an oligonucleotide or polynucleotide comprising a second nucleotide or nucleoside sequence over the entire length of one or both nucleotide or nucleoside sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via an RNase H-mediated pathway or reduction of expression via a RISC pathway. “Substantially complementary” can also refer to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding MLH1). For example, a polynucleotide is complementary to at least a part of a MLH1 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding MLH1. However, where two oligonucleotides of a dsRNA are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide of 21 linked nucleosides in length and another oligonucleotide of 23 nucleosides in length, wherein the longer oligonucleotide comprises a sequence of 21 linked nucleosides that is fully complementary to the shorter oligonucleotide, can be referred to as “fully complementary” for the purposes described herein.

As used herein, the term “region of complementarity” refers to the region on the antisense oligonucleotide or the antisense strand of the dsRNA that is substantially complementary to all or a portion of a gene, primary transcript, a sequence (e.g., a target sequence, e.g., an MLH1 nucleotide sequence), or processed mRNA, so as to interfere with expression of the endogenous gene (e.g., MLH1). Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′- and/or 3′-terminus of the antisense oligonucleotide or the antisense strand of the dsRNA.

As used herein, an “agent that reduces the level and/or activity of MLH1” refers to any polynucleotide agent (e.g., an antisense oligonucleotide or a dsRNA, e.g., siRNA or shRNA) that reduces the level of or inhibits expression of MLH1 in a cell or subject. The phrase “inhibiting expression of MLH1,” as used herein, includes inhibition of expression of any MLH1 gene (such as, e.g., a mouse MLH1 gene, a rat MLH1 gene, a monkey MLH1 gene, or a human MLH1 gene) as well as variants or mutants of a MLH1 gene that encode a MLH1 protein. Thus, the MLH1 gene can be a wild-type MLH1 gene, a mutant MLH1 gene, or a transgenic MLH1 gene in the context of a genetically manipulated cell, group of cells, or organism.

By “reducing the activity of MLH1” is meant decreasing the level of an activity related to MLH1 (e.g., by reducing the amount of trinucleotide repeats in a gene associated with a trinucleotide repeat expansion disorder that is related to MLH1 activity). The activity level of MLH1 can be measured using any method known in the art (e.g., by directly sequencing a gene associated with a trinucleotide repeat expansion disorder to measure the levels of trinucleotide repeats).

By “reducing the level of MLH1” is meant decreasing the level of MLH1 in a cell or subject, e.g., by administering an antisense oligonucleotide or dsRNA to the cell or subject. The level of MLH1 can be measured using any method known in the art (e.g., by measuring the levels of MLH1 mRNA or levels of MLH1 protein in a cell or a subject).

By “modulating the activity of a MutLa heterodimer comprising MLH1” is meant altering the level of an activity related to a MutLa heterodimer, or a related downstream effect. The activity level of a MutLa heterodimer can be measured using any method known in the art.

As used herein, the term “inhibitor” refers to any agent which reduces the level and/or activity of a protein (e.g., MLH1). Non-limiting examples of inhibitors include polynucleotides (e.g., antisense oligonucleotide or dsRNA, e.g., siRNA or shRNA). The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing,” and other similar terms, and includes any level of inhibition/reduction.

The phrases “contacting a cell with an antisense oligonucleotide,” such as an antisense oligonucleotide, and “contacting a cell with a dsRNA,” such as dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an antisense oligonucleotide or a dsRNA includes contacting a cell in vitro with the antisense oligonucleotide or dsRNA or contacting a cell in vivo with the antisense oligonucleotide or dsRNA. The contacting can be done directly or indirectly. Thus, for example, the antisense oligonucleotide or dsRNA can be put into physical contact with the cell by the individual performing the method, or alternatively, the antisense oligonucleotide or dsRNA agent can be put into a situation that will permit or cause it to subsequently come into contact with the cell.

Contacting a cell in vitro can be done, for example, by incubating the cell with the antisense oligonucleotide or dsRNA. Contacting a cell in vivo can be done, for example, by injecting the antisense oligonucleotide or dsRNA into or near the tissue where the cell is located, or by injecting the antisense oligonucleotide or dsRNA agent into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the antisense oligonucleotide or dsRNA can contain and/or be coupled to a ligand, e.g., GalNAc3 coupled to the antisense oligonucleotide, that directs the antisense oligonucleotide or dsRNA to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell can be contacted in vitro with an antisense oligonucleotide or dsRNA and subsequently transplanted into a subject.

In one aspect, contacting a cell with an antisense oligonucleotide or dsRNA includes “introducing” or “delivering the antisense oligonucleotide or dsRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an antisense oligonucleotide or dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an antisense oligonucleotide or dsRNA into a cell can be in vitro and/or in vivo. For example, for in vivo introduction, antisense oligonucleotides or dsRNAs can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.

As used herein, “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an antisense oligonucleotide or a dsRNA or plasma from which a dsRNA is transcribed. LNP refers to a stable nucleic acid-lipid particle. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). LNPs are described in, for example, U.S. Pat. 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.

As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the antisense oligonucleotide or dsRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the antisense oligonucleotide or dsRNA composition, although in some examples, it can. Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.

“Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

As used herein, the terms “effective amount,” “therapeutically effective amount,” and “a “sufficient amount” of an agent that reduces the level and/or activity of MLH1 (e.g., in a cell or a subject) described herein refer to a quantity sufficient to, when administered to the subject, including a human, effect beneficial or desired results, including clinical results, and, as such, an “effective amount” or synonym thereto depends on the context in which it is being applied. For example, in the context of treating a trinucleotide repeat expansion disorder, it is an amount of the agent that reduces the level and/or activity of MLH1 sufficient to achieve a treatment response as compared to the response obtained without administration of the agent that reduces the level and/or activity of MLH1. The amount of a given agent that reduces the level and/or activity of MLH1 described herein that will correspond to such an amount will vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, and/or weight) or host being treated, and the like, but can nevertheless be routinely determined by one of skill in the art. Also, as used herein, a “therapeutically effective amount” of an agent that reduces the level and/or activity of MLH1 of the present disclosure is an amount which results in a beneficial or desired result in a subject as compared to a control. As defined herein, a therapeutically effective amount of an agent that reduces the level and/or activity of MLH1 of the present disclosure can be readily determined by one of ordinary skill by routine methods known in the art. Dosage regimen can be adjusted to provide the optimum therapeutic response.

“Prophylactically effective amount,” as used herein, is intended to include the amount of an antisense oligonucleotide or a dsRNA that, when administered to a subject having or predisposed to have a trinucleotide repeat expansion disorder, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” can vary depending on the antisense oligonucleotide or dsRNA, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated. A prophylactically effective amount can refer to, for example, an amount of the agent that reduces the level and/or activity of MLH1 (e.g., in a cell or a subject) described herein or can refer to a quantity sufficient to, when administered to the subject, including a human, delay the onset of one or more of the trinucleotide repeat disorders described herein by at least 120 days, for example, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years or more, when compared with the predicted onset.

A “therapeutically-effective amount” or “prophylactically effective amount” also includes an amount (either administered in a single or in multiple doses) of an antisense oligonucleotide or a dsRNA that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. The antisense oligonucleotides or dsRNAs employed in the methods described herein can be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

As used herein, the term “region of complementarity” refers to the region on the antisense oligonucleotide or antisense strands of the dsRNA that is substantially complementary to all or a portion of a gene, primary transcript, a sequence (e.g., a target sequence, e.g., an MLH1 nucleotide sequence), or processed mRNA, so as to interfere with expression of the endogenous gene (e.g., MLH1). Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′- and/or 3′-terminus of the antisense oligonucleotide or dsRNA.

An “amount effective to reduce trinucleotide repeat expansion” of a particular gene refers to an amount of the agent that reduces the level and/or activity of MLH1 (e.g., in a cell or a subject) described herein, or to a quantity sufficient to, when administered to the subject, including a human, to reduce the trinucleotide repeat expansion of a particular gene (e.g., a gene associated with a trinucleotide repeat expansion disorder described herein).

As used herein, the term “a subject identified as having a trinucleotide repeat expansion disorder” refers to a subject identified as having a molecular or pathological state, disease or condition of or associated with a trinucleotide repeat expansion disorder, such as the identification of a trinucleotide repeat expansion disorder or symptoms thereof, or to identification of a subject having or suspected of having a trinucleotide repeat expansion disorder who can benefit from a particular treatment regimen.

As used herein, “trinucleotide repeat expansion disorder,” refers to a class of genetic diseases or disorders characterized by excessive trinucleotide repeats (e.g., trinucleotide repeats such as CAG) in a gene or intron in the subject which exceed the normal, stable threshold, for the gene or intron. Trinucleotide repeats are common in the human genome and are not normally associated with disease. In some cases, however, the number of trinucleotide repeats expands beyond a stable threshold and can lead to disease, with the severity of symptoms generally correlated with the number of trinucleotide repeats. Trinucleotide repeat expansion disorders include “polyglutamine” and “non-polyglutamine” disorders.

By “determining the level of a protein” is meant the detection of a protein, or an mRNA encoding the protein, by methods known in the art either directly or indirectly. “Directly determining” means performing a process (e.g., performing an assay or test on a sample or “analyzing a sample” as that term is defined herein) to obtain the physical entity or value. “Indirectly determining” refers to receiving the physical entity or value from another party or source (e.g., a third-party laboratory that directly acquired the physical entity or value). Methods to measure protein level generally include, but are not limited to, western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, liquid chromatography (LC)-mass spectrometry, microcytometry, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry, as well as assays based on a property of a protein including, but not limited to, enzymatic activity or interaction with other protein partners. Methods to measure mRNA levels are known in the art.

“Percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values can be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:

100 multiplied by (the fraction X/Y)

where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

By “level” is meant a level or activity of a protein, or mRNA encoding the protein (e.g., MLH1), optionally as compared to a reference. The reference can be any useful reference, as defined herein. By a “decreased level” or an “increased level” of a protein is meant a decrease or increase in protein level, as compared to a reference (e.g., a decrease or an increase by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, or more; a decrease or an increase of more than about 10%, about 15%, about 20%, about 50%, about 75%, about 100%, or about 200%, as compared to a reference; a decrease or an increase by less than about 0.01-fold, about 0.02-fold, about 0.1-fold, about 0.3-fold, about 0.5-fold, about 0.8-fold, or less; or an increase by more than about 1.2-fold, about 1.4-fold, about 1.5-fold, about 1.8-fold, about 2.0-fold, about 3.0-fold, about 3.5-fold, about 4.5-fold, about 5.0-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 1000-fold, or more). A level of a protein can be expressed in mass/vol (e.g., g/dL, mg/mL, μg/mL, ng/mL) or percentage relative to total protein or mRNA in a sample.

The term “pharmaceutical composition,” as used herein, represents a composition containing a compound described herein formulated with a pharmaceutically acceptable excipient, and can be manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); for intrathecal injection; for intracerebroventricular injections; for intraparenchymal injection; or in any other pharmaceutically acceptable formulation.

A “pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients can include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

As used herein, the term “pharmaceutically acceptable salt” means any pharmaceutically acceptable salt of the compound of any of the compounds described herein. For example, pharmaceutically acceptable salts of any of the compounds described herein include those that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P. H. Stahl and C. G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting a free base group with a suitable organic acid.

The compounds described herein can have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts. These salts can be acid addition salts involving inorganic or organic acids or the salts can, in the case of acidic forms of the compounds described herein, be prepared from inorganic or organic bases. Frequently, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases and methods for preparation of the appropriate salts are well-known in the art. Salts can be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

By a “reference” is meant any useful reference used to compare protein or mRNA levels or activity. The reference can be any sample, standard, standard curve, or level that is used for comparison purposes. The reference can be a normal reference sample or a reference standard or level. A “reference sample” can be, for example, a control, e.g., a predetermined negative control value such as a “normal control” or a prior sample taken from the same subject; a sample from a normal healthy subject, such as a normal cell or normal tissue; a sample (e.g., a cell or tissue) from a subject not having a disease; a sample from a subject that is diagnosed with a disease, but not yet treated with a compound described herein; a sample from a subject that has been treated by a compound described herein; or a sample of a purified protein (e.g., any described herein) at a known normal concentration. By “reference standard or level” is meant a value or number derived from a reference sample. A “normal control value” is a pre-determined value indicative of non-disease state, e.g., a value expected in a healthy control subject. Typically, a normal control value is expressed as a range (“between X and Y”), a high threshold (“no higher than X”), or a low threshold (“no lower than X”). A subject having a measured value within the normal control value for a particular biomarker is typically referred to as “within normal limits” for that biomarker. A normal reference standard or level can be a value or number derived from a normal subject not having a disease or disorder (e.g., a trinucleotide repeat expansion disorder); a subject that has been treated with a compound described herein. In some aspects, the reference sample, standard, or level is matched to the sample subject sample by at least one of the following criteria: age, weight, sex, disease stage, and overall health. A standard curve of levels of a purified protein, e.g., any described herein, within the normal reference range can also be used as a reference.

As used herein, the term “subject” refers to any organism to which a composition can be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject can seek or be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, or be a human or animal who is under care by a trained professional for a particular disease or condition.

As used herein, the terms “treat,” “treated,” and “treating” mean both therapeutic treatment and prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder, or disease, or obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease; stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

As used herein, the terms “variant” and “derivative” are used interchangeably and refer to naturally-occurring, synthetic, and semi-synthetic analogues of a compound, peptide, protein, or other substance described herein. A variant or derivative of a compound, peptide, protein, or other substance described herein can retain or improve upon the biological activity of the original material.

The details of one or more aspects are set forth in the description below. Other features, objects, and advantages will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a distribution plot showing the somatic expansion of the human HTT transgene in the striatum as measured by the instability index in R6/2 mice at 4, 8, 12, and 16 weeks of age (4 male and 4 female per age group). The bars are mean values and error bars indicate standard deviation.

FIG. 2 is a distribution plot showing the somatic expansion of the human HTT transgene in the cerebellum as measured by the instability index in R6/2 mice at 4, 8, 12, and 16 weeks of age (4 male and 4 female per age group).

DETAILED DESCRIPTION

The present inventors have found that inhibition, reduction, or depletion of MLH1 level and/or activity in a cell is effective in the treatment of a trinucleotide repeat expansion disorder. Accordingly, useful compositions and methods to treat trinucleotide repeat expansion disorders, e.g., in a subject in need thereof are provided herein.

1. Trinucleotide Repeat Expansion Disorders

Trinucleotide repeat expansion disorders are a family of genetic disorders characterized by the pathogenic expansion of a repeat region within a genomic region. In such disorders, the number of repeats exceeds that of a gene's normal, stable threshold, expanding into a diseased range.

Trinucleotide repeat expansion disorders generally can be categorized as “polyglutamine” or “non-polyglutamine.” Polyglutamine disorders, including Huntington's disease (HD) and several spinocerebellar ataxias, are caused by a CAG (glutamine) repeats in the protein-coding regions of specific genes. Non-polyglutamine disorders are more heterogeneous and can be caused by CAG trinucleotide repeat expansions in non-coding regions, as in Myotonic dystrophy, or by the expansion of trinucleotide repeats other than CAG that can be in coding or non-coding regions such as the CGG repeat expansion responsible for Fragile X Syndrome.

Trinucleotide repeat expansion disorders are dynamic in the sense that the number of repeats can vary from generation-to-generation, or even from cell-to-cell in the same individual. Repeat expansion is believed to be caused by polymerase “slipping” during DNA replication. Tandem repeats in the DNA sequence can “loop out” while maintaining complementary base pairing between the parent strand and daughter strands. If the loop structure is formed from the daughter strand, the number of repeats will increase.

Conversely, if the loop structure is formed from the parent strand, the number of repeats will decrease. It appears that expansion is more common than reduction. In general, the length of repeat expansion is negatively correlated with prognosis; longer repeats are correlated with an earlier age of onset and worsened disease severity. Thus, trinucleotide repeat expansion disorders are subject to “anticipation,” meaning the severity of symptoms and/or age of onset worsen through successive generations of affected families due to the expansion of these repeats from one generation to the next.

Trinucleotide repeat expansion disorders are well known in the art. Exemplary trinucleotide repeat expansion disorders and the nucleotide repeats of the genes commonly associated with them are included in Table 1.

TABLE 1

Exemplary Trinucleotide Repeat Expansion Disorders

Nucleotide

Disease
Gene
Repeat

ARX-nonsyndromic X-linked mental
ARX
GCG

retardation (XLMR)

Baratela-Scott Syndrome
XYLT1
GGC

Blepharophimosis/Ptosis/Epicanthus
FOXL2
GCG

inversus syndrome type II

Cleidocranial dysplasia (CCD)
RUNX2
GCG

Congenital central hypoventilation
PHOX-2B
GCG

Congenital central hypoventilation
PHOX2B
GCG

syndrome (CCHS)

Creutzfeldt-Jakob disease
PRNP

Dentatorubral-pallidoluysian atrophy
ATN1
CAG

(DRPLA)/Haw River syndrome

Early infantile epileptic encephalopathy
ARX
GCG

(Ohtahara syndrome)

FRA2A syndrome
AFF3
CGC

FRA7A syndrome
ZNF713
CGG

Fragile X mental retardation (FRAX-E)
AFF2/FMR2
GCC

Fragile X Syndrome (FXS)
FMR1
CGG

Fragile X-associated Primary Ovarian
FMR1
CGG

Insufficiency (FXPOI)

Fragile X-associated Tremor Ataxia
FMR1
CGG

Syndrome (FXTAS)

Friedreich ataxia (FRDA)
FXN
GAA

Fuchs' Corneal Endothelial Dystrophy
TCF4
CTG

(FECD)

Hand-foot genital syndrome (HFGS)
HOXA13
GCG

Holoprosencephaly disorder (HPE)
ZIC2
GCG

Huntington disease-like 2 (HDL2)
JPH3
CTG

Huntington's Disease (HD)
HTT
CAG

Infantile spasm syndrome/West
ARX
GCG

syndrome (ISS)

Jacobsen syndrome

KCNN3-associated (e.g., schizophrenia)
KCNN3
CAG

Multiple Skeletal dysplasias
COMP
GAC

Myotonic Dystrophy type 1 (DM1)
DMPK
CTG

Myotonic Dystrophy type 2 (DM2)
CNBP
CCTG

NCOA3-associated (e.g., increased risk
NCOA3
CAG

of prostate cancer)

Neuronal intranuclear inclusion disease
NOTCH2NLC
GGC

(NIID)

Oculopharyngeal Muscular Dystrophy
PABPN1
GCG

(OPMD)

Spastic ataxia - Charlevoix-Saguenay

Spinal Muscular Bulbar Atrophy (SMBA)
AR
CAG

Spinocerebellar ataxia type 1 (SCA1)
ATXN1
CAG

Spinocerebellar ataxia type 10 (SCA10)
ATXN10
ATTCT

Spinocerebellar ataxia type 12 (SCA12)
PPP2R2B
CAG

Spinocerebellar ataxia type 17 (SCA17)
TBP/ATXN17
CAG

Spinocerebellar ataxia type 2 (SCA2)
ATXN2
CAG

Spinocerebellar ataxia type 3 (SCA3)/
ATXN3
CAG

Machado-Joseph Disease

Spinocerebellar ataxia type 45 (SCA45)
FAT2
CAG

Spinocerebellar ataxia type 6 (SCA6)
CACNA1A
CAG

Spinocerebellar ataxia type 7 (SCA7)
ATXN7
CAG

Spinocerebellar ataxia type 8 (SCA8)
ATXN8
CTG

Syndromic neurodevelopmental
MAB21L1
CAG

disorder with cerebellar, ocular,

craniofacial, and genital features (COFG

syndrome)

Synpolydactyly (SPD I)
HOXD13
GCG

Synpolydactyly (SPD II)
HOXD12
GCG

The proteins associated with trinucleotide repeat expansion disorders are typically selected based on an experimental association of the protein associated with a trinucleotide repeat expansion disorder to a trinucleotide repeat expansion disorder. For example, the production rate or circulating concentration of a protein associated with a trinucleotide repeat expansion disorder can be elevated or depressed in a population having a trinucleotide repeat expansion disorder relative to a population lacking the trinucleotide repeat expansion disorder. Differences in protein levels can be assessed using proteomic techniques including but not limited to Western blot, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, the proteins associated with trinucleotide repeat expansion disorders can be identified by obtaining gene expression profiles of the genes encoding the proteins using genomic techniques including, but not limited to, DNA microarray analysis, serial analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (qPCR).

II. Evidence for the Involvement of Mismatch Repair Pathway in Trinucleotide Repeat Expansion

There is growing evidence that DNA repair pathways, particularly mismatch repair (MMR), are involved in the expansion of trinucleotide repeats (Liu & Wilson (2012) Trends Biochem Sci. 37: 162-172). A recent genome-wide association (GWA) analysis led to the identification of loci harboring genetic variations that alter the age at neurological onset of Huntington's disease (HD) (GEM-HD Consortium, Cell. 2015 Jul. 30; 162(3):516-26). The study identified MLH1, the human homolog of the E. coli DNA mismatch repair gene mutL. A subsequent GWA study in polyglutamine disease patients found significant association of age at onset when grouping all polyglutamine diseases (HD and SCAs) with DNA repair genes as a group, as well as significant associations for specific SNPs in FAN1 and PMS2 with the diseases (Bettencourt et al., (2016) Ann. Neurol., 79: 983-990). These results were consistent with those from an earlier study comparing differences in repeat expansion in two different mouse models of Huntington's Disease, which identified Mlh1 and Mlh3 as novel critical modifiers of CAG instability (Pinto et al., (2013) Mismatch Repair Genes Mlh1 and MIh3 Modify CAG Instability in Huntington's Disease Mice: Genome-Wide and Candidate Approaches. PLoS Genet 9(10): e1003930). Likewise, mice lacking MSH2 or MSH3 have attenuated expansion in the human HD gene (Manley et al., (1999) Nat. Genet. 23, 471-473), the human myotonic dystrophy 1 protein kinase transgene (van den Broek et al. (2002) Hum. Mol. Genet. 11, 191-198), the FAX gene in Friedreich's ataxia (FRDA) (Bourn et al. (2012) PLoS One 7, e47085) and the fragile mental retardation gene in fragile X syndrome (FXS) (Lokanga et al., (2012) Hum. Mutat. 35, 129-136). Another member of the mismatch repair pathway, 8-oxo-guanine glycosylase (OGG1) has also been implicated in expansion, as somatic expansion was found to be reduced in transgenic mice lacking OGG1 (Kovtun I. V. et al. (2007) Nature 447, 447-452). However, another study found that human subjects containing a Ser326Cys polymorphism in hOGG1, which results in reduced OGG1 activity, results in increased mutant huntingtin (Coppede et al., (2009) Toxicol., 278: 199-203). Likewise, complete inactivation of Fan1, another component of the DNA repair pathway, in a mouse HD model produces somatic CAG expansions (Long et al. (2018) J. Hum Genet., 103: 1-9). MLH1, another component of the mismatch repair pathway, has been reported to be linked somatic expansion: polymorphisms in MLH1 was associated with somatic instability of the expanded CTG trinucleotide repeat in myotonic dystrophy type 1 (DM1) patients (Morales et al., (2016) DNA Repair 40: 57-66). Furthermore, natural polymorphisms in MLH1 and Mlh1 have been revealed as mediators of mouse strain specific differences in CTG⋅CAG repeat instability (Pinto et al. (2013) ibid; Tome et al., (2013) PLoS Genet. 9 el 003280). Further evidence of Msh2 and MLH1's involvement in expansion repeats was reported in a study in which short hairpin RNA (shRNA) knockdown of either MSH2 or MLH1 slowed, and ectopic expression of either MSH2 or MLH1 induced GAA trinucleotide repeat expansion of the Friedreich Ataxia (FRDA) gene in fibroblasts derived from FRDA patients (Halabi et al., (2012) J. Biol. Chem. 287, 29958-29967). In spite of some inconsistent results provided above, there is strong evidence that the MMR pathway plays some role in the expansion of trinucleotide repeats in various disorders. Moreover, they are the first to recognize that the inhibition of the MMR pathway provides for the treatment or prevention of these repeat expansion disorders; however, no therapy is currently available or in development which modulates MMR for purposes of treating or preventing these repeat expansion disorders.

III. Agents

Agents described herein that reduce the level and/or activity of MLH1 in a cell can be, for example, a polynucleotide, e.g., an antisense oligonucleotide or a dsRNA. These agents reduce the level of an activity related to MLH1, or a related downstream effect, or reduce the level of MLH1 in a cell or subject.

A. Antisense Oligonucleotide Agents

In some aspects, the agent that reduces the level and/or activity of MLH1 is a polynucleotide. In some aspects, the polynucleotide is a single-stranded antisense oligonucleotide, e.g., that acts by way of an RNase H-mediated pathway. Antisense oligonucleotides include DNA and DNA/RNA chimeric molecules, typically about 10 to 30 nucleotides in length, which recognize polynucleotide target sequences or sequence portions through hydrogen bonding interactions with the nucleotide bases of the target sequence (e.g., MLH1). An antisense oligonucleotide molecule can decrease the expression level (e.g., protein level or mRNA level) of MLH1. For example, an antisense oligonucleotide includes oligonucleotides that targets full-length MLH1. In some aspects, the antisense oligonucleotide molecule recruits an RNase H enzyme, leading to target mRNA degradation.

In some aspects, the antisense oligonucleotide decreases the level and/or activity of a positive regulator of function. In other aspects, the antisense oligonucleotide increases the level and/or activity of an inhibitor of a positive regulator of function. In some aspects, the antisense oligonucleotide increases the level and/or activity of a negative regulator of function.

In some aspects, the antisense oligonucleotide decreases the level and/or activity or function of MLH1. In some aspects, the antisense oligonucleotide inhibits expression of MLH1. In other aspects, the antisense oligonucleotide increases degradation of MLH1 and/or decreases the stability (i.e., half-life) of MLH1. The antisense oligonucleotide can be chemically synthesized.

The antisense oligonucleotide includes an oligonucleotide having a region of complementarity (e.g., a contiguous nucleobase region) which is complementary to at least a part of an mRNA formed in the expression of MLH1. The region of complementarity can be about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the MLH1 gene, the antisense oligonucleotide can inhibit the expression of the MLH1 gene (e.g., a human, a primate, a non-primate, or a bird MLH1 gene) by at least about 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, Western Blotting or flowcytometric techniques.

Similarly, the region of complementarity to the target sequence can be between 10 and 30 linked nucleosides in length, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or between 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 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 linked nucleosides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated.

An antisense oligonucleotide can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

The antisense oligonucleotide compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the antisense oligonucleotide comprising unnatural or alternative nucleotides can be easily prepared. Single-stranded antisense oligonucleotides can be prepared using solution-phase or solid-phase organic synthesis or both.

In one aspect, an antisense oligonucleotide includes a region of at least 10 contiguous nucleobases having at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) complementary to at least 10 contiguous nucleotides of a MLH1 gene. In some aspects, the antisense oligonucleotide comprises a sequence complementary to at least 17 contiguous nucleotides, 19-23 contiguous nucleotides, 19 contiguous nucleotides, or 20 contiguous nucleotides of a MLH1 gene. The antisense oligonucleotide sequence can be selected from the group of sequences provided in any one of SEQ ID NOs: 6-1393.

In one aspect, the sequence is substantially complementary to a sequence of an mRNA generated in the expression of MLH1. In some aspects, the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at is one or more of positions 193-258, 289-607, 629-734, 757-836, 865-1125, 1177-1206, 1218-1286, 1324-1408, 1433-1747, 1759-1814, 1852-1901, 1959-2029, 2053-2240, 2250-2356, 2382-2479, 2510-2546, and 2573-2598 of the MLH1 gene. In some aspects, the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 193-251, 289-607, 629-734, 757-836, 865-1125, 1177-1206, 1218-286, 1324-1408, 1433-1747, 1759-1814, 1852-1901, 1959-2029, 2053-2240, 2250-2356, 2382-2479, 2510-2546, and 2573-2598 of the MLH1 gene. In some aspects, the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at is one or more of positions 193-251, 289-607, 629-734, 758-836, 865-1125, 1177-1206, 1218-1286, 1324-1408, 1433-1747, 1759-1814, 1852-1901, 1959-2029, 2053-2240, 2250-2356, 2382-2479, 2510-2546, and 2573-2598 of the MLH1 gene. In some aspects, the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 312-391, 410-508, 522-607, 629-726, 759-1125, 1177-1206, 1221-1286, 1324-1407, 1433-1747, 1764-1814, 1854-1901, 1959-2029, 2053-2113, 2184-2240, 2251-2283, 2303-2351, 2384-2479, 2510-2546 of the MLH1 gene. In some aspects, the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 575-602, 662-724, 805-830, 891-960, 1002-1027, 1056-1081, 1100-1125, 1342-1384, 1443-1498, 1513-1561, 1600-1625, 1652-1747, 1876-1901, 2001-2026, and 2430-2459 of the MLH1 gene. In some aspects, the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 307-332, 458-500, 571-602, 758-787, 865-890, 892-917, 1045-1084, 1624-1649, 1786-1813, 1871-1901, 2053-2081, 2086-2114, and 2149-2176 of the MLH1 gene. In some aspects, the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 575-602, 1056-1081, and 1876-1901 of the MLH1 gene.

In some aspects, the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 6-1393. In some aspects, the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 81-84, 86-87, 90, 99-101, 106-107, 111, 113-114, 117, 122-126, 129-131, 137-138, 140, 144, 146-160, 172, 188-191, 211, 215-220, 222-226, 229, 231-239, 242-249, 270-271, 274-279, 286-293, 295-298, 310-320, 322-328, 332-335, 337, 345, 383-388, 397-402, 405-413, 415-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-598, 600-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699, 701-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 1118-1123, 1131-1134, 1136, 1138-1139, 1140-1144, 1151-1152, 1161-1163, 1187-1192, 1194-1195, 1197, 1199-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, and 1343. In some aspects, the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 81-84, 86-87, 99-101, 106, 113-114, 122-126, 129-131, 137-138, 140, 144, 146-159, 172, 188-191, 211, 215-217, 219, 223-226, 229, 232-239, 242-249, 270-271, 274-279, 286-293, 295-298, 310-320, 322-328, 332-335, 337, 345, 384-388, 397-402, 405-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 1118-1123, 1131-1134, 1136, 1138-1144, 1151-1152, 1161-1163, 1187-1191, 1194-1195, 1197, 1199-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, and 1343. In some aspects, the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 81-87, 99-101, 106, 113-114, 122-126, 129-131, 137-138, 140, 144, 146, 147, 148-151, 153-159, 172, 188-191, 211215-217, 219, 223-226, 229, 232-239, 242-245, 248-249, 270-271274-276, 278-279, 286-293, 295-298, 310-320, 322-338, 332-335, 337, 345, 384-386, 387-388, 397-402, 405-413, 415-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-598, 600-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699, 701-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 1118-1123, 1131-1134, 1136, 1138-1144, 1151-1152, 1161-1163, 1187-1191, 1194-1195, 1197, 1199, 1200-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, and 1343. In some aspects, the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 122, 123, 125-126, 129-130, 131, 137, 146-147, 153-156, 158, 188-191, 211, 216, 223, 226, 235, 237, 245, 248, 270-271, 276, 278-279, 286, 289-293, 297-298, 310, 312-320, 323-328, 332, 334-335, 337, 385-386, 397-402, 407, 410-412, 415-417, 419-420, 476, 478, 482-487, 490-491, 493-494, 497-501, 522-523, 525-526, 528-530, 546-548, 557, 563-565, 586, 603, 605-610, 613, 619-622, 631-632, 634, 639-641, 645-646, 650-651, 655, 699, 701, 704-709, 714, 729-731, 733-734, 736-740, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 847, 850, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 928-931, 936-939, 977, 981-986, 1019, 1024-1026, 1034, 1036-1041, 1083-1085, 1087, 1111, 1132-1134, 1136, 1138-1140, 1142, 1161-1163, 1188-1190, 1194-1195, 1207, 1218, 1241, 1244, 1247, 1257-1259, 1262-1269, 1277-1278, and 1314-1315. In some aspects, the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 291-293, 313, 316-317, 325-326, 334-335, 415, 483, 485-486, 499-500, 523, 525, 564, 607, 622, 704-705, 707-709, 739, 752, 768-769, 788, 827, 861, 871-872, 877-879, 882, 885, 886, 901, 986, 1038, 1263, and 1265-1267. In some aspects, the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 117, 215-226, 229-232, 287-293, 384-385, 387-388, 458, 484, 596-610, 850, 936-938, 981-986, 1083-1086, 1109-1112, and 1121-1123.

In some aspects, the nucleobase sequence of the antisense oligonucleotide consists of any one of SEQ ID NOs: 6-1393. In some aspects, the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 81-87, 90, 99-101, 106-107, 111, 113-114, 117, 122-126, 129-131, 137-138, 140, 144, 146-160, 172, 188-191, 211, 215-220, 22-226, 229, 231-239, 242-249, 270-271, 274-279, 286-293, 295-297, 298, 310-320, 322-328, 332-335, 337, 345, 383-388, 397-402, 405-413, 415-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-598, 600-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699, 701-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 118-1123, 1131-1134, 1136, 1138-1144, 1151-1152, 1161-1163, 1187-1192, 1194-1195, 1197, 1199-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1222, 1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, and 1343. In some aspects, the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 81-84, 86-87, 99-101, 106, 113-114, 122-126, 129-131, 137-138, 140, 144, 146-159, 172, 188-191, 211, 215-217, 219, 223-226, 229, 232-239, 242-249, 270-271, 274-279, 286-293, 295-298, 310-320, 322-328, 332-335, 337, 345, 384-388, 397-402, 405-413, 415-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-598, 600-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699, 701-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 1118-1123, 1131-1134, 1136, 1138-1144, 1151-1152, 1161-1163, 1187-1191, 1194-1195, 1197, 1199-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, and 1343. In some aspects, the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 81-84, 86-87, 99-101, 106, 113-114, 122-126, 129-131, 137-138, 140, 144, 146-151, 153-159, 172, 188-191, 211, 215-217, 219223-226, 229, 232-239, 242-245, 248-249, 270-271, 274-276, 278-279, 286-293, 295-298, 310-320, 322-328, 332-335, 337, 345, 384-388, 397-402, 405-413, 415-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-525, 526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-598, 600-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699, 701-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 1118-1123, 1131-1134, 1136, 1138-1144, 1151-1152, 1161-1163, 1187-1191, 1194-1195, 1197, 1199-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, and 1343. In some aspects, the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 122-123, 125-126, 129-131, 137, 146-147, 153-156, 158, 188-191, 211, 216, 223, 226, 235, 237, 245, 248, 270-271, 276, 278-279, 286, 289-293, 297-298, 310, 312-320, 323-328, 332, 334-335, 337, 385-386, 397-402, 407, 410-412, 415-417, 419-420, 476, 478, 482-487, 490-491, 493-494, 497-501, 522-523, 525-526, 528-530, 546-548, 557, 563-565, 586, 603, 605-610, 613, 619-622, 631-632, 634, 639-641, 645-646, 650-651, 655, 699, 701, 704-709, 714, 729-731, 733-734, 736-740, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 847, 850, 859, 861, 866-867, 868-872, 877-882, 884-887, 889-890, 893-894, 901, 928-931, 936-939, 977, 981-986, 1019, 1024-1026, 1034, 1036-1041, 1083-1085, 1087, 1111, 1132-1134, 1136, 1138-1140, 1142, 1161-1163, 1188-1190, 1194-1195, 1207, 1218, 1241, 1244, 1247, 1257-1259, 1262-1269, 1277-1278, and 1314-1315. In some aspects, wherein the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 291-293, 313, 316-317, 325-326, 334-335, 415, 483, 485-486, 499-500, 523, 525, 564, 607, 622, 704-705, 707-709, 739, 752, 768-769, 788, 827, 861, 871-872, 877-879, 882, 885, 886, 901, 986, 1038, 1263, and 1265-1267. In some aspects, wherein the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 117, 215-226, 229-232, 287-293, 384-385, 387-388, 458-484, 596-610, 850, 936-938, 981-986, 1083-1086, 1109-1112 and 1121-1123.

In some aspects, the antisense oligonucleotide exhibits at least 50% mRNA inhibition at 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell. In some aspects, the antisense oligonucleotide exhibits at least 60% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell. In some aspects, the antisense oligonucleotide exhibits at least 70% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell. In some aspects, the antisense oligonucleotide exhibits at least 85% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell. In some aspects, the antisense oligonucleotide exhibits at least 50% mRNA inhibition at 2 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell. In some aspects, the antisense oligonucleotide exhibits at least 60% mRNA inhibition at a 2 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell. In some aspects, the antisense oligonucleotide exhibits at least 70% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell. In some aspects, the antisense oligonucleotide exhibits at least 85% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell.

The cell assay can comprise transfecting mammalian cells, such as HEK293, NIH3T3, or HeLa cells, with the desired concentration of oligonucleotide (e.g., 2 nM or 20 nM) using Lipofectamine 2000 (Invitrogen) and comparing MLH1 mRNA levels of transfected cells to MLH1 levels of control cells. Control cells can be transfected with oligonucleotides not specific to MLH1 or mock transfected. mRNA levels can be determined using RT-qPCR and MLH1 mRNA levels can be normalized to GAPDH mRNA levels. The percent inhibition can be calculated as the percent of MLH1 mRNA concentration relative to the MLH1 concentration of the control cells.

In some aspects, the antisense oligonucleotide, or contiguous nucleotide region thereof, has a gapmer design or structure also referred herein merely as “gapmer.” In a gapmer structure the antisense oligonucleotide comprises at least three distinct structural regions a 5′-flanking sequence (also known as a 5′-wing), a DNA core sequence (also known as a gap) and a 3′-flanking sequence (also known as a 3′-wing), in ‘5->3’ orientation. In this design, the 5′ and 3′ flanking sequences comprise at least one alternative nucleoside which is adjacent to a DNA core sequence, and can in some aspects, comprise a contiguous stretch of 2-7 alternative nucleosides, or a contiguous stretch of alternative and DNA nucleosides (mixed flanking sequences comprising both alternative and DNA nucleosides). The length of the 5′-flanking sequence region can be at least two nucleosides in length (e.g., at least at least 2, at least 3, at least 4, at least 5, or more nucleosides in length). The length of the 3′-flanking sequence region can be at least two nucleosides in length (e.g., at least 2, at least 3, at least at least 4, at least 5, or more nucleosides in length). The 5′ and 3′ flanking sequences can be symmetrical or asymmetrical with respect to the number of nucleosides they comprise. In some aspects, the DNA core sequence comprises about 10 nucleosides flanked by a 5′ and a 3′ flanking sequence each comprising about 5 nucleosides, also referred to as a 5-10-5 gapmer.

Consequently, the nucleosides of the 5′ flanking sequence and the 3′ flanking sequence which are adjacent to the DNA core sequence are alternative nucleosides, such as 2′ alternative nucleosides. The DNA core sequence comprises a contiguous stretch of nucleotides which are capable of recruiting RNase H, when the oligonucleotide is in duplex with the MLH1 target nucleic acid. In some aspects, the DNA core sequence comprises a contiguous stretch of 5-16 DNA nucleosides. In other aspects, the DNA core sequence comprises a region of at least 10 contiguous nucleobases having at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) complementarity to an MLH1 gene. In some aspects, the gapmer comprises a region complementary to at least 17 contiguous nucleotides, 19-23 contiguous nucleotides, or 19 contiguous nucleotides of an MLH1 gene. The gapmer is complementary to the MLH1 target nucleic acid, and can therefore be the contiguous nucleoside region of the oligonucleotide.

The 5′ and 3′ flanking sequences, flanking the 5′ and 3′ ends of the DNA core sequence, can comprise one or more affinity enhancing alternative nucleosides. In some aspects, the 5′ and/or 3′ flanking sequence comprises at least one 2′-O-methoxyethyl (MOE) nucleoside. In some aspects, the 5′ and/or 3′ flanking sequences, contain at least two MOE nucleosides. In some aspects, the 5′ flanking sequence comprises at least one MOE nucleoside. In some aspects both the 5′ and 3′ flanking sequence comprise a MOE nucleoside. In some aspects, all the nucleosides in the flanking sequences are MOE nucleosides. In other aspects, the flanking sequence can comprise both MOE nucleosides and other nucleosides (mixed flanking sequence), such as DNA nucleosides and/or non-MOE alternative nucleosides, such as bicyclic nucleosides (BNAs) (e.g., LNA nucleosides or cET nucleosides), or other 2′ substituted nucleosides. In this case the DNA core sequence is defined as a contiguous sequence of at least 5 RNase H recruiting nucleosides (such as 5-16 DNA nucleosides) flanked at the 5′ and 3′ end by an affinity enhancing alternative nucleoside, such as an MOE nucleoside.

In other aspects, the 5′ and/or 3′ flanking sequence comprises at least one BNA (e.g., at least one LNA nucleoside or cET nucleoside). In some aspects, 5′ and/or 3′ flanking sequence comprises at least 2 bicyclic nucleosides. In some aspects, the 5′ flanking sequence comprises at least one BNA. In some aspects both the 5′ and 3′ flanking sequence comprise a BNA. In some aspects, all the nucleosides in the flanking sequences are BNAs. In other aspects, the flanking sequence can comprise both BNAs and other nucleosides (mixed flanking sequences), such as DNA nucleosides and/or non-BNA alternative nucleosides, such as 2′ substituted nucleosides. In this case, the DNA core sequence is defined as a contiguous sequence of at least five RNase H recruiting nucleosides (such as 5-16 DNA nucleosides) flanked at the 5′ and 3′ end by an affinity enhancing alternative nucleoside, such as a BNA, such as an LNA, such as beta-D-oxy-LNA.

The 5′ flank attached to the 5′ end of the DNA core sequence comprises, contains, or consists of at least one alternative sugar moiety (e.g., at least three, at least four, at least five, at least six, at least seven, or more alternative sugar moieties). In some aspects the flanking sequence comprises or consists of from 1 to 7 alternative nucleobases, such as from 2 to 6 alternative nucleobases, such as from 2 to 5 alternative nucleobases, such as from 2 to 4 alternative nucleobases, such as from 1 to 3 alternative nucleobases, such as one, two, three or four alternative nucleobases. In some aspects, the flanking sequence comprises or consists of at least one alternative internucleoside linkage (e.g., at least three, at least four, at least five, at least six, at least seven, or more alternative internucleoside linkages).

The 3′ flank attached to the 3′ end of the DNA core sequence comprises, contains, or consists of at least one alternative sugar moiety (e.g., at least three, at least four, at least five, at least six, at least seven, or more alternative sugar moieties). In some aspects the flanking sequence comprises or consists of from 1 to 7 alternative nucleobases, such as from 2 to 6 alternative nucleobases, such as from 2 to 5 alternative nucleobases, such as from 2 to 4 alternative nucleobases, such as from 1 to 3 alternative nucleobases, such as one, two, three, or four alternative nucleobases. In some aspects, the flanking sequence comprises or consists of at least one alternative internucleoside linkage (e.g., at least three, at least four, at least five, at least six, at least seven, or more alternative internucleoside linkages).

In an aspect, one or more or all of the alternative sugar moieties in the flanking sequence are 2′ alternative sugar moieties.

In a further aspect, one or more of the 2′ alternative sugar moieties in the wing regions are selected from 2′-O-alkyl-sugar moieties, 2′-O-methyl-sugar moieties, 2′-amino-sugar moieties, 2′-fluoro-sugar moieties, 2′-alkoxy-sugar moieties, MOE sugar moieties, LNA sugar moieties, arabino nucleic acid (ANA) sugar moieties, and 2′-fluoro-ANA sugar moieties.

In one aspect all the alternative nucleosides in the flanking sequences are bicyclic nucleosides. In a further aspect, the bicyclic nucleosides in the flanking sequences are independently selected from the group consisting of oxy-LNA, thio-LNA, amino-LNA, cET, and/or ENA, in either the beta-D or alpha-L configurations or combinations thereof.

In some aspects, the one or more alternative internucleoside linkages in the flanking sequences are phosphorothioate internucleoside linkages. In some aspects, the phosphorothioate linkages are stereochemically pure phosphorothioate linkages. In some aspects the phosphorothioate linkages are Sp phosphorothioate linkages. In other aspects, the phosphorothioate linkages are Rp phosphorothioate linkages. In some aspects, the alternative internucleoside linkages are 2′-alkoxy internucleoside linkages. In other aspects, the alternative internucleoside linkages are alkyl phosphate internucleoside linkages.

The DNA core sequence can comprise, contain, or consist of at least 5-16 consecutive DNA nucleosides capable of recruiting RNase H. In some aspects, all of the nucleosides of the DNA core sequence are DNA units. In further aspects the DNA core region can consist of a mixture of DNA and other nucleosides capable of mediating RNase H cleavage. In some aspects, at least 50% of the nucleosides of the DNA core sequence are DNA, such as at least 60%, at least 70% or at least 80%, or at least 90% DNA. In some aspects, all of the nucleosides of the DNA core sequence are RNA units.

The antisense oligonucleotide can comprise a contiguous region which is complementary to the target nucleic acid. In some aspects, the antisense oligonucleotide can further comprise additional linked nucleosides positioned 5′ and/or 3′ to either the 5′ and 3′ flanking sequences. These additional linked nucleosides can be attached to the 5′ end of the 5′ flanking sequence or the 3′ end of the 3′ flanking sequence, respectively. The additional nucleosides can, in some aspects, form part of the contiguous sequence which is complementary to the target nucleic acid, or in other aspects, can be non-complementary to the target nucleic acid.

The inclusion of the additional nucleosides at either, or both of the 5′ and 3′ flanking sequences can independently comprise one, two, three, four, or five additional nucleotides, which can be complementary or non-complementary to the target nucleic acid. In this respect the antisense oligonucleotide, can in some aspects, comprise a contiguous sequence capable of modulating the target which is flanked at the 5′ and/or 3′ end by additional nucleotides. Such additional nucleosides can serve as a nuclease susceptible biocleavable linker, and can therefore be used to attach a functional group such as a conjugate moiety to the antisense oligonucleotide. In some aspects the additional 5′ and/or 3′ end nucleosides are linked with phosphodiester linkages, and can be DNA or RNA. In another aspect, the additional 5′ and/or 3′ end nucleosides are alternative nucleosides which can for example be included to enhance nuclease stability or for ease of synthesis.

In other aspects, the antisense oligonucleotides can utilize “altimer” design and comprise alternating 2′-fluoro-ANA and DNA regions that are alternated every three nucleosides. Altimer oligonucleotides are discussed in more detail in Min, et al., Bioorganic & Medicinal Chemistry Letters, 2002, 12(18): 2651-2654 and Kalota, et al., Nuc. Acid Res. 2006, 34(2): 451-61 (herein incorporated by reference).

In other aspects, the antisense oligonucleotides can utilize “hemimer” design and comprise a single 2′-modified flanking sequence adjacent to (on either side of the 5′ or the 3′ side of) a DNA core sequence. Hemimer oligonucleotides are discussed in more detail in Geary et al., 2001, J. Pharm. Exp. Therap., 296: 898-904 (herein incorporated by reference).

In some aspects, an antisense oligonucleotide has a nucleic acid sequence with at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the nucleic acid sequence any one of SEQ ID NOs: 6-1393. In some aspects, an antisense oligonucleotide has a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 6-1393.

It will be understood that, although the sequences in SEQ ID NOs: 6-1393 are described as unmodified and/or un-conjugated sequences, the nucleosides of the antisense oligonucleotide can comprise any one of the sequences set forth in any one of SEQ ID NOs: 6-1393 that is an alternative nucleoside and/or conjugated as described in detail below.

The skilled person is well aware that antisense oligonucleotides having a structure of between about 18-20 base pairs can be particularly effective in inducing RNase H-mediated degradation. However, one can appreciate that shorter or longer antisense oligonucleotides can be effective. In the aspects described above, by virtue of the nature of the antisense oligonucleotide sequences provided herein, antisense oligonucleotides described herein can include shorter or longer antisense oligonucleotide sequences. It can be reasonably expected that shorter antisense oligonucleotides minus only a few linked nucleosides on one or both ends can be similarly effective as compared to the antisense oligonucleotides described above. Hence, antisense oligonucleotides having a sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous linked nucleosides derived from one of the sequences provided herein, and differing in their ability to inhibit the expression of MLH1 by not more than about 5, 10, 15, 20, 25, or 30% inhibition from an antisense oligonucleotide comprising the full sequence, are contemplated.

The antisense oligonucleotides described herein can function via nuclease mediated degradation of the target nucleic acid, where the antisense oligonucleotides are capable of recruiting a nuclease, such as an endonuclease like endoribonuclease (RNase) (e.g., RNase H). Examples of antisense oligonucleotide designs which operate via nuclease mediated mechanisms are antisense oligonucleotides which typically comprise a region of at least 5 or 6 DNA nucleosides and are flanked on one side or both sides by affinity enhancing alternative nucleosides, for example gapmers, headmers, and tailmers.

The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO01/23613 provides in vitro methods for determining RNase H activity, which can be used to determine the ability to recruit RNase H. Typically an antisense oligonucleotide is deemed capable of recruiting RNase H if it, when provided with a complementary target nucleic acid sequence, has an initial rate, as measured in pmol/l/min, of at least 5%, such as at least 10% or more than 20% of the of the initial rate determined when using an antisense oligonucleotide having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers, with phosphorothioate linkages between all monomers in the antisense oligonucleotide, and using the methodology provided by Example 91-95 of WO01/23613 (hereby incorporated by reference).

Furthermore, the antisense oligonucleotides described herein identify a site(s) in a MLH1 transcript that is susceptible to RNase H-mediated cleavage. As used herein, an antisense oligonucleotide is said to target within a particular site of an RNA transcript if the antisense oligonucleotide promotes cleavage of the transcript anywhere within that particular site. Such an antisense oligonucleotide will generally include at least about 5-10 contiguous linked nucleosides from one of the sequences provided herein coupled to additional linked nucleoside sequences taken from the region contiguous to the selected sequence in a MLH1 gene.

Inhibitory antisense oligonucleotides can be designed by methods well known in the art. While a target sequence is generally about 10-30 linked nucleosides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA.

Antisense oligonucleotides with homology sufficient to provide sequence specificity required to uniquely degrade any RNA can be designed using programs known in the art

Systematic testing of several designed species for optimization of the antisense oligonucleotide sequence can be undertaken in accordance with the teachings provided herein. Considerations when designing antisense oligonucleotides include, but are not limited to, biophysical, thermodynamic, and structural considerations, base preferences at specific positions, and homology. The making and use of inhibitory therapeutic agents based on non-coding antisense oligonucleotides are also known in the art.

Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an antisense oligonucleotide agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified herein represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified herein, further optimization could be achieved by systematically either adding or removing linked nucleosides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of antisense oligonucleotides based on those target sequences in an inhibition assay as known in the art and/or as described herein can lead to further improvements in the efficiency of inhibition.

Further still, such optimized sequences can be adjusted by, e.g., the introduction of alternative nucleosides, alternative sugar moieties, and/or alternative internucleosidic linkages as described herein or as known in the art, including alternative nucleosides, alternative sugar moieties, and/or alternative internucleosidic linkages as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor. An antisense oligonucleotide agent as described herein can contain one or more mismatches to the target sequence. In one aspect, an antisense oligonucleotide as described herein contains no more than 3 mismatches. If the antisense oligonucleotide contains mismatches to a target sequence, in some aspects, the area of mismatch is not located in the center of the region of complementarity. If the antisense oligonucleotide contains mismatches to the target sequence, in some aspects, the mismatch should be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, fora 30-linked nucleoside antisense oligonucleotide agent, the contiguous nucleobase region which is complementary to a region of a MLH1 gene, generally does not contain any mismatch within the central 5-10 linked nucleosides. The methods described herein or methods known in the art can be used to determine whether an antisense oligonucleotide containing a mismatch to a target sequence is effective in inhibiting the expression of MLH1. Consideration of the efficacy of antisense oligonucleotides with mismatches in inhibiting expression of MLH1 is important, especially if the particular region of complementarity in a MLH1 gene is known to have polymorphic sequence variation within the population.

Construction of vectors for expression of polynucleotides can be accomplished using conventional techniques which do not require detailed explanation to one of ordinary skill in the art. For generation of efficient expression vectors, it is necessary to have regulatory sequences that control the expression of the polynucleotide. These regulatory sequences include promoter and enhancer sequences and are influenced by specific cellular factors that interact with these sequences, and are well known in the art.

B. dsRNA Agents

In some aspects, the agent that reduces the level and/or activity of MLH1 is a polynucleotide. In some aspects, the polynucleotide is an inhibitory RNA molecule, e.g., that acts by way of the RNA interference (RNAi) pathway. An inhibitory RNA molecule can decrease the expression level (e.g., protein level or mRNA level) of MLH1. Inhibitory RNA molecules can be double stranded (dsRNA) molecules. For example, a dsRNA includes a short interfering RNA (siRNA) that targets full-length MLH1. A siRNA is a double-stranded RNA molecule that typically has a length of about 19-25 base pairs. In other aspects, the dsRNA is a short hairpin RNA (shRNA) that targets full-length MLH1. A shRNA is a dsRNA molecule including a hairpin turn that decreases expression of target genes via the RNAi pathway. In some aspects, the dsRNA molecule recruits an RNAse H enzyme. Degradation is caused by an enzymatic, RNA-induced silencing complex (RISC).

In some aspects, the dsRNA decreases the level and/or activity of a positive regulator of function. In other aspects, the dsRNA increases the level and/or activity of an inhibitor of a positive regulator of function. In some aspects, the dsRNA increases the level and/or activity of a negative regulator of function.

In some aspects, the dsRNA decreases the level and/or activity or function of MLH1. In some aspects, the dsRNA reduces expression of MLH1. In other aspects, the dsRNA increases degradation of MLH1 and/or decreases the stability (i.e., half-life) of MLH1. The dsRNA can be chemically synthesized or transcribed in vitro.

The dsRNA includes an antisense strand having a region of complementarity (e.g., a contiguous nucleobase region) which is complementary to at least a part of an mRNA formed in the expression of a MLH1 gene. The region of complementarity can be about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the MLH1 gene, the dsRNA can reduce the expression of MLH1 (e.g., a human, a primate, a non-primate, or a bird MLH1) by at least about 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, Western Blotting or flowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA can be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of a MLH1 gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides. Generally, the duplex structure is between 15 and 30 linked nucleosides in length, e.g., 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, 18-22, 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 linked nucleosides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated.

Similarly, the region of complementarity to the target sequence is between 15 and 30 linked nucleosides in length, e.g., 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, 18-22, 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 linked nucleosides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated.

In some aspects, the dsRNA is between about 15 and about 23 linked nucleosides in length, or between about 25 and about 30 linked nucleosides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 linked nucleosides can serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA. Thus, in one aspect, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 linked nucleosides, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 linked nucleosides is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one aspect, a dsRNA is not a naturally occurring dsRNA. In another aspect, a dsRNA agent useful to target MLH1 expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucleoside overhangs e.g., 1, 2, 3, or 4 linked nucleosides. dsRNAs having at least one nucleoside overhang can have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. A nucleoside overhang can comprise or consist of a deoxyribonucleoside. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleoside(s) of an overhang can be present on the 5′-end, 3′-end, or both ends of either an antisense or sense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

dsRNA compounds can be prepared using a two-step procedure. For example, the individual strands of the dsRNA are prepared separately. Then, the component strands are annealed. The individual strands of the dsRNA can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or alternative nucleotides can be easily prepared. Double-stranded oligonucleotides can be prepared using solution-phase or solid-phase organic synthesis or both.

In one aspect, a dsRNA includes at least two nucleobase sequences, a sense sequence and an antisense sequence. In some aspects, the antisense strand comprises a nucleobase sequence of an antisense strand in Table 4, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T), and the sense strand comprises a nucleobase sequence complementary to the nucleobase sequence of the antisense strand. In other aspects, the sense strand comprises a nucleobase sequence of a sense strand in Table 4, and the antisense strand comprises a nucleobase sequence complementary to the nucleobase sequence of the sense strand. In some aspects, the antisense strand consists of a nucleobase sequence of an antisense strand in Table 4, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T), and the sense strand consists of a nucleobase sequence complementary to the nucleobase sequence of the antisense strand. In other aspects, the sense strand consists of a nucleobase sequence of a sense strand in Table 4, and the antisense strand consists of a nucleobase sequence complementary to the nucleobase sequence of the sense strand. In some aspects, the sense strand comprises a nucleobase sequence of a sense strand in any one of Tables 5-11, and the antisense strand comprises a nucleobase sequence complementary to the nucleobase sequence of the sense strand. In some aspects, the sense strand consists of a nucleobase sequence of a sense strand in any one of Tables 5-11, and the antisense strand consists of a nucleobase sequence complementary to the nucleobase sequence of the sense strand. In some aspects, the antisense strand comprises a nucleobase sequence of an antisense strand in Table 13, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T), and the sense strand comprises a nucleobase sequence complementary to the nucleobase sequence of the antisense strand. In other aspects, the antisense strand consists of a nucleobase sequence of an antisense strand in Table 13, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T), and the sense strand consists of a nucleobase sequence complementary to the nucleobase sequence of the antisense strand. In some aspects, the sense strand comprises a nucleobase sequence of a sense strand in Table 13, and the antisense strand comprises a nucleobase sequence complementary to the nucleobase sequence of the sense strand. In other aspects, the sense strand consists of a nucleobase sequence of a sense strand in Table 13, and the antisense strand consists of a nucleobase sequence complementary to the nucleobase sequence of the sense strand. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of MLH1. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand (passenger strand) in Table 4, and the second oligonucleotide is described as the corresponding antisense strand (guide strand) of the sense strand in Table 4, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In one aspect, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another aspect, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

In one aspect, the antisense or sense strand of the dsRNA includes a region of at least 15 contiguous nucleobases having at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) complementary to at least 15 contiguous nucleotides of an MLH1 gene. In some aspects, the region of at least 15 contiguous that is complementary to an MLH1 gene corresponding to reference mRNA NM_000249.3 is one or more of positions 153-176, 267-388, 417-545, 792-995, 1639-1727, 1849-1900, 2105-2207, 2337-2387, 2426-2479 and 2508-2600 of the MLH1 gene. In some aspects, the region of at least 15 contiguous that is complementary to an MLH1 gene corresponding to reference mRNA NM_000249.3 is one or more of positions 326-388, 459-511, 805-878, 903-926, 1639-1720, and 2141-2192 of the MLH1 gene. In some aspects, the region of at least 15 contiguous that is complementary to an MLH1 gene corresponding to reference mRNA NM_000249.3 is one or more of positions 267-388, 417-545, 805-878, 903-995, 1639-1727, 1849-1900, 2141-2207, 2337-2387, and 2426-2479 of the MLH1 gene. In some aspects, the region of at least 15 contiguous that is complementary to an MLH1 gene corresponding to reference mRNA NM_000249.3 is one or more of positions 153-176, 267-388, 417-545, 792-995, 1639-1727, 1849-1900, 2105-2207, 2337-2387, and 2426-2479 of the MLH1 gene. In some aspects, the region of at least 15 contiguous that is complementary to an MLH1 gene corresponding to reference mRNA NM_000249.3 is one or more of positions 332-355, 459-545, 836-859, 1849-1900, 2141-2164, and 2426-2449 of the MLH1 gene. In some aspects, the region of at least 15 contiguous that is complementary to an MLH1 gene corresponding to reference mRNA NM_000249.3 is one or more of positions 267-388, 417-545, 805-995, 1639-1722, 1849-1900, 2105-2207, 2337-2387, 2426-2479, and 2508-2600 of the MLH1 gene.

In some aspects, a dsRNA having a sense strand or an antisense strand comprises the nucleobase sequence of any one of SEQ ID NOs: 1394-3353, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

In some aspects, the sense strand of the dsRNA comprises nucleobase sequence of any one of SEQ ID NOs: 1486, 1492, 1494, 1496, 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, 3164, 3210, 3212, 3216, 3248, 3272, 3274, 3276, 3278, and 3288. In some aspects, the sense strand of the dsRNA comprises nucleobase sequence of any one of SEQ ID NOs: 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, and 2966. In some aspects, the sense strand of the dsRNA comprises nucleobase sequence of any one of SEQ ID NOs: 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, and 3164. In some aspects, the sense strand of the dsRNA comprises nucleobase sequence of any one of SEQ ID NOs: 1486, 1492, 1494, 1496, 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, and 3164. In some aspects, the sense strand of the dsRNA comprises nucleobase sequence of any one of SEQ ID NOs: 1584, 1598, 1604, 1608, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1998, 2008, 2012, 2024, 2720, 2744, 2746, 2748, 2752, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 3120, 3122, 3124, 3130, 3134, 3136, and 3148. In some aspects, the sense strand of the dsRNA comprises nucleobase sequence of any one of SEQ ID NOs: 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, 3164, 3210, 3212, 3216, 3248, 3272, 3274, 3276, 3278, and 3288.

In some aspects, the antisense strand of the dsRNA comprises nucleobase sequence of any one of SEQ ID NOs: 1487, 1493, 1495, 1497, 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, 3165, 3211, 3213, 3217, 3249, 3273, 3275, 3277, 3279, and 3289, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand of the dsRNA comprises nucleobase sequence of any one of SEQ ID NOs: 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, and 2967, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand of the dsRNA comprises nucleobase sequence of any one of SEQ ID NOs: 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, and 3165, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand of the dsRNA comprises nucleobase sequence of any one of SEQ ID NOs: 1487, 1493, 1495, 1497, 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, and 3165, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand of the dsRNA comprises nucleobase sequence of any one of SEQ ID NOs: 1585, 1599, 1605, 1609, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1999, 2009, 2013, 2025, 2721, 2745, 2747, 2749, 2753, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 3121, 3123, 3125, 3131, 3135, 3137, and 3149, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand of the dsRNA comprises nucleobase sequence of any one of SEQ ID NOs: 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, 3165, 3211, 3213, 3217, 3249, 3273, 3275, 3277, 3279, and 3289, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

In some aspects, a dsRNA having a sense strand or an antisense strand consists of the nucleobase sequence of any one of SEQ ID NOs: 1394-3353, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

In some aspects, the sense strand of the dsRNA consists of nucleobase sequence of any one of SEQ ID NOs: 1486, 1492, 1494, 1496, 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, 3164, 3210, 3212, 3216, 3248, 3272, 3274, 3276, 3278, and 3288. In some aspects, the sense strand of the dsRNA consists of nucleobase sequence of any one of SEQ ID NOs: 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, and 2966. In some aspects, the sense strand of the dsRNA consists of nucleobase sequence of any one of SEQ ID NOs: 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, and 3164. In some aspects, the sense strand of the dsRNA consists of nucleobase sequence of any one of SEQ ID NOs: 1486, 1492, 1494, 1496, 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, and 3164. In some aspects, the sense strand of the dsRNA consists of nucleobase sequence of any one of SEQ ID NOs: 1584, 1598, 1604, 1608, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1998, 2008, 2012, 2024, 2720, 2744, 2746, 2748, 2752, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 3120, 3122, 3124, 3130, 3134, 3136, and 3148. In some aspects, the sense strand of the dsRNA consists of nucleobase sequence of any one of SEQ ID NOs: 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, 3164, 3210, 3212, 3216, 3248, 3272, 3274, 3276, 3278, and 3288.

In some aspects, the antisense strand of the dsRNA consists of nucleobase sequence of any one of SEQ ID NOs: 1487, 1493, 1495, 1497, 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, 3165, 3211, 3213, 3217, 3249, 3273, 3275, 3277, 3279, and 3289, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand of the dsRNA consists of nucleobase sequence of any one of SEQ ID NOs: 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, and 2967, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand of the dsRNA consists of nucleobase sequence of any one of SEQ ID NOs: 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, and 3165, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand of the dsRNA consists of nucleobase sequence of any one of SEQ ID NOs: 1487, 1493, 1495, 1497, 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, and 3165, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand of the dsRNA consists of nucleobase sequence of any one of SEQ ID NOs: 1585, 1599, 1605, 1609, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1999, 2009, 2013, 2025, 2721, 2745, 2747, 2749, 2753, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 3121, 3123, 3125, 3131, 3135, 3137, and 3149, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, the antisense strand of the dsRNA consists of nucleobase sequence of any one of SEQ ID NOs: 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, 3165, 3211, 3213, 3217, 3249, 3273, 3275, 3277, 3279, and 3289, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

In some aspects, the dsRNA exhibits at least 50% mRNA inhibition at 0.5 nM dsRNA concentration when determined using a cell assay when compared with a control cell. In some aspects, the dsRNA exhibits at least 40% mRNA inhibition at 0.5 nM dsRNA concentration when determined using a cell assay when compared with a control cell. In some aspects, the dsRNA exhibits at least 30% mRNA inhibition at 0.5 nM dsRNA concentration when determined using a cell assay when compared with a control cell. In some aspects, the dsRNA exhibits at least 60% mRNA inhibition at 10 nM dsRNA concentration when determined using a cell assay when compared with a control cell. In some aspects, the dsRNA exhibits at least 50% mRNA inhibition at 10 nM dsRNA concentration when determined using a cell assay when compared with a control cell.

In some aspects, the dsRNA comprises an antisense strand that is complementary to at least 17 contiguous nucleotides of an MLH1 gene. In other aspects, the dsRNA comprises an antisense strand that is complementary to at least 19 contiguous nucleotides of an MLH1 gene. In other aspects, the dsRNA comprises an antisense strand that is complementary to 19 contiguous nucleotides of an MLH1 gene.

Multiple dsRNAs can be joined together by a linker. The linker can be cleavable or non-cleavable. The dsRNAs can be the same or different.

In some aspects, a dsRNA has a sense strand or an antisense strand having a nucleobase sequence with at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the nucleobase sequence any one of SEQ ID NOs: 1394-3353, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). In some aspects, a dsRNA has a sense strand or an antisense strand having a nucleobase sequence with at least 85% sequence identity to the nucleobase sequence of any one of SEQ ID NOs: 1394-3353, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

It will be understood that, although the sequences in SEQ ID NOs: 1394-3353 are described as unmodified and/or un-conjugated sequences, the RNA of the dsRNA] can comprise any one of the sequences set forth in any one of SEQ ID NOs: 1394-3353 that is an alternative nucleoside and/or conjugated as described in detail below.

The skilled person is well aware that dsRNAs having a duplex structure of between about 20 and 23 linked nucleosides, e.g., 21 linked nucleosides, have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the aspects described above, by virtue of the nature of the oligonucleotide sequences provided herein, dsRNAs described herein can include at least one strand of a length of minimally 21 linked nucleosides. It can be reasonably expected that shorter duplexes minus only a few linked nucleosides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous linked nucleosides derived from one of the sequences provided herein, and differing in their ability to reduce the expression of MLH1 by not more than about 5, 10, 15, 20, 25, or 30% reduction from a dsRNA comprising the full sequence, are contemplated.

In addition, the RNAs described herein identify a site(s) in a MLH1 transcript that is susceptible to RISC-mediated cleavage. As used herein, a dsRNA is said to target within a particular site of an RNA transcript if the dsRNA promotes cleavage of the transcript anywhere within that particular site. Such a dsRNA will generally include at least about 15 contiguous linked nucleosides from one of the sequences provided herein coupled to additional linked nucleoside sequences taken from the region contiguous to the selected sequence in a MLH1 gene.

Inhibitory dsRNAs can be designed by methods well known in the art. While a target sequence is generally about 15-30 linked nucleosides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA.

dsRNAs (e.g., siRNA and shRNA molecules) with homology sufficient to provide sequence specificity required to uniquely degrade any RNA can be designed using programs known in the art.

Systematic testing of several designed species for optimization of the inhibitory dsRNA sequence can be undertaken in accordance with the teachings provided herein. Considerations when designing interfering oligonucleotides include, but are not limited to, biophysical, thermodynamic, and structural considerations, base preferences at specific positions in the sense strand, and homology. The making and use of inhibitory therapeutic agents based on non-coding RNA such as siRNAs and shRNAs are also known in the art, for example, as described in Sioud, RNA Therapeutics: Function, Design, and Delivery (Methods in Molecular Biology). Humana Press 2010.

Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with a dsRNA agent, mediate the best reduction of target gene expression. Thus, while the sequences identified herein represent effective target sequences, it is contemplated that further optimization of reduction efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better reduction characteristics.

Further, it is contemplated that for any sequence identified herein, further optimization could be achieved by systematically either adding or removing linked nucleosides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of dsRNAs based on those target sequences in an inhibition assay as known in the art and/or as described herein can lead to further improvements in the efficiency of inhibition.

Further still, such optimized sequences can be adjusted by, e.g., addition or changes in overhang, the introduction of alternative nucleosides, alternative sugar moieties, and/or alternative internucleosidic linkages as described herein or as known in the art, including alternative nucleosides, alternative sugar moieties, and/or alternative internucleosidic linkages as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor. A dsRNA agent as described herein can contain one or more mismatches to the target sequence. In one aspect, a dsRNA as described herein contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not located in the center of the region of complementarity.

If the antisense strand of the dsRNA contains mismatches to the target sequence, the mismatch can be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, for a 23-nucleotide dsRNA, the strand which is complementary to a region of a MLH1 gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in reducing the expression of MLH1. Consideration of the efficacy of dsRNAs with mismatches in reducing expression of MLH1 is important, especially if the particular region of complementarity in MLH1 is known to have polymorphic sequence variation within the population.

C. Alternative Antisense Oligonucleosides or dsRNA

In one aspect, one or more of the linked nucleosides or internucleosidic linkages of the antisense oligonucleotide or dsRNA, is naturally occurring, and does not comprise, e.g., chemical modifications and/or conjugations known in the art and described herein. In another aspect, one or more of the linked nucleosides or internucleosidic linkages of an antisense oligonucleotide or dsRNA, is chemically modified to enhance stability or other beneficial characteristics. Without being bound by theory, it is believed that certain modifications can increase nuclease resistance and/or serum stability, or decrease immunogenicity. For example, antisense oligonucleotides or dsRNAs can contain nucleotides found to occur naturally in DNA or RNA (e.g., adenine, thymidine, guanosine, cytidine, uridine, or inosine) or can contain alternative nucleosides or internucleosidic linkages which have one or more chemical modifications to one or more components of the nucleotide (e.g., the nucleobase, sugar, or phospho-linker moiety). Antisense oligonucleotides or dsRNAs can be linked to one another through naturally occurring phosphodiester bonds, or can contain alternative linkages (e.g., covalently linked through phosphorothioate (e.g., Sp phosphorothioate or Rp phosphorothioate), 3′-methylenephosphonate, 5′-methylenephosphonate, 3′-phosphoamidate, 2′-5′ phosphodiester, guanidinium, S-methylthiourea, 2′-alkoxy, alkyl phosphate, and/or peptide bonds).

In certain aspects, substantially all of the nucleosides or internucleosidic linkages of an antisense oligonucleotide or dsRNA are alternative nucleosides. In other aspects, all of the nucleosides or internucleosidic linkages of an antisense oligonucleotide or dsRNA are alternative nucleosides. Antisense oligonucleotides or dsRNAs in which “substantially all of the nucleosides are alternative nucleosides” are largely but not wholly modified and can include not more than five, four, three, two, or one naturally-occurring nucleosides. In still other aspects, antisense oligonucleotides or dsRNAs can include not more than five, four, three, two, or one alternative nucleosides.

The nucleic acids can be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Alternative nucleotides and nucleosides include those with modifications including, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; and/or backbone modifications, including modification or replacement of the phosphodiester linkages. The nucleobase can be an isonucleoside in which the nucleobase is moved from the C1 position of the sugar moiety to a different position (e.g. C2, C3, C4, or C5). Specific examples of antisense oligonucleotide or dsRNA compounds useful in the aspects described herein include, but are not limited to alternative nucleosides containing modified backbones or no natural internucleoside linkages. Nucleotides and nucleosides having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, alternative RNAs that do not have a phosphorus atom in their internucleoside backbone can be considered to be oligonucleosides. In some aspects, an antisense oligonucleotide or dsRNA will have a phosphorus atom in its internucleoside backbone.

Alternative internucleoside linkages include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boronophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts, and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. 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; 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; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

Alternative internucleoside linkages that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH₂component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. 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; 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.

In other aspects, suitable antisense oligonucleotides or dsRNAs include those in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, a mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar of a nucleoside is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the antisense oligonucleotides or dsRNAs are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some aspects include antisense oligonucleotides or dsRNAs with phosphorothioate backbones and antisense oligonucleotides or dsRNAs with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂-[known as a 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 above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some aspects, the antisense oligonucleotides or dsRNA featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506. In other aspects, the antisense oligonucleotides or dsRNAs described herein include phosphorodiamidate morpholino oligomers (PMO), in which the deoxyribose moiety is replaced by a morpholine ring, and the charged phosphodiester inter-subunit linkage is replaced by an uncharged phophorodiamidate linkage, as described in Summerton, et al., Antisense Nucleic Acid Drug Dev. 1997, 7:63-70.

Alternative nucleosides and nucleotides can contain one or more substituted sugar moieties. The antisense oligonucleotides or dsRNAs, e.g., siRNAs and shRNAs, featured herein can 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 can be substituted or unsubstituted C₁to C₁₀alkyl or C₂to C₁₀alkenyl and alkynyl. Exemplary suitable modifications include —O[(CH₂)_nO]_mCH₃, —O(CH₂)_nOCH₃, —O(CH₂)_n—NH₂, —O(CH₂)_nCH₃, —O(CH₂)_n—ONH₂, and —O(CH₂)_n—ON[(CH₂)_nCH₃]₂, where n and m are from 1 to about 10. In other aspects, antisense oligonucleotides or dsRNAs include one of the following at the 2′ position: C₁to C₁₀lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an antisense oligonucleotide or dsRNA, or a group for improving the pharmacodynamic properties of an antisense oligonucleotide or dsRNA, and other substituents having similar properties. In some aspects, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chin. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. MOE nucleosides confer several beneficial properties to antisense oligonucleotides or dsRNAs including, but not limited to, increased nuclease resistance, improved pharmacokinetics properties, reduced non-specific protein binding, reduced toxicity, reduced immunostimulatory properties, and enhanced target affinity as compared to unmodified antisense oligonucleotides or dsRNAs.

Another exemplary alternative contains 2′-dimethylaminooxyethoxy, i.e., a —O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—(CH₂)₂—O—(CH₂)₂—N(CH₃)₂. Further exemplary alternatives include: 5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides, 5′-Me-2′-deoxynucleotides, (both R and S isomers in these three families); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other alternatives include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can be made at other positions on the nucleosides and nucleotides of an antisense oligonucleotide or dsRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked antisense oligonucleotides or dsRNAs and the 5′ position of 5′ terminal nucleotide. Antisense oligonucleotides or dsRNAs can have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. 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, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

An antisense oligonucleotide or dsRNA can include nucleobase (often referred to in the art simply as “base”) alternatives (e.g., modifications or substitutions). Unmodified or natural nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Alternative nucleobases include other synthetic and natural nucleobases such as 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, pyrrolocytidine, dideoxycytidine, uridine, 5-methoxyuridine, 5-hydroxydeoxyuridine, dihydrouridine, 4-thiourdine, pseudouridine, 1-methyl-pseudouridine, deoxyuridine, 5-hydroxybutynl-2′-deoxyuridine, xanthine, hypoxanthine, 7-deaza-xanthine, thienoguanine, 8-aza-7-deazaguanosine, 7-methylguanosine, 7-deazaguanosine, 6-aminomethyl-7-deazaguanosine, 8-aminoguanine, 2,2,7-trimethylguanosine, 8-methyladenine, 8-azidoadenine, 7-methyladenine, 7-deazaadenine, 3-deazaadenine, 2,6-diaminopurine, 2-aminopurine, 7-deaza-8-aza-adenine, 8-amino-adenine, thymine, dideoxythymine, 5-nitroindole, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouridine, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uridine and cytidine, 6-azo uridine, cytidine and thymine, 4-thiouridine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uridines and cytidines, 8-azaguanine and 8-azaadenine, and 3-deazaguanine. Further nucleobases include those disclosed in U.S. Pat. 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, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the antisense oligonucleotides or dsRNAs. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted alternative nucleobases as well as other alternative nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 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; 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.

In other aspects, the sugar moiety in the nucleotide can be a ribose molecule, optionally having a 2′-O-methyl, 2′-O-MOE, 2′-F, 2′-amino, 2′-O-propyl, 2′-aminopropyl, or 2′-OH modification.

An antisense oligonucleotide or dsRNA can include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In some aspects, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some aspects an antisense oligonucleotide or dsRNA can include one or more locked nucleosides. A locked nucleoside is a nucleoside having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. In other words, a locked nucleoside is a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′ bridge. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleosides to antisense oligonucleotides or dsRNAs has been shown to increase antisense oligonucleotide or dsRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. 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 for use in the polynucleotides include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In some aspects, the antisense polynucleotide agents include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, 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 referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH₂—O—N(CH₃)₂-2′ (see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH₂—N(R)—O-2′, wherein R is H, C1-C12 alkyl, ora protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.

Additional representative U.S. Patents and US Patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. 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.

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and 13-D-ribofuranose (see WO 99/14226).

An antisense oligonucleotide or dsRNA can be modified to include one or more constrained ethyl nucleosides. As used herein, a “constrained ethyl nucleoside” or “cEt” is a locked nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge. In one aspect, a constrained ethyl nucleoside is in the S conformation referred to herein as “S-cEt.”

An antisense oligonucleotide or dsRNA can include one or more “conformationally restricted nucleosides” (“CRN”). CRN are nucleoside analogs with a linker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to 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.

Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US Patent Publication No. 2013/0190383; and PCT publication WO 2013/036868, the entire contents of each of which are hereby incorporated herein by reference.

In some aspects, an antisense oligonucleotide or dsRNA comprises one or more monomers that are UNA (unlocked nucleoside) nucleosides. UNA is unlocked acyclic nucleoside, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated by reference).

Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference.

The ribose molecule can be modified with a cyclopropane ring to produce a tricyclodeoxynucleic acid (tricyclo DNA). The ribose moiety can be substituted for another sugar such as 1,5,-anhydrohexitol, threose to produce a threose nucleoside (TNA), or arabinose to produce an arabino nucleoside. The ribose molecule can also be replaced with non-sugars such as cyclohexene to produce cyclohexene nucleoside or glycol to produce glycol nucleosides.

Potentially stabilizing modifications to the ends of nucleoside molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.

Other alternatives chemistries of an antisense oligonucleotide or a dsRNA include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic of an antisense oligonucleotide or on the antisense strand of a dsRNA. Suitable phosphate mimics are disclosed in, for example US Patent Publication No. 2012/0157511, the entire contents of which are incorporated herein by reference.

Exemplary antisense oligonucleotides or dsRNA comprise nucleosides with alternative sugar moieties and can comprise DNA or RNA nucleosides. In some aspects, the antisense oligonucleotide or dsRNA comprises nucleosides comprising alternative sugar moieties and DNA nucleosides. Incorporation of alternative nucleosides into the antisense oligonucleotide or dsRNA can enhance the affinity of the antisense oligonucleotide or dsRNA for the target nucleic acid. In that case, the alternative nucleosides can be referred to as affinity enhancing alternative nucleotides.

In some aspects, the antisense oligonucleotide or dsRNA comprises at least 1 alternative nucleoside, such as 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 11, at least 12, at least 13, at least 14, at least 15 or at least 16 alternative nucleosides. In other aspects, the antisense oligonucleotides or dsRNAs comprise from 1 to 10 alternative nucleosides, such as from 2 to 9 alternative nucleosides, such as from 3 to 8 alternative nucleosides, such as from 4 to 7 alternative nucleosides, such as 6 or 7 alternative nucleosides. In an aspect, the antisense oligonucleotide or dsRNA can comprise alternatives, which are independently selected from these three types of alternative (alternative sugar moiety, alternative nucleobase, and alternative internucleoside linkage), or a combination thereof. Preferably the oligonucleotide comprises one or more nucleosides comprising alternative sugar moieties, e.g., 2′ sugar alternative nucleosides. In some aspects, the antisense oligonucleotide or dsRNA comprises the one or more 2′ sugar alternative nucleoside independently selected from the group consisting of 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, arabino nucleic acid (ANA), 2′-fluoro-ANA, and BNA (e.g., LNA) nucleosides. In some aspects, the one or more alternative nucleoside is a BNA.

In some aspects, at least 1 of the alternative nucleosides is a BNA (e.g., an LNA), such as at least 2, such as at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 of the alternative nucleosides are BNAs. In a still further aspect, all the alternative nucleosides are BNAs.

In a further aspect, the antisense oligonucleotide or dsRNA comprises at least one alternative internucleoside linkage. In some aspects, the internucleoside linkages within the contiguous nucleotide sequence are phosphorothioate or boronophosphate internucleoside linkages. In some aspects, all the internucleotide linkages in the contiguous sequence of the antisense oligonucleotide or dsRNA are phosphorothioate linkages. In some aspects the phosphorothioate linkages are stereochemically pure phosphorothioate linkages. In some aspects, the phosphorothioate linkages are Sp phosphorothioate linkages. In other aspects, the phosphorothioate linkages are Rp phosphorothioate linkages.

In some aspects, the antisense oligonucleotide or dsRNA comprises at least one alternative nucleoside which is a 2′-MOE-RNA, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 2′-MOE-RNA nucleoside units. In some aspects, the 2′-MOE-RNA nucleoside units are connected by phosphorothioate linkages. In some aspects, at least one of said alternative nucleoside is 2′-fluoro DNA, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 2′-fluoro-DNA nucleoside units. In some aspects, the antisense oligonucleotide or dsRNA comprises at least one BNA unit and at least one 2′ substituted modified nucleoside. In some aspects, the antisense oligonucleotide or dsRNA comprises both 2′ sugar modified nucleosides and DNA units. In some aspects, the antisense oligonucleotide or contiguous nucleotide region thereof is a gapmer oligonucleotide.

D. Antisense Oligonucleotides or dsRNAs Conjugated to Ligands

Antisense oligonucleotides or dsRNAs can be chemically linked to one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide or dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86: 6553-6556), cholic acid (Manoharan et al., (1994) Biorg. Med. Chem. Let., 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., (1992) Ann. N.Y. Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), a thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids Res., 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), a palmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

In one aspect, a ligand alters the distribution, targeting, or lifetime of an antisense oligonucleotide or dsRNA agent into which it is incorporated. In some aspects, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ, or region of the body, as, e.g., compared to a species absent such a ligand. Some ligands will not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can include hormones and hormone receptors. They can include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the antisense oligonucleotide or dsRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some aspects, a ligand attached to an antisense oligonucleotide or dsRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Antisense nucleotides or dsRNAs that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short antisense oligonucleotides or dsRNAs, e.g., antisense oligonucleotides or dsRNAs of about 5 bases, 10 bases, 15 bases, or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the aspects described herein.

Ligand-conjugated antisense oligonucleotides or dsRNAs can be synthesized by the use of an antisense oligonucleotide or dsRNA that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the antisense oligonucleotide or dsRNA (described below). This reactive antisense oligonucleotide or dsRNA can be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The antisense oligonucleotides or dsRNA used in the conjugates can be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art can additionally or alternatively be employed. It is also known to use similar techniques to prepare other antisense oligonucleotides or dsRNAs, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated antisense oligonucleotides or dsRNAs, such as the ligand-molecule bearing sequence-specific linked nucleosides, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated antisense oligonucleotide or dsRNA. In some aspects, the antisense oligonucleotides or dsRNAs are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

i. Lipid Conjugates

In one aspect, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule can bind a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. Exemplary vitamins include vitamin A, E, and K.

ii. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, such a helical cell-permeation agent. In one aspect, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. In one aspect, the helical agent is an alpha-helical agent which can have a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to antisense oligonucleotide or dsRNA agents can affect pharmacokinetic distribution of the antisense oligonucleotide or dsRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP. An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP containing a hydrophobic MTS can be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to an antisense oligonucleotide or dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods described herein can be linear or cyclic, and can be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics can include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Some conjugates of this ligand target PECAM-1 or VEGF.

A cell permeation peptide is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin, or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

iii. Carbohydrate Conjugates

In some aspects of the compositions and methods described herein, an antisense oligonucleotide or dsRNA further comprises a carbohydrate. The carbohydrate conjugated antisense oligonucleotides are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In one aspect, a carbohydrate conjugate for use in the compositions and methods described herein is a monosaccharide.

In some aspects, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.

Additional carbohydrate conjugates (and linkers) suitable for use include those described in PCT Publication Nos. WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

iv. Linkers

In some aspects, the conjugate or ligand described herein can be attached to an antisense oligonucleotide or a dsRNA with various linkers that can be cleavable or non-cleavable.

Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR⁸, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R⁸), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R⁸is hydrogen, acyl, aliphatic or substituted aliphatic. In one aspect, the linker is between about 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-17, 8-16 or 1, 2, 3, 4 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1516, 17, 18, 19, 20, 21, 22 23, or 24 atoms.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In some aspects, the cleavable linking group is cleaved at least about 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential, or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selective for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between at least two conditions, where at least one condition is selected to be indicative of cleavage in a target cell and another condition is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In some aspects, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

a. Redox Cleavable Linking Groups

In one aspect, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular antisense oligonucleotide or dsRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can be evaluated under conditions which are selected to mimic blood or serum conditions. In one aspect, candidate compounds are cleaved by at most about 10% in the blood. In other aspects, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

b. Phosphate-Based Cleavable Linking Groups

In another aspect, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(OR^k)—O—, —O—P(S)(OR^k)—O—, —O—P(S)(SR^k)—O—, —S—P(O)(OR^k)—O—, —O—P(O)(OR^k)—S—, —S—P(O)(OR^k)—S—, —O—P(S)(OR^k)—S—, —S—P(S)(OR^k)—O—, —O—P(O)(R^k)—O—, —O—P(S)(R^k)—O—, —S—P(O)(R^k)—O—, —S—P(S)(R^k)—O—, —S—P(O)(R^k)—S—, —O—P(S)(R^k)—S—. These candidates can be evaluated using methods analogous to those described above.

c. Acid Cleavable Linking Groups

In another aspect, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In some aspects, acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). In one aspect, the carbon is attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

d. Ester-Based Linking Groups

In another aspect, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

e. Peptide-Based Cleaving Groups

In yet another aspect, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHR^AC(O)NHCHR^BC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

In one aspect, an oligonucleotide or dsRNA is conjugated to a carbohydrate through a linker. Linkers include bivalent and trivalent branched linker groups. Linkers for antisense oligonucleotide or dsRNA carbohydrate conjugates include, but are not limited to, those described in formulas 24-35 of PCT Publication No. WO 2018/195165.

Representative U.S. patents that teach the preparation of antisense oligonucleotide or dsRNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an antisense oligonucleotide or dsRNA. Antisense oligonucleotide or dsRNA compounds that are chimeric compounds are also contemplated. Chimeric antisense oligonucleotides or chimeric dsRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the antisense oligonucleotide or dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the antisense oligonucleotide or dsRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense oligonucleotide inhibition or dsRNA reduction of gene expression. Consequently, comparable results can often be obtained with shorter antisense oligonucleotides or dsRNAs when chimeric antisense oligonucleotides or chimeric dsRNAs are used, compared to phosphorothioate deoxy antisense oligonucleotides or dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In some instances, the nucleotides of an antisense oligonucleotide or nucleosides of a dsRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to antisense oligonucleotides or dsRNAs to enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide or dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm, 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such antisense oligonucleotide or dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an antisense oligonucleotide or dsRNA bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the antisense oligonucleotide or dsRNA still bound to the solid support or following cleavage of the antisense oligonucleotide or dsRNA, in solution phase. Purification of the antisense oligonucleotide or dsRNA conjugate by HPLC typically affords the pure conjugate.

IV. Pharmaceutical Uses

The antisense oligonucleotide or dsRNA compositions described herein are useful in the methods described herein and, while not bound by theory, are believed to exert their desirable effects through their ability to modulate the level, status, and/or activity of a MutLa heterodimer comprising MLH1, e.g., by inhibiting or reducing the activity or level of the MLH1 protein in a cell in a mammal.

An aspect relates to methods of treating disorders related to DNA mismatch repair such as trinucleotide repeat expansion disorders in a subject in need thereof. Another aspect includes reducing the level of MLH1 in a cell of a subject identified as having a trinucleotide repeat expansion disorder. Still another aspect includes a method of inhibiting or reducing expression of MLH1 in a cell in a subject. Further aspects include methods of decreasing trinucleotide repeat expansion in a cell. The methods include contacting a cell with an antisense oligonucleotide or dsRNA, in an amount effective to inhibit or reduce expression of MLH1 in the cell, thereby inhibiting or reducing expression of MLH1 in the cell.

Based on the above methods, an antisense oligonucleotide or dsRNA, or a composition comprising such an antisense oligonucleotide or dsRNA, for use in therapy, or for use as a medicament, or for use in treating disorders related to DNA mismatch repair such as trinucleotide repeat expansion disorders in a subject in need thereof, or for use in reducing the level of MLH1 in a cell of a subject identified as having a trinucleotide repeat expansion disorder, or for use in inhibiting or reducing expression of MLH1 in a cell in a subject, or for use in decreasing trinucleotide repeat expansion in a cell is contemplated. The uses include the contacting of a cell with the antisense oligonucleotide or dsRNA, in an amount effective to inhibit or reduce expression of MLH1 in the cell, thereby inhibiting or reducing expression of MLH1 in the cell. Aspects described below in relation to the methods described herein are also applicable to these further aspects.

Contacting of a cell with an antisense oligonucleotide or dsRNA can be done in vitro or in vivo. Contacting a cell in vivo with the antisense oligonucleotide or dsRNA includes contacting a cell or group of cells within a subject, e.g., a human subject, with the antisense oligonucleotide or dsRNA. Combinations of in vitro and in vivo methods of contacting a cell are also possible. Contacting a cell can be direct or indirect, as discussed above. Furthermore, contacting a cell can be accomplished via a targeting ligand, including any ligand described herein or known in the art. In some aspects, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc3 ligand, or any other ligand that directs the antisense oligonucleotide or dsRNA to a site of interest. Cells can include those of the central nervous system, or muscle cells.

Inhibiting or reducing expression of MLH1 includes any level of inhibition or reduction of MLH1, e.g., at least partial suppression of the expression of MLH1, such as an inhibition or reduction by at least about 20%. In some aspects, inhibition or reduction is by 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%.

The expression of MLH1 can be assessed based on the level of any variable associated with MLH1 gene expression, e.g., MLH1 mRNA level or MLH1 protein level.

Inhibition or reduction can be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level can be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

In some aspects, surrogate markers can be used to detect inhibition or reduction of MLH1. For example, effective treatment of a trinucleotide repeat expansion disorder, as demonstrated by acceptable diagnostic and monitoring criteria with an agent to reduce MLH1 expression can be understood to demonstrate a clinically relevant reduction in MLH1.

In some aspects of the methods, expression of MLH1 is inhibited or reduced by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay. In some aspects, the methods include a clinically relevant inhibition or reduction of expression of MLH1, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of MLH1.

Inhibition or reduction of the expression of MLH1 can be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells can be present, for example, in a sample derived from a subject) in which MLH1 is transcribed and which has or have been treated (e.g., by contacting the cell or cells with an antisense oligonucleotide or dsRNA, or by administering an antisense oligonucleotide or dsRNA to a subject in which the cells are or were present) such that the expression of MLH1 is inhibited or reduced, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s) not treated with an antisense oligonucleotide or dsRNA or not treated with an antisense oligonucleotide or dsRNA targeted to the gene of interest). The degree of inhibition or reduction can be expressed in terms of:

$\frac{(mRNA in control cells) - (mRNA in treated cells)}{(mRNA in control cells)} \times 1 0 0 %$

In other aspects, inhibition or reduction of the expression of MLH1 can be assessed in terms of a reduction of a parameter that is functionally linked to MLH1 gene expression, e.g., MLH1 protein expression or MLH1 signaling pathways. MLH1 silencing can be determined in any cell expressing MLH1, either endogenous or heterologous from an expression construct, and by any assay known in the art.

Inhibition or reduction of the expression of a MLH1 protein can be manifested by a reduction in the level of the MLH1 protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above, for the assessment of mRNA suppression, the inhibition or reduction of protein expression levels in a treated cell or group of cells can similarly be expressed as a percentage of the level of protein in a control cell or group of cells.

A control cell or group of cells that can be used to assess the inhibition or reduction of the expression of MLH1 includes a cell or group of cells that has not yet been contacted with an antisense oligonucleotide or dsRNA. For example, the control cell or group of cells can be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an antisense oligonucleotide or dsRNA.

The level of MLH1 mRNA that is expressed by a cell or group of cells can be determined using any method known in the art for assessing mRNA expression. In one aspect, the level of expression of MLH1 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the MLH1 gene. RNA can be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNEASY™ RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis. Circulating MLH1 mRNA can be detected using methods the described in PCT Publication WO2012/177906, the entire contents of which are hereby incorporated herein by reference. In some aspects, the level of expression of MLH1 is determined using a nucleic acid probe. The term “probe,” as used herein, refers to any molecule that is capable of selectively binding to a specific MLH1 sequence, e.g. to an mRNA or polypeptide. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes can be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or northern analyses, polymerase chain reaction (PCR) analyses, and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to MLH1 mRNA. In one aspect, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative aspect, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an AFFYMETRIX gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of MLH1 mRNA.

An alternative method for determining the level of expression of MLH1 in a sample involves the process of nucleic acid amplification and/or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental aspect set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In some aspects, the level of expression of MLH1 is determined by quantitative fluorogenic RT-PCR (i.e., the TAQMAN™ System) or the DUAL-GLO® Luciferase assay.

The expression levels of MLH1 mRNA can be monitored using a membrane blot (such as used in hybridization analysis such as northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722; 5,874,219; 5,744,305; 5,677,195; and 5,445,934, which are incorporated herein by reference. The determination of MLH1 expression level can comprise using nucleic acid probes in solution.

In some aspects, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of this PCR method is described and exemplified in the Examples presented herein. Such methods can be used for the detection of MLH1 nucleic acids.

The level of MLH1 protein expression can be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like. Such assays can be used for the detection of proteins indicative of the presence or replication of MLH1 proteins.

In some aspects of the methods described herein, the antisense oligonucleotide or dsRNA is administered to a subject such that the antisense oligonucleotide or dsRNA is delivered to a specific site within the subject. The inhibition or reduction of expression of MLH1 can be assessed using measurements of the level or change in the level of MLH1 mRNA or MLH1 protein in a sample derived from a specific site within the subject. In certain aspects, the methods include a clinically relevant inhibition of expression of MLH1, e.g., as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of MLH1.

In other aspects, the antisense oligonucleotide or dsRNA is administered in an amount and for a time effective to result in one of (or more, e.g., two or more, three or more, four or more op: (a) decrease the number of trinucleotide repeats, (b) decrease the level of polyglutamine, (c) decreased cell death (e.g., CNS cell death and/or muscle cell death), (d) delayed onset of the disorder, (e) increased survival of subject, and (f) increased progression free survival of a subject.

Treating trinucleotide repeat expansion disorders can result in an increase in average survival time of an individual or a population of subjects treated with an antisense oligonucleotide or dsRNA described herein in comparison to a population of untreated subjects. For example, the survival time of an individual or average survival time of a population is increased by more than 30 days (more than 60 days, 90 days, or 120 days). An increase in survival time of an individual or in average survival time of a population can be measured by any reproducible means. An increase in survival time of an individual can be measured, for example, by calculating for an individual the length of survival time following the initiation of treatment with the compound described herein. An increase in average survival time of a population can be measured, for example, by calculating for the average length of survival time following initiation of treatment with the compound described herein. An increase in survival time of an individual can be measured, for example, by calculating for an individual length of survival time following completion of a first round of treatment with a compound or pharmaceutically acceptable salt of a compound described herein. An increase in average survival time of a population can be measured, for example, by calculating for a population the average length of survival time following completion of a first round of treatment with a compound or pharmaceutically acceptable salt of a compound described herein.

Treating trinucleotide repeat expansion disorders can result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. For example, the mortality rate is decreased by more than 2% (e.g., more than 5%, 10%, or 25%). A decrease in the mortality rate of a population of treated subjects can be measured by any reproducible means, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with a compound or pharmaceutically acceptable salt of a compound described herein. A decrease in the mortality rate of a population can be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with a compound or pharmaceutically acceptable salt of a compound described herein.

A. Delivery of anti-MLH1 Agents

The delivery of an antisense oligonucleotide or dsRNA to a cell e.g., a cell within a subject, such as a human subject e.g., a subject in need thereof, such as a subject having a trinucleotide repeat expansion disorder can be achieved in a number of different ways. For example, delivery can be performed by contacting a cell with an antisense oligonucleotide or dsRNA either in vitro or in vivo.

In vivo delivery can also be performed directly by administering a composition comprising an antisense oligonucleotide or a dsRNA, e.g., a siRNA or a shRNA to a subject. These alternatives are discussed further below. In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an antisense oligonucleotide or dsRNA (see e.g., Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider to deliver an antisense oligonucleotide or dsRNA include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an antisense oligonucleotide or dsRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the antisense oligonucleotide or dsRNA to be administered.

For administering an antisense oligonucleotide or dsRNA systemically for the treatment of a disease, the antisense oligonucleotide or dsRNA can include alternative nucleobases, alternative sugar moieties, and/or alternative internucleoside linkages, or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the antisense oligonucleotide or dsRNA by endo- and exo-nucleases in vivo. Modification of the antisense oligonucleotide or dsRNA or the pharmaceutical carrier can permit targeting of the antisense oligonucleotide or dsRNA composition to the target tissue and avoid undesirable off-target effects. Antisense oligonucleotides or ds RNAs can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, a dsRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of a dsRNA to an aptamer has been shown to reduce tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O. et al., (2006) Nat. Biotechnol. 24:1005-1015). In an alternative aspect, the antisense oligonucleotide or dsRNA can be delivered using drug delivery systems such as a nanoparticle, a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an antisense oligonucleotide or dsRNA (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an antisense oligonucleotide or dsRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an oligonucleotide, or induced to form a vesicle or micelle (see e.g., Kim S H. et al., (2008) Journal of Controlled Release 129(2):107-116) that encases an antisense oligonucleotide or dsRNA. The formation of vesicles or micelles further prevents degradation of the antisense oligonucleotide or dsRNA when administered systemically. In general, any methods of delivery of nucleic acids known in the art can be adaptable to the delivery of the antisense oligonucleotides or dsRNAs. Methods for making and administering cationic antisense oligonucleotide or dsRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al., (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of antisense oligonucleotides or dsRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N. et al., (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S. et al., (2006) Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E. et al., (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some aspects, an antisense oligonucleotide or dsRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of antisense oligonucleotides or dsRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety. In some aspects, the antisense oligonucleotides or dsRNAs are delivered by polyplex or lipoplex nanoparticles. Methods for administration and pharmaceutical compositions of antisense oligonucleotides or dsRNAs and polyplex nanoparticles and lipoplex nanoparticles can be found in U.S. Patent Application Nos. 2017/0121454; 2016/0369269; 2016/0279256; 2016/0251478; 2016/0230189; 2015/0335764; 2015/0307554; 2015/0174549; 2014/0342003; 2014/0135376; and 2013/0317086, which are herein incorporated by reference in their entirety.

i. Vector Delivery Methods

dsRNA targeting MLH1 can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).

The individual strand or strands of a dsRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively, each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

dsRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, such as those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of a dsRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

In some embodiments, the dsRNA agent that reduces the level and/or activity of MLH1 is delivered by a viral vector (e.g., a viral vector expressing an anti-MLH1 agent). Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into a mammalian cell. Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of a mammalian cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors include a retrovirus (e.g., Retroviridae family viral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus, replication deficient herpes virus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology (Third Edition) Lippincott-Raven, Philadelphia, 1996). Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in U.S. Pat. No. 5,801,030, the vectors of which are incorporated herein by reference.

Exemplary viral vectors include lentiviral vectors, AAVs, and retroviral vectors. Lentiviral vectors and AAVs can integrate into the genome without cell divisions, and both types have been tested in pre-clinical animal studies. Methods for preparation of AAVs are described in the art e.g., in U.S. Pat. Nos. 5,677,158, 6,309,634, and 6,683,058, the methods of which is incorporated herein by reference. Methods for preparation and in vivo administration of lentiviruses are described in US 20020037281, the methods of which are incorporated herein by reference. In one aspect, a lentiviral vector is a replication-defective lentivirus particle. Such a lentivirus particle can be produced from a lentiviral vector comprising a 5′ lentiviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to a polynucleotide signal encoding the fusion protein, an origin of second strand DNA synthesis and a 3′ lentiviral LTR.

Retroviruses are most commonly used in human clinical trials, as they carry 7-8 kb, and have the ability to infect cells and have their genetic material stably integrated into the host cell with high efficiency (see, e.g., WO 95/30761; WO 95/24929, the retroviruses of which is incorporated herein by reference). In one aspect, a retroviral vector is replication defective. This prevents further generation of infectious retroviral particles in the target tissue. Thus, the replication defective virus becomes a “captive” transgene stable incorporated into the target cell genome. This is typically accomplished by deleting the gag, env, and pol genes (along with most of the rest of the viral genome). Heterologous nucleic acids are inserted in place of the deleted viral genes. The heterologous genes can be under the control of the endogenous heterologous promoter, another heterologous promoter active in the target cell, or the retroviral 5′ LTR (the viral LTR is active in diverse tissues).

These delivery vectors described herein can be made target-specific by attaching, for example, a sugar, a glycolipid, or a protein (e.g., an antibody to a target cell receptor).

Reversible delivery expression systems can be used. The Cre-loxP or FLP/FRT system and other similar systems can be used for reversible delivery-expression of one or more of the above-described nucleic acids. See WO2005/112620, WO2005/039643, US20050130919, US20030022375, US20020022018, US20030027335, and US20040216178, the systems of which are herein incorporated by reference. In particular, the reversible delivery-expression system described in US20100284990, the systems of which are herein incorporated by reference, can be used to provide a selective or emergency shut-off.

ii. Membranous Molecular Assembly Delivery Methods

The antisense oligonucleotides and dsRNAs can be delivered using a variety of membranous molecular assembly delivery methods including polymeric, biodegradable microparticle, or microcapsule delivery devices known in the art. For example, a colloidal dispersion system can be used for targeted delivery of an antisense oligonucleotide or dsRNA agent described herein. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the antisense oligonucleotide or dsRNA are delivered into the cell where the antisense oligonucleotide or dsRNA can specifically bind to a target RNA and can mediate RNase H-mediated gene silencing. In some cases, the liposomes are also specifically targeted, e.g., to direct the oligonucleotide to particular cell types. The composition of the liposome is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids can be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

A liposome containing an antisense oligonucleotide or dsRNA can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The antisense oligonucleotide or dsRNA preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the antisense oligonucleotide or dsRNA and condense around the antisense oligonucleotide or dsRNA to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of antisense oligonucleotide or dsRNA.

If necessary, a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH can be adjusted to favor condensation.

Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as a structural component of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can include one or more aspects of exemplary methods described in Feigner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham et al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75: 4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161. Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169). These methods are readily adapted to packaging antisense oligonucleotide or dsRNA preparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).

Liposomes, which are pH-sensitive or negatively charged, entrap nucleic acids rather than complex with them. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Feigner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising NOVASOME™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and NOVASOME™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) S.T.P. Pharma. Sci., 4(6):466).

Liposomes can be sterically stabilized liposomes, comprising one or more specialized lipids that result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_M1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64) reported the ability of monosialoganglio G^M1, galactocerebroside sulfate, and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., (1988), 85:6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

In one aspect, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver antisense oligonucleotides or dsRNAs to macrophages.

Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated antisense oligonucleotides or dsRNAs in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of antisense oligonucleotide or dsRNA (see, e.g., Feigner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. LIPOFECTIN™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (TRANSFECTAM™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of antisense oligonucleotides or dsRNAs are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer an antisense oligonucleotide or a dsRNA into the skin. In some implementations, liposomes are used for delivering an antisense oligonucleotide or dsRNA to epidermal cells and also to enhance the penetration of the antisense oligonucleotide or dsRNA into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2,405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. and Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising NOVASOME I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and NOVASOME II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with antisense oligonucleotides or dsRNAs are useful for treating a dermatological disorder.

The targeting of liposomes is also possible based on, for example, organ-specificity, cell-specificity, and organelle-specificity and is known in the art. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand. Additional methods are known in the art and are described, for example in U.S. Patent Application Publication No. 20060058255, the linking groups of which are herein incorporated by reference.

Liposomes that include antisense oligonucleotides or dsRNAs can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include antisense oligonucleotides or dsRNAs can be delivered, for example, subcutaneously by infection to deliver antisense oligonucleotides or dsRNAs to keratinocytes in the skin. To cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transfersomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Other formulations amenable are described in U.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008; 61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCT application No. PCT/US2007/080331, filed Oct. 3, 2007 also describes suitable formulations.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

The antisense oligonucleotides or dsRNAs for use in the methods described herein can be provided as micellar formulations. Micelles are a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

Lipid Nanoparticle-Based Delivery Methods

The antisense oligonucleotides or dsRNAs can be fully encapsulated in a lipid formulation, e.g., a lipid nanoparticle (LNP), or other nucleic acid-lipid particle. LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one aspect, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to antisense oligonucleotide or dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated.

Non-limiting examples of cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleywry-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.CI), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyetetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-(((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yeethylazanediyedidodecan-2-01 (Tech G1), or a mixture thereof. The cationic lipid can comprise, for example, from about 20 mol to about 50 mol % or about 40 mol % of the total lipid present in the particle.

The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be, for example, from about 5 mol % to about 90 mol %, about 10 mol %, or about 60 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits or reduces aggregation of particles can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (Cm), or a PEG-distearyloxypropyl (Cm). The conjugated lipid that prevents aggregation of particles can be, for example, from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some aspects, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 50 mol % of the total lipid present in the particle.

Additional exemplary lipid-dsRNA formulations are described in Table 1 of WO 2018/195165, herein incorporated by reference.

B. Combination Therapies

An antisense oligonucleotide or dsRNA can be used alone or in combination with at least one additional therapeutic agent, e.g., other agents that treat trinucleotide repeat expansion disorders or symptoms associated therewith, or in combination with other types of therapies to treat trinucleotide repeat expansion disorders. In combination treatments, the dosages of one or more of the therapeutic compounds can be reduced from standard dosages when administered alone. For example, doses can be determined empirically from drug combinations and permutations or can be deduced by isobolographic analysis (e.g., Black et al., Neurology 65:S3-S6 (2005)). In this case, dosages of the compounds when combined should provide a therapeutic effect.

In some aspects, the antisense oligonucleotide or dsRNA agents described herein can be used in combination with at least one an additional therapeutic agent to treat a trinucleotide repeat expansion disorder associated with gene having a trinucleotide repeat (e.g., any of the trinucleotide repeat expansion disorders and associated genes having a trinucleotide repeat listed in Table 1). In some aspects, at least one of the additional therapeutic agents can be an antisense oligonucleotide or a dsRNA (e.g., siRNA or shRNA) that hybridizes with the mRNA of gene associated with a trinucleotide repeat expansion disorder (e.g., any of the genes listed in Table 1). In some aspects, the trinucleotide repeat expansion disorder is Huntington's disease (HD). In some aspects, the gene associated with a trinucleotide repeat expansion disorder is Huntingtin (HTT). Several allelic variants of the Huntingtin gene have been implicated in the etiology of Huntington's disease. In some cases, these variants are identified on the basis of having unique HD-associated single nucleotide polymorphisms (SNPs). In some aspects, the antisense oligonucleotide or dsRNA that is an additional therapeutic agent hybridizes to an mRNA of the Huntingtin gene containing any of the HD-associated SNPs known in the art (e.g., any of the HD-associated SNPs described in Skotte et al., PLoS One 2014, 9(9): e107434, Carroll et al., Mol. Ther. 2011, 19(12): 2178-85, Warby et al., Am. J. Hum. Gen. 2009, 84(3): 351-66 (herein incorporated by reference)). In some aspects, the antisense oligonucleotide or dsRNA that is an additional therapeutic agent hybridizes to an mRNA of the Huntingtin gene lacking any of the HD-associated SNPs. In some of the aspects, the antisense oligonucleotide or dsRNA that is an additional therapeutic agent hybridizes to an mRNA of the Huntingtin gene having any of the SNPs selected from the group of rs362307 and rs365331. In some aspects, the antisense oligonucleotide or dsRNA that is an additional therapeutic agent can be a modified oligonucleotide or dsRNA (e.g., an antisense oligonucleotide or dsRNA including any of the modifications described herein). In some aspects, the modified oligonucleotide or dsRNA that is an additional therapeutic agent comprises one or more phosphorothioate internucleoside linkages. In some aspects, the modified oligonucleotide or dsRNA that is an additional therapeutic agent comprises one or more 2′-MOE moieties. In some aspects, the antisense oligonucleotide or dsRNA that is an additional therapeutic agent that hybridizes to the mRNA of the Huntingtin gene has a sequence selected from the SEQ ID NOs. 6-285 of U.S. Pat. No. 9,006,198; SEQ ID NOs. 6-8 of US Patent Application Publication No. 2017/0044539; SEQ ID NOs. 1-1565 of US Patent Application Publication 2018/0216108; and SEQ ID NO. 1-2432 of PCT Publication WO 2017/192679, the sequences of which are hereby incorporated by reference.

In some aspects, at least one of the additional therapeutic agents is a chemotherapeutic agent (e.g., a cytotoxic agent or other chemical compound useful in the treatment of a trinucleotide repeat expansion disorder).

In some aspects, at least one of the additional therapeutic agents can be a therapeutic agent which is a non-drug treatment. For example, at least one of the additional therapeutic agents is physical therapy.

In any of the combination aspects described herein, the two or more therapeutic agents can be administered simultaneously or sequentially, in either order. For example, a first therapeutic agent can be administered immediately, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to, 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, 14 hours, up to hours 16, up to 17 hours, up 18 hours, up to 19 hours up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours up to 24 hours or up to 1-7, 1-14, 1-21 or 1-30 days before or one or more of the additional therapeutic agents.

V. Pharmaceutical Compositions

The antisense oligonucleotides or dsRNAs described herein can be formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.

The compounds described herein can be used in the form of the free base, in the form of salts, solvates, and as prodrugs. All forms are within the methods described herein. In accordance with the methods described herein, the antisense oligonucleotides or dsRNAs or salts, solvates, or prodrugs thereof can be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compounds described herein can be administered, for example, by oral, parenteral, intrathecal, intracerebroventricular, intraparenchymal, buccal, sublingual, nasal, rectal, patch, pump, or transdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, intracerebroventricular, intraparenchymal, rectal, and topical modes of administration. Parenteral administration can be by continuous infusion over a selected period of time.

A compound described herein can be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it can be enclosed in hard or soft shell gelatin capsules, or it can be compressed into tablets, or it can be incorporated directly with the food of the diet. For oral therapeutic administration, a compound described herein can be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers. A compound described herein can be administered parenterally. Solutions of a compound described herein can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can be prepared in glycerol, liquid polyethylene glycols, DMSO, and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2012, 22nd ed.) and in The United States Pharmacopeia: The National Formulary (USP 41 NF 36), published in 2018. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that can be easily administered via syringe. Compositions for nasal administration can conveniently be formulated as aerosols, drops, gels, and powders. Aerosol formulations typically include a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device. Alternatively, the sealed container can be a unitary dispensing device, such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form includes an aerosol dispenser, it will contain a propellant, which can be a compressed gas, such as compressed air or an organic propellant, such as fluorochlorohydrocarbon. The aerosol dosage forms can take the form of a pump-atomizer. Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, where the active ingredient is formulated with a carrier, such as sugar, acacia, tragacanth, gelatin, and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base, such as cocoa butter

The compounds described herein can be administered to an animal, e.g., a human, alone or in combination with pharmaceutically acceptable carriers, as noted herein, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration, and standard pharmaceutical practice.

VI. Dosages

The dosage of the compositions (e.g., a composition including an antisense oligonucleotide or dsRNA) described herein, can vary depending on many factors, such as the pharmacodynamic properties of the compound; the mode of administration; the age, health, and weight of the recipient; the nature and extent of the symptoms; the frequency of the treatment, and the type of concurrent treatment, if any; and the clearance rate of the compound in the animal to be treated. The compositions described herein can be administered initially in a suitable dosage that can be adjusted as required, depending on the clinical response. In some aspects, the dosage of a composition (e.g., a composition including an antisense oligonucleotide or dsRNA) is a prophylactically or a therapeutically effective amount.

VII. Kits

Kits including (a) a pharmaceutical composition including an antisense oligonucleotide or dsRNA agent that reduces the level and/or activity of MLH1 in a cell or subject described herein, and (b) a package insert with instructions to perform any of the methods described herein are contemplated. In some aspects, the kit includes (a) a pharmaceutical composition including an antisense oligonucleotide or dsRNA agent that reduces the level and/or activity of MLH1 in a cell or subject described herein, (b) an additional therapeutic agent, and (c) a package insert with instructions to perform any of the methods described herein.

EXAMPLES
Example 1. Design and Selection of Antisense Oligonucleotides or dsRNA Agents

Identification and selection of target transcripts: Target transcript selection and off-target scoring (below) utilized NCBI RefSeq sequences, downloaded from NCBI 21 Nov. 2018. Experimentally validated “NM” transcript models were used except for cynomolgus monkey, which only has “XM” predicted models for the large majority of genes. The longest human, mouse, rat, and cynomolgus monkey MLH1 transcript that contained all mapped internal exons was selected (SEQ IDs 1, 3, 4, and 5 for human, mouse, rat, and cynomolgus monkey, respectively, SEQ ID NO:2 is the protein sequence).

TABLE 2

Exemplary Human, Cyno, Mouse, and Rat MLH1 Transcripts

Human (SEQ
Cyno (SEQ
Mouse (SEQ
Rat (SEQ

ID NO: 1)
ID NO: 3)
ID NO: 4)
ID NO: 5)

NM_000249.3
XM_005546623.2
NM_026810.2
NM_031053.1

Antisense Oligonucleotide

Selection of 20mer antisense oligonucleotide sequences: All antisense 20mer sub-sequences per transcript were generated. Candidate antisense oligonucleotides (“ASOs”) were selected that met the following thermodynamic and physical characteristics determined by the inventors: predicted melting temperature of ASO:target duplex (“Tm”) of 30-65° C., predicted melting temperature of hairpins (“T_hairpin”)<35° C., predicted melting temperature of homopolymer formation (“T_homo”)<25° C., GC content of 20-60%, no G homopolymers 4 or longer, and no A, T, or C homopolymers of 6 or longer. These selected antisense oligonucleotides were further evaluated for specificity (off-target scoring, below).

Off-target scoring: The specificity of the preferred ASOs was evaluated via alignment to all unspliced RefSeq transcripts (“NM” models for human, mouse, and rat; “NM” and “XM” models for cynomolgus monkey), using the FASTA algorithm with an E value cutoff of 1000. The number of mismatches between each ASO and each transcript (per species) was tallied. An “off-target score” for each ASO in each species was calculated as the lowest number of mismatches to any transcript other than those encoded by the MLH1 gene.

Selection of ASOs for screening: A set of 480 preferred ASOs was selected for screening according to both specificity and ASO:mRNA (target) hybridization energy maximization information as follows. All candidate ASOs were evaluated for delta G of hybridization with the predicted target mRNA secondary structure (ΔG^overall) according to Xu and Mathews (Methods Mol Biol. 1490:15-34 (2016)). Next, two subsets of ASOs were chosen: First, 70 ASOs that matched human, cyno, and mouse target transcripts, had off-target scores of at least 1 in three species, and negative ΔG^overall; second, 410 ASOs that matched human and cyno target transcripts, had off-target scores of at least 2 in both species, and ΔG^overallless than −9.5 degrees Celsius.

The sequences, positions in human transcript, conservation in other species and species-specific off-target scores of each ASO are given in Table 3. Wherever indicated as “NC”, the ASO does not match the MLH1 gene in that species, and therefore off-target scores were not generated.

ASOs were synthesized as 5-10-5 “flanking sequence-DNA core sequence-flanking sequence” antisense oligonucleotides, with ribonucleotides at positions 1-5 and 16-20 and deoxyribonucleotides at positions 6-15, and with the following generic structure:

5′-NmsNmsNmsNmsNmsNsNsNsNsNsNsNsNsNsNsNmsNmsNmsNmsNm-3′ wherein:

- Nm: 2′-MOE residues (including 5methyl-2′-MOE-C and 5methyl-2′-MOE-U)
- N: DNA/RNA residues
- s: phosphorothioate (the backbone is fully phosphorothioate-modified)
- All “C” within the DNA core (positions 6-15) are 5′-Methyl-2′-MOE-dC
- All “T” in positions 1-5 or 16-20 are 5′-methyl-2′-MOE-U.

For primary screens at 2 nM and 20 nM, desalted antisense oligonucleotides were used. For detailed characterization of a subset of antisense oligonucleotides, antisense oligonucleotides were further purified by HPLC.

TABLE 3

Exemplary Antisense Oligonucleotides

SEQ

ID

Off-target Score

NO
Position
Sequence
Human
Cyno
Mouse
Rat

6
2
TGTGGGTTGCTGGGTCTCTT
2
NC
NC
NC

7
7
AACTCTGTGGGTTGCTGGGT
2
2
NC
NC

8
14
ATTTCTCAACTCTGTGGGTT
2
NC
NC
NC

9
15
AATTTCTCAACTCTGTGGGT
2
1
NC
NC

10
16
AAATTTCTCAACTCTGTGGG
2
2
NC
NC

11
17
CAAATTTCTCAACTCTGTGG
1
3
NC
NC

12
18
TCAAATTTCTCAACTCTGTG
2
2
NC
NC

13
19
GTCAAATTTCTCAACTCTGT
2
2
NC
NC

14
20
AGTCAAATTTCTCAACTCTG
1
2
NC
NC

15
21
CAGTCAAATTTCTCAACTCT
1
2
NC
NC

16
22
CCAGTCAAATTTCTCAACTC
1
NC
NC
NC

17
23
GCCAGTCAAATTTCTCAACT
1
NC
NC
NC

18
24
TGCCAGTCAAATTTCTCAAC
1
2
NC
NC

19
25
ATGCCAGTCAAATTTCTCAA
2
2
NC
NC

20
26
AATGCCAGTCAAATTTCTCA
2
2
NC
NC

21
27
GAATGCCAGTCAAATTTCTC
3
1
NC
NC

22
28
TGAATGCCAGTCAAATTTCT
2
2
NC
NC

23
29
TTGAATGCCAGTCAAATTTC
2
2
NC
NC

24
30
CTTGAATGCCAGTCAAATTT
2
2
NC
NC

25
31
GCTTGAATGCCAGTCAAATT
2
2
NC
NC

26
32
AGCTTGAATGCCAGTCAAAT
2
2
NC
NC

27
33
CAGCTTGAATGCCAGTCAAA
2
2
NC
NC

28
34
ACAGCTTGAATGCCAGTCAA
2
NC
NC
NC

29
35
GACAGCTTGAATGCCAGTCA
2
NC
NC
NC

30
36
GGACAGCTTGAATGCCAGTC
1
NC
NC
NC

31
37
TGGACAGCTTGAATGCCAGT
2
NC
NC
NC

32
38
TTGGACAGCTTGAATGCCAG
2
NC
NC
NC

33
39
ATTGGACAGCTTGAATGCCA
1
NC
NC
NC

34
40
GATTGGACAGCTTGAATGCC
2
NC
NC
NC

35
41
TGATTGGACAGCTTGAATGC
2
NC
NC
NC

36
42
TTGATTGGACAGCTTGAATG
2
NC
NC
NC

37
43
ATTGATTGGACAGCTTGAAT
1
NC
NC
NC

38
44
TATTGATTGGACAGCTTGAA
1
NC
NC
NC

39
45
CTATTGATTGGACAGCTTGA
2
NC
NC
NC

40
46
GCTATTGATTGGACAGCTTG
2
NC
NC
NC

41
47
AGCTATTGATTGGACAGCTT
2
NC
NC
NC

42
49
GCAGCTATTGATTGGACAGC
2
NC
NC
NC

43
50
GGCAGCTATTGATTGGACAG
2
NC
NC
NC

44
51
CGGCAGCTATTGATTGGACA
3
NC
NC
NC

45
83
TGTAGCTTACGCCATCCAGC
2
NC
NC
NC

46
84
CTGTAGCTTACGCCATCCAG
2
NC
NC
NC

47
98
CGTTCTTCCTTCAGCTGTAG
2
NC
NC
NC

48
99
ACGTTCTTCCTTCAGCTGTA
2
NC
NC
NC

49
100
CACGTTCTTCCTTCAGCTGT
2
NC
NC
NC

50
101
TCACGTTCTTCCTTCAGCTG
2
NC
NC
NC

51
102
CTCACGTTCTTCCTTCAGCT
2
NC
NC
NC

52
103
GCTCACGTTCTTCCTTCAGC
2
NC
NC
NC

53
104
TGCTCACGTTCTTCCTTCAG
1
NC
NC
NC

54
105
GTGCTCACGTTCTTCCTTCA
2
2
NC
NC

55
130
CTTCAGCCAATCACCTCAGT
2
NC
NC
NC

56
131
CCTTCAGCCAATCACCTCAG
2
NC
NC
NC

57
132
GCCTTCAGCCAATCACCTCA
2
NC
NC
NC

58
153
GTCTAGATGCTCAACGGAAG
3
1
NC
NC

59
154
CGTCTAGATGCTCAACGGAA
2
1
NC
NC

60
155
ACGTCTAGATGCTCAACGGA
2
2
NC
NC

61
156
AACGTCTAGATGCTCAACGG
3
2
NC
NC

62
158
GAAACGTCTAGATGCTCAAC
3
2
NC
NC

63
159
GGAAACGTCTAGATGCTCAA
2
2
NC
NC

64
160
AGGAAACGTCTAGATGCTCA
2
NC
NC
NC

65
161
AAGGAAACGTCTAGATGCTC
2
NC
NC
NC

66
162
CAAGGAAACGTCTAGATGCT
3
NC
NC
NC

67
163
CCAAGGAAACGTCTAGATGC
2
NC
NC
NC

68
164
GCCAAGGAAACGTCTAGATG
3
NC
NC
NC

69
165
AGCCAAGGAAACGTCTAGAT
2
NC
NC
NC

70
166
GAGCCAAGGAAACGTCTAGA
2
NC
NC
NC

71
167
AGAGCCAAGGAAACGTCTAG
2
NC
NC
NC

72
168
AAGAGCCAAGGAAACGTCTA
2
NC
NC
NC

73
169
GAAGAGCCAAGGAAACGTCT
2
NC
NC
NC

74
170
AGAAGAGCCAAGGAAACGTC
2
NC
NC
NC

75
171
CAGAAGAGCCAAGGAAACGT
2
NC
NC
NC

76
172
CCAGAAGAGCCAAGGAAACG
1
NC
NC
NC

77
173
GCCAGAAGAGCCAAGGAAAC
1
NC
NC
NC

78
174
CGCCAGAAGAGCCAAGGAAA
2
NC
NC
NC

79
194
TGCCACGAACGACATTTTGG
2
1
NC
NC

80
195
CTGCCACGAACGACATTTTG
2
2
NC
NC

81
196
CCTGCCACGAACGACATTTT
2
2
NC
NC

82
197
CCCTGCCACGAACGACATTT
3
1
NC
NC

83
202
ATAACCCCTGCCACGAACGA
3
2
NC
NC

84
203
AATAACCCCTGCCACGAACG
2
2
NC
NC

85
204
GAATAACCCCTGCCACGAAC
2
NC
NC
NC

86
205
CGAATAACCCCTGCCACGAA
2
2
NC
NC

87
229
TTCACCACTGTCTCGTCCAG
2
2
NC
NC

88
230
GTTCACCACTGTCTCGTCCA
1
2
NC
NC

89
235
ATGCGGTTCACCACTGTCTC
2
NC
NC
NC

90
236
GATGCGGTTCACCACTGTCT
3
2
NC
NC

91
284
CATCTCTTTGATAGCATTAG
1
2
NC
NC

92
285
TCATCTCTTTGATAGCATTA
1
2
NC
NC

93
286
ATCATCTCTTTGATAGCATT
1
2
1
NC

94
287
AATCATCTCTTTGATAGCAT
2
2
NC
NC

95
288
CAATCATCTCTTTGATAGCA
2
2
NC
NC

96
289
TCAATCATCTCTTTGATAGC
2
2
NC
NC

97
290
CTCAATCATCTCTTTGATAG
2
2
NC
NC

98
291
TCTCAATCATCTCTTTGATA
1
2
NC
NC

99
292
TTCTCAATCATCTCTTTGAT
2
1
NC
NC

100
293
GTTCTCAATCATCTCTTTGA
2
2
NC
NC

101
294
AGTTCTCAATCATCTCTTTG
2
2
NC
NC

102
295
CAGTTCTCAATCATCTCTTT
1
1
NC
NC

103
296
ACAGTTCTCAATCATCTCTT
1
NC
NC
NC

104
297
AACAGTTCTCAATCATCTCT
2
NC
NC
NC

105
298
AAACAGTTCTCAATCATCTC
1
NC
NC
NC

106
299
TAAACAGTTCTCAATCATCT
2
2
NC
NC

107
300
CTAAACAGTTCTCAATCATC
2
2
NC
NC

108
301
TCTAAACAGTTCTCAATCAT
2
1
NC
NC

109
302
ATCTAAACAGTTCTCAATCA
2
2
NC
NC

110
303
CATCTAAACAGTTCTCAATC
2
2
NC
NC

111
304
GCATCTAAACAGTTCTCAAT
2
2
NC
NC

112
305
TGCATCTAAACAGTTCTCAA
1
2
NC
NC

113
306
TTGCATCTAAACAGTTCTCA
2
3
NC
NC

114
307
TTTGCATCTAAACAGTTCTC
2
2
NC
NC

115
308
TTTTGCATCTAAACAGTTCT
1
2
NC
NC

116
309
ATTTTGCATCTAAACAGTTC
1
2
NC
NC

117
310
GATTTTGCATCTAAACAGTT
1
1
2
2

118
311
GGATTTTGCATCTAAACAGT
2
2
NC
NC

119
312
TGGATTTTGCATCTAAACAG
2
2
NC
NC

120
313
GTGGATTTTGCATCTAAACA
3
2
NC
NC

121
314
TGTGGATTTTGCATCTAAAC
2
2
NC
NC

122
315
TTGTGGATTTTGCATCTAAA
2
2
NC
NC

123
316
CTTGTGGATTTTGCATCTAA
2
2
NC
NC

124
317
ACTTGTGGATTTTGCATCTA
2
2
NC
NC

125
318
TACTTGTGGATTTTGCATCT
2
2
NC
NC

126
319
ATACTTGTGGATTTTGCATC
2
2
NC
NC

127
320
AATACTTGTGGATTTTGCAT
2
NC
NC
NC

128
321
GAATACTTGTGGATTTTGCA
1
NC
NC
NC

129
322
TGAATACTTGTGGATTTTGC
2
2
NC
NC

130
323
TTGAATACTTGTGGATTTTG
2
2
NC
NC

131
324
CTTGAATACTTGTGGATTTT
2
2
NC
NC

132
325
ACTTGAATACTTGTGGATTT
2
NC
NC
NC

133
326
CACTTGAATACTTGTGGATT
2
2
NC
NC

134
327
TCACTTGAATACTTGTGGAT
1
3
NC
NC

135
328
ATCACTTGAATACTTGTGGA
2
2
NC
NC

136
329
AATCACTTGAATACTTGTGG
2
1
NC
NC

137
330
CAATCACTTGAATACTTGTG
2
2
NC
NC

138
331
ACAATCACTTGAATACTTGT
2
2
NC
NC

139
332
AACAATCACTTGAATACTTG
1
2
NC
NC

140
333
TAACAATCACTTGAATACTT
2
1
NC
NC

141
334
TTAACAATCACTTGAATACT
1
2
NC
NC

142
335
TTTAACAATCACTTGAATAC
2
2
NC
NC

143
336
CTTTAACAATCACTTGAATA
2
2
NC
NC

144
337
TCTTTAACAATCACTTGAAT
2
2
NC
NC

145
338
CTCTTTAACAATCACTTGAA
2
NC
NC
NC

146
339
CCTCTTTAACAATCACTTGA
2
2
NC
NC

147
341
TCCCTCTTTAACAATCACTT
2
2
NC
NC

148
342
CTCCCTCTTTAACAATCACT
2
2
NC
NC

149
343
CCTCCCTCTTTAACAATCAC
2
2
NC
NC

150
344
GCCTCCCTCTTTAACAATCA
2
2
NC
NC

151
345
GGCCTCCCTCTTTAACAATC
2
2
NC
NC

152
346
AGGCCTCCCTCTTTAACAAT
2
2
NC
NC

153
357
GAATCAACTTCAGGCCTCCC
2
2
NC
NC

154
358
TGAATCAACTTCAGGCCTCC
2
3
NC
NC

155
366
CTTGGATCTGAATCAACTTC
2
2
NC
NC

156
367
TCTTGGATCTGAATCAACTT
2
2
NC
NC

157
368
GTCTTGGATCTGAATCAACT
2
1
NC
NC

158
369
TGTCTTGGATCTGAATCAAC
2
3
NC
NC

159
370
TTGTCTTGGATCTGAATCAA
2
2
NC
NC

160
371
ATTGTCTTGGATCTGAATCA
2
2
NC
NC

161
372
CATTGTCTTGGATCTGAATC
1
NC
NC
NC

162
382
ATCCCGGTGCCATTGTCTTG
2
NC
NC
NC

163
383
GATCCCGGTGCCATTGTCTT
2
NC
NC
NC

164
384
TGATCCCGGTGCCATTGTCT
3
NC
NC
NC

165
388
TTCCTGATCCCGGTGCCATT
3
NC
NC
NC

166
389
TTTCCTGATCCCGGTGCCAT
3
NC
NC
NC

167
392
TTCTTTCCTGATCCCGGTGC
2
NC
NC
NC

168
393
CTTCTTTCCTGATCCCGGTG
2
NC
NC
NC

169
394
TCTTCTTTCCTGATCCCGGT
2
NC
NC
NC

170
395
ATCTTCTTTCCTGATCCCGG
3
NC
NC
NC

171
396
GATCTTCTTTCCTGATCCCG
3
NC
NC
NC

172
397
AGATCTTCTTTCCTGATCCC
2
2
NC
NC

173
398
CAGATCTTCTTTCCTGATCC
2
NC
NC
NC

174
399
CCAGATCTTCTTTCCTGATC
2
NC
NC
NC

175
400
TCCAGATCTTCTTTCCTGAT
1
3
NC
NC

176
401
ATCCAGATCTTCTTTCCTGA
2
2
NC
NC

177
402
TATCCAGATCTTCTTTCCTG
2
1
NC
NC

178
403
ATATCCAGATCTTCTTTCCT
1
2
NC
NC

179
404
AATATCCAGATCTTCTTTCC
2
2
NC
NC

180
405
CAATATCCAGATCTTCTTTC
1
2
NC
NC

181
406
ACAATATCCAGATCTTCTTT
1
2
NC
NC

182
407
TACAATATCCAGATCTTCTT
2
2
NC
NC

183
408
ATACAATATCCAGATCTTCT
2
2
NC
NC

184
409
CATACAATATCCAGATCTTC
2
2
NC
NC

185
410
ACATACAATATCCAGATCTT
2
2
NC
NC

186
411
CACATACAATATCCAGATCT
2
2
NC
NC

187
412
TCACATACAATATCCAGATC
2
2
NC
NC

188
413
TTCACATACAATATCCAGAT
2
1
NC
NC

189
414
TTTCACATACAATATCCAGA
2
2
NC
NC

190
415
CTTTCACATACAATATCCAG
2
1
NC
NC

191
416
CCTTTCACATACAATATCCA
2
2
NC
NC

192
417
ACCTTTCACATACAATATCC
2
2
NC
NC

193
418
AACCTTTCACATACAATATC
2
2
NC
NC

194
419
GAACCTTTCACATACAATAT
2
2
NC
NC

195
420
TGAACCTTTCACATACAATA
2
2
NC
NC

196
430
TTACTAGTAGTGAACCTTTC
2
NC
NC
NC

197
431
TTTACTAGTAGTGAACCTTT
2
NC
NC
NC

198
432
GTTTACTAGTAGTGAACCTT
2
NC
NC
NC

199
436
TGCAGTTTACTAGTAGTGAA
1
NC
NC
NC

200
437
CTGCAGTTTACTAGTAGTGA
2
NC
NC
NC

201
438
ACTGCAGTTTACTAGTAGTG
2
NC
NC
NC

202
439
GACTGCAGTTTACTAGTAGT
2
NC
NC
NC

203
440
GGACTGCAGTTTACTAGTAG
2
NC
NC
NC

204
441
AGGACTGCAGTTTACTAGTA
1
NC
NC
NC

205
442
AAGGACTGCAGTTTACTAGT
1
NC
NC
NC

206
443
AAAGGACTGCAGTTTACTAG
2
NC
NC
NC

207
444
CAAAGGACTGCAGTTTACTA
1
NC
NC
NC

208
445
TCAAAGGACTGCAGTTTACT
2
2
NC
NC

209
446
CTCAAAGGACTGCAGTTTAC
2
2
NC
NC

210
447
CCTCAAAGGACTGCAGTTTA
2
2
NC
NC

211
448
TCCTCAAAGGACTGCAGTTT
2
2
NC
NC

212
458
ACTGGCTAAATCCTCAAAGG
2
2
NC
NC

213
459
TACTGGCTAAATCCTCAAAG
2
2
NC
NC

214
460
ATACTGGCTAAATCCTCAAA
2
2
2
NC

215
461
AATACTGGCTAAATCCTCAA
2
2
2
NC

216
462
AAATACTGGCTAAATCCTCA
2
1
2
NC

217
463
GAAATACTGGCTAAATCCTC
3
2
2
NC

218
464
AGAAATACTGGCTAAATCCT
2
2
2
NC

219
465
TAGAAATACTGGCTAAATCC
2
2
2
NC

220
466
GTAGAAATACTGGCTAAATC
2
2
2
NC

221
467
GGTAGAAATACTGGCTAAAT
2
2
2
NC

222
468
AGGTAGAAATACTGGCTAAA
1
2
1
NC

223
469
TAGGTAGAAATACTGGCTAA
1
2
2
NC

224
470
ATAGGTAGAAATACTGGCTA
2
3
2
NC

225
471
CATAGGTAGAAATACTGGCT
2
2
2
NC

226
472
CCATAGGTAGAAATACTGGC
2
3
2
NC

227
473
GCCATAGGTAGAAATACTGG
1
2
2
NC

228
474
AGCCATAGGTAGAAATACTG
1
NC
2
NC

229
475
AAGCCATAGGTAGAAATACT
2
2
2
NC

230
476
AAAGCCATAGGTAGAAATAC
2
3
2
NC

231
477
GAAAGCCATAGGTAGAAATA
2
1
1
NC

232
478
CGAAAGCCATAGGTAGAAAT
2
2
1
NC

233
479
TCGAAAGCCATAGGTAGAAA
2
2
NC
NC

234
480
CTCGAAAGCCATAGGTAGAA
2
2
NC
NC

235
481
CCTCGAAAGCCATAGGTAGA
2
2
NC
NC

236
485
CTCACCTCGAAAGCCATAGG
2
2
NC
NC

237
486
CCTCACCTCGAAAGCCATAG
2
2
NC
NC

238
487
GCCTCACCTCGAAAGCCATA
2
1
NC
NC

239
488
AGCCTCACCTCGAAAGCCAT
2
2
NC
NC

240
489
AAGCCTCACCTCGAAAGCCA
2
2
NC
NC

241
490
AAAGCCTCACCTCGAAAGCC
2
1
NC
NC

242
491
CAAAGCCTCACCTCGAAAGC
2
1
NC
NC

243
492
CCAAAGCCTCACCTCGAAAG
2
2
NC
NC

244
493
GCCAAAGCCTCACCTCGAAA
2
2
NC
NC

245
525
TAATAGTAACATGAGCCACA
2
2
NC
NC

246
526
GTAATAGTAACATGAGCCAC
2
1
NC
NC

247
527
TGTAATAGTAACATGAGCCA
2
3
NC
NC

248
528
TTGTAATAGTAACATGAGCC
2
2
NC
NC

249
529
GTTGTAATAGTAACATGAGC
2
2
NC
NC

250
530
CGTTGTAATAGTAACATGAG
3
NC
NC
NC

251
531
TCGTTGTAATAGTAACATGA
2
NC
NC
NC

252
532
TTCGTTGTAATAGTAACATG
2
NC
NC
NC

253
533
TTTCGTTGTAATAGTAACAT
2
NC
NC
NC

254
534
TTTTCGTTGTAATAGTAACA
3
NC
NC
NC

255
535
GTTTTCGTTGTAATAGTAAC
2
NC
NC
NC

256
536
TGTTTTCGTTGTAATAGTAA
2
NC
NC
NC

257
537
CTGTTTTCGTTGTAATAGTA
2
NC
NC
NC

258
538
GCTGTTTTCGTTGTAATAGT
2
NC
NC
NC

259
539
AGCTGTTTTCGTTGTAATAG
2
NC
NC
NC

260
540
CAGCTGTTTTCGTTGTAATA
2
NC
NC
NC

261
541
TCAGCTGTTTTCGTTGTAAT
2
NC
NC
NC

262
542
ATCAGCTGTTTTCGTTGTAA
2
NC
NC
NC

263
543
CATCAGCTGTTTTCGTTGTA
3
NC
NC
NC

264
544
CCATCAGCTGTTTTCGTTGT
3
NC
NC
NC

265
545
TCCATCAGCTGTTTTCGTTG
2
NC
NC
NC

266
546
TTCCATCAGCTGTTTTCGTT
2
NC
NC
NC

267
547
TTTCCATCAGCTGTTTTCGT
2
NC
NC
NC

268
548
CTTTCCATCAGCTGTTTTCG
2
NC
NC
NC

269
549
ACTTTCCATCAGCTGTTTTC
2
NC
NC
NC

270
550
CACTTTCCATCAGCTGTTTT
2
2
NC
NC

271
551
ACACTTTCCATCAGCTGTTT
2
2
NC
NC

272
552
CACACTTTCCATCAGCTGTT
1
2
NC
NC

273
553
GCACACTTTCCATCAGCTGT
2
NC
NC
NC

274
554
TGCACACTTTCCATCAGCTG
2
2
NC
NC

275
555
ATGCACACTTTCCATCAGCT
2
2
NC
NC

276
556
TATGCACACTTTCCATCAGC
2
1
NC
NC

277
557
GTATGCACACTTTCCATCAG
2
2
NC
NC

278
558
TGTATGCACACTTTCCATCA
2
1
NC
NC

279
559
CTGTATGCACACTTTCCATC
2
2
NC
NC

280
560
TCTGTATGCACACTTTCCAT
1
2
NC
NC

281
561
CTCTGTATGCACACTTTCCA
1
NC
NC
NC

282
562
GCTCTGTATGCACACTTTCC
1
1
NC
NC

283
563
TGCTCTGTATGCACACTTTC
2
NC
NC
NC

284
571
GAGTAACTTGCTCTGTATGC
2
2
NC
NC

285
572
TGAGTAACTTGCTCTGTATG
2
2
NC
NC

286
573
CTGAGTAACTTGCTCTGTAT
2
2
NC
NC

287
574
TCTGAGTAACTTGCTCTGTA
1
2
2
2

288
575
ATCTGAGTAACTTGCTCTGT
2
2
2
2

289
576
CATCTGAGTAACTTGCTCTG
2
2
2
2

290
577
CCATCTGAGTAACTTGCTCT
2
2
2
2

291
578
TCCATCTGAGTAACTTGCTC
2
2
2
3

292
579
TTCCATCTGAGTAACTTGCT
2
2
2
3

293
580
TTTCCATCTGAGTAACTTGC
2
2
2
3

294
581
TTTTCCATCTGAGTAACTTG
1
2
NC
NC

295
582
GTTTTCCATCTGAGTAACTT
2
2
NC
NC

296
583
AGTTTTCCATCTGAGTAACT
2
2
NC
NC

297
584
CAGTTTTCCATCTGAGTAAC
2
2
NC
NC

298
585
TCAGTTTTCCATCTGAGTAA
2
2
NC
NC

299
590
GGCTTTCAGTTTTCCATCTG
1
NC
NC
NC

300
591
GGGCTTTCAGTTTTCCATCT
1
NC
NC
NC

301
609
CAGCACATGGTTTAGGAGGG
2
NC
NC
NC

302
620
CCCTTGATTGCCAGCACATG
2
2
NC
NC

303
621
TCCCTTGATTGCCAGCACAT
2
2
NC
NC

304
622
GTCCCTTGATTGCCAGCACA
2
2
NC
NC

305
627
TCTGGGTCCCTTGATTGCCA
2
2
NC
NC

306
628
ATCTGGGTCCCTTGATTGCC
2
NC
NC
NC

307
629
GATCTGGGTCCCTTGATTGC
3
2
NC
NC

308
630
TGATCTGGGTCCCTTGATTG
2
2
NC
NC

309
631
GTGATCTGGGTCCCTTGATT
2
NC
NC
NC

310
632
CGTGATCTGGGTCCCTTGAT
2
2
NC
NC

311
663
TCGTGGCTATGTTGTAAAAA
3
2
NC
NC

312
664
CTCGTGGCTATGTTGTAAAA
2
1
NC
NC

313
665
CCTCGTGGCTATGTTGTAAA
2
2
NC
NC

314
666
TCCTCGTGGCTATGTTGTAA
2
2
NC
NC

315
667
CTCCTCGTGGCTATGTTGTA
3
2
NC
NC

316
668
TCTCCTCGTGGCTATGTTGT
2
3
NC
NC

317
669
TTCTCCTCGTGGCTATGTTG
2
2
NC
NC

318
670
TTTCTCCTCGTGGCTATGTT
2
2
NC
NC

319
671
TTTTCTCCTCGTGGCTATGT
2
2
NC
NC

320
672
CTTTTCTCCTCGTGGCTATG
2
2
NC
NC

321
673
GCTTTTCTCCTCGTGGCTAT
2
NC
NC
NC

322
674
AGCTTTTCTCCTCGTGGCTA
2
2
NC
NC

323
675
AAGCTTTTCTCCTCGTGGCT
2
2
NC
NC

324
676
AAAGCTTTTCTCCTCGTGGC
2
1
NC
NC

325
677
TAAAGCTTTTCTCCTCGTGG
2
2
NC
NC

326
678
TTAAAGCTTTTCTCCTCGTG
3
2
NC
NC

327
679
TTTAAAGCTTTTCTCCTCGT
2
2
NC
NC

328
680
TTTTAAAGCTTTTCTCCTCG
2
2
NC
NC

329
681
TTTTTAAAGCTTTTCTCCTC
1
1
NC
NC

330
697
TATTCTTCACTTGGATTTTT
1
2
NC
NC

331
698
ATATTCTTCACTTGGATTTT
1
1
NC
NC

332
699
CATATTCTTCACTTGGATTT
2
2
NC
NC

333
700
CCATATTCTTCACTTGGATT
2
2
NC
NC

334
701
CCCATATTCTTCACTTGGAT
2
2
NC
NC

335
702
TCCCATATTCTTCACTTGGA
2
3
NC
NC

336
703
TTCCCATATTCTTCACTTGG
2
2
NC
NC

337
704
TTTCCCATATTCTTCACTTG
2
2
NC
NC

338
705
TTTTCCCATATTCTTCACTT
1
2
NC
NC

339
706
ATTTTCCCATATTCTTCACT
1
NC
NC
NC

340
707
AATTTTCCCATATTCTTCAC
1
2
NC
NC

341
708
AAATTTTCCCATATTCTTCA
1
2
NC
NC

342
709
AAAATTTTCCCATATTCTTC
2
2
NC
NC

343
710
CAAAATTTTCCCATATTCTT
1
1
NC
NC

344
711
CCAAAATTTTCCCATATTCT
1
2
NC
NC

345
712
TCCAAAATTTTCCCATATTC
2
2
NC
NC

346
713
TTCCAAAATTTTCCCATATT
1
1
NC
NC

347
714
CTTCCAAAATTTTCCCATAT
2
NC
NC
NC

348
715
ACTTCCAAAATTTTCCCATA
2
NC
NC
NC

349
716
AACTTCCAAAATTTTCCCAT
1
NC
NC
NC

350
717
CAACTTCCAAAATTTTCCCA
2
NC
NC
NC

351
718
ACAACTTCCAAAATTTTCCC
1
NC
NC
NC

352
719
AACAACTTCCAAAATTTTCC
2
NC
NC
NC

353
720
CAACAACTTCCAAAATTTTC
2
NC
NC
NC

354
721
CCAACAACTTCCAAAATTTT
1
NC
NC
NC

355
722
GCCAACAACTTCCAAAATTT
1
NC
NC
NC

356
723
TGCCAACAACTTCCAAAATT
1
NC
NC
NC

357
724
CTGCCAACAACTTCCAAAAT
1
NC
NC
NC

358
725
CCTGCCAACAACTTCCAAAA
1
NC
NC
NC

359
726
ACCTGCCAACAACTTCCAAA
1
NC
NC
NC

360
727
TACCTGCCAACAACTTCCAA
2
NC
NC
NC

361
728
ATACCTGCCAACAACTTCCA
1
NC
NC
NC

362
729
AATACCTGCCAACAACTTCC
1
NC
NC
NC

363
730
GAATACCTGCCAACAACTTC
1
NC
NC
NC

364
731
TGAATACCTGCCAACAACTT
2
NC
NC
NC

365
732
CTGAATACCTGCCAACAACT
1
NC
NC
NC

366
733
ACTGAATACCTGCCAACAAC
1
NC
NC
NC

367
734
TACTGAATACCTGCCAACAA
2
NC
NC
NC

368
735
GTACTGAATACCTGCCAACA
2
NC
NC
NC

369
736
TGTACTGAATACCTGCCAAC
1
NC
NC
NC

370
737
GTGTACTGAATACCTGCCAA
2
NC
NC
NC

371
738
TGTGTACTGAATACCTGCCA
1
NC
NC
NC

372
739
TTGTGTACTGAATACCTGCC
2
NC
NC
NC

373
740
ATTGTGTACTGAATACCTGC
2
NC
NC
NC

374
741
CATTGTGTACTGAATACCTG
2
NC
NC
NC

375
742
GCATTGTGTACTGAATACCT
2
NC
NC
NC

376
743
TGCATTGTGTACTGAATACC
2
NC
NC
NC

377
744
CTGCATTGTGTACTGAATAC
2
NC
NC
NC

378
745
CCTGCATTGTGTACTGAATA
2
NC
NC
NC

379
746
GCCTGCATTGTGTACTGAAT
1
NC
NC
NC

380
747
TGCCTGCATTGTGTACTGAA
1
NC
NC
NC

381
748
ATGCCTGCATTGTGTACTGA
2
NC
NC
NC

382
759
CTGAGAAACTAATGCCTGCA
1
2
NC
NC

383
760
ACTGAGAAACTAATGCCTGC
2
2
NC
NC

384
761
AACTGAGAAACTAATGCCTG
2
2
2
NC

385
762
TAACTGAGAAACTAATGCCT
2
2
2
NC

386
763
TTAACTGAGAAACTAATGCC
1
NC
2
NC

387
764
TTTAACTGAGAAACTAATGC
1
1
2
NC

388
765
TTTTAACTGAGAAACTAATG
1
2
2
NC

389
782
TACTGTCTCTCCTTGTTTTT
2
NC
NC
NC

390
783
CTACTGTCTCTCCTTGTTTT
2
NC
NC
NC

391
784
GCTACTGTCTCTCCTTGTTT
2
NC
NC
NC

392
785
AGCTACTGTCTCTCCTTGTT
2
NC
NC
NC

393
786
CAGCTACTGTCTCTCCTTGT
2
NC
NC
NC

394
787
TCAGCTACTGTCTCTCCTTG
2
NC
NC
NC

395
788
ATCAGCTACTGTCTCTCCTT
2
NC
NC
NC

396
789
CATCAGCTACTGTCTCTCCT
2
NC
NC
NC

397
790
ACATCAGCTACTGTCTCTCC
2
3
NC
NC

398
791
AACATCAGCTACTGTCTCTC
2
2
NC
NC

399
792
TAACATCAGCTACTGTCTCT
2
2
NC
NC

400
793
CTAACATCAGCTACTGTCTC
2
2
NC
NC

401
794
CCTAACATCAGCTACTGTCT
2
2
NC
NC

402
795
TCCTAACATCAGCTACTGTC
2
2
NC
NC

403
796
GTCCTAACATCAGCTACTGT
2
NC
NC
NC

404
797
TGTCCTAACATCAGCTACTG
2
NC
NC
NC

405
798
GTGTCCTAACATCAGCTACT
2
2
NC
NC

406
799
AGTGTCCTAACATCAGCTAC
2
2
NC
NC

407
800
TAGTGTCCTAACATCAGCTA
3
3
NC
NC

408
801
GTAGTGTCCTAACATCAGCT
3
1
NC
NC

409
802
GGTAGTGTCCTAACATCAGC
2
2
NC
NC

410
803
GGGTAGTGTCCTAACATCAG
2
2
NC
NC

411
804
TGGGTAGTGTCCTAACATCA
2
2
NC
NC

412
805
TTGGGTAGTGTCCTAACATC
2
1
NC
NC

413
806
ATTGGGTAGTGTCCTAACAT
2
1
NC
NC

414
807
CATTGGGTAGTGTCCTAACA
2
NC
NC
NC

415
808
GCATTGGGTAGTGTCCTAAC
3
1
NC
NC

416
809
GGCATTGGGTAGTGTCCTAA
2
2
NC
NC

417
810
AGGCATTGGGTAGTGTCCTA
2
3
NC
NC

418
811
GAGGCATTGGGTAGTGTCCT
2
3
NC
NC

419
812
TGAGGCATTGGGTAGTGTCC
2
3
NC
NC

420
813
TTGAGGCATTGGGTAGTGTC
2
2
NC
NC

421
814
GTTGAGGCATTGGGTAGTGT
2
2
NC
NC

422
815
GGTTGAGGCATTGGGTAGTG
2
NC
NC
NC

423
816
CGGTTGAGGCATTGGGTAGT
3
NC
NC
NC

424
817
ACGGTTGAGGCATTGGGTAG
3
NC
NC
NC

425
818
CACGGTTGAGGCATTGGGTA
2
NC
NC
NC

426
822
TGTCCACGGTTGAGGCATTG
2
NC
NC
NC

427
823
TTGTCCACGGTTGAGGCATT
3
NC
NC
NC

428
824
ATTGTCCACGGTTGAGGCAT
2
NC
NC
NC

429
825
TATTGTCCACGGTTGAGGCA
3
NC
NC
NC

430
826
ATATTGTCCACGGTTGAGGC
3
NC
NC
NC

431
827
AATATTGTCCACGGTTGAGG
2
NC
NC
NC

432
828
GAATATTGTCCACGGTTGAG
3
NC
NC
NC

433
829
CGAATATTGTCCACGGTTGA
3
NC
NC
NC

434
830
GCGAATATTGTCCACGGTTG
3
NC
NC
NC

435
831
AGCGAATATTGTCCACGGTT
3
NC
NC
NC

436
833
GGAGCGAATATTGTCCACGG
2
NC
NC
NC

437
834
TGGAGCGAATATTGTCCACG
3
NC
NC
NC

438
837
AGATGGAGCGAATATTGTCC
2
2
NC
NC

439
838
AAGATGGAGCGAATATTGTC
1
NC
NC
NC

440
839
AAAGATGGAGCGAATATTGT
1
2
NC
NC

441
840
CAAAGATGGAGCGAATATTG
2
1
NC
NC

442
852
TAACAGCATTTCCAAAGATG
1
2
NC
NC

443
853
CTAACAGCATTTCCAAAGAT
2
1
NC
NC

444
854
ACTAACAGCATTTCCAAAGA
2
2
NC
NC

445
855
GACTAACAGCATTTCCAAAG
2
1
NC
NC

446
856
CGACTAACAGCATTTCCAAA
2
2
NC
NC

447
857
TCGACTAACAGCATTTCCAA
2
2
NC
NC

448
858
CTCGACTAACAGCATTTCCA
2
2
NC
NC

449
859
TCTCGACTAACAGCATTTCC
2
2
NC
NC

450
860
TTCTCGACTAACAGCATTTC
1
1
NC
NC

451
861
GTTCTCGACTAACAGCATTT
2
2
NC
NC

452
862
AGTTCTCGACTAACAGCATT
2
NC
NC
NC

453
863
CAGTTCTCGACTAACAGCAT
2
2
NC
NC

454
864
TCAGTTCTCGACTAACAGCA
2
2
NC
NC

455
865
ATCAGTTCTCGACTAACAGC
3
1
NC
NC

456
866
TATCAGTTCTCGACTAACAG
2
2
NC
NC

457
867
CTATCAGTTCTCGACTAACA
2
NC
NC
NC

458
868
TCTATCAGTTCTCGACTAAC
2
3
1
NC

459
869
TTCTATCAGTTCTCGACTAA
3
2
1
NC

460
870
TTTCTATCAGTTCTCGACTA
2
2
NC
NC

461
872
AATTTCTATCAGTTCTCGAC
1
1
NC
NC

462
873
CAATTTCTATCAGTTCTCGA
2
2
NC
NC

463
874
CCAATTTCTATCAGTTCTCG
2
3
NC
NC

464
875
TCCAATTTCTATCAGTTCTC
2
2
NC
NC

465
876
ATCCAATTTCTATCAGTTCT
2
1
NC
NC

466
877
CATCCAATTTCTATCAGTTC
3
1
NC
NC

467
878
ACATCCAATTTCTATCAGTT
2
2
NC
NC

468
879
CACATCCAATTTCTATCAGT
1
1
NC
NC

469
880
TCACATCCAATTTCTATCAG
1
2
NC
NC

470
881
CTCACATCCAATTTCTATCA
1
1
NC
NC

471
882
CCTCACATCCAATTTCTATC
1
2
NC
NC

472
883
TCCTCACATCCAATTTCTAT
1
2
NC
NC

473
884
ATCCTCACATCCAATTTCTA
2
2
NC
NC

474
885
TATCCTCACATCCAATTTCT
2
2
NC
NC

475
886
TTATCCTCACATCCAATTTC
2
NC
NC
NC

476
887
TTTATCCTCACATCCAATTT
2
1
NC
NC

477
888
TTTTATCCTCACATCCAATT
2
2
NC
NC

478
889
GTTTTATCCTCACATCCAAT
2
2
NC
NC

479
890
GGTTTTATCCTCACATCCAA
1
1
NC
NC

480
891
GGGTTTTATCCTCACATCCA
1
1
NC
NC

481
892
AGGGTTTTATCCTCACATCC
1
2
NC
NC

482
893
TAGGGTTTTATCCTCACATC
2
2
NC
NC

483
894
CTAGGGTTTTATCCTCACAT
2
2
NC
NC

484
895
GCTAGGGTTTTATCCTCACA
3
2
2
2

485
896
GGCTAGGGTTTTATCCTCAC
2
2
NC
NC

486
897
AGGCTAGGGTTTTATCCTCA
2
2
NC
NC

487
898
AAGGCTAGGGTTTTATCCTC
2
3
NC
NC

488
900
TGAAGGCTAGGGTTTTATCC
1
2
NC
NC

489
901
TTGAAGGCTAGGGTTTTATC
1
1
NC
NC

490
902
TTTGAAGGCTAGGGTTTTAT
2
1
NC
NC

491
903
TTTTGAAGGCTAGGGTTTTA
2
3
NC
NC

492
904
ATTTTGAAGGCTAGGGTTTT
1
2
NC
NC

493
905
CATTTTGAAGGCTAGGGTTT
2
2
NC
NC

494
906
TCATTTTGAAGGCTAGGGTT
2
2
NC
NC

495
907
TTCATTTTGAAGGCTAGGGT
1
2
NC
NC

496
908
ATTCATTTTGAAGGCTAGGG
1
2
NC
NC

497
909
CATTCATTTTGAAGGCTAGG
2
3
NC
NC

498
910
CCATTCATTTTGAAGGCTAG
2
2
NC
NC

499
911
ACCATTCATTTTGAAGGCTA
2
2
NC
NC

500
912
AACCATTCATTTTGAAGGCT
2
2
NC
NC

501
913
TAACCATTCATTTTGAAGGC
2
1
NC
NC

502
914
GTAACCATTCATTTTGAAGG
1
NC
NC
NC

503
915
TGTAACCATTCATTTTGAAG
1
NC
NC
NC

504
917
TATGTAACCATTCATTTTGA
2
NC
NC
NC

505
918
ATATGTAACCATTCATTTTG
1
NC
NC
NC

506
919
GATATGTAACCATTCATTTT
2
NC
NC
NC

507
920
GGATATGTAACCATTCATTT
2
NC
NC
NC

508
921
TGGATATGTAACCATTCATT
2
NC
NC
NC

509
922
TTGGATATGTAACCATTCAT
2
NC
NC
NC

510
923
ATTGGATATGTAACCATTCA
2
NC
NC
NC

511
924
CATTGGATATGTAACCATTC
2
NC
NC
NC

512
925
GCATTGGATATGTAACCATT
2
NC
NC
NC

513
926
TGCATTGGATATGTAACCAT
2
NC
NC
NC

514
927
TTGCATTGGATATGTAACCA
2
NC
NC
NC

515
928
TTTGCATTGGATATGTAACC
2
NC
NC
NC

516
929
GTTTGCATTGGATATGTAAC
3
NC
NC
NC

517
930
AGTTTGCATTGGATATGTAA
2
NC
NC
NC

518
931
TAGTTTGCATTGGATATGTA
2
NC
NC
NC

519
932
GTAGTTTGCATTGGATATGT
3
NC
NC
NC

520
933
AGTAGTTTGCATTGGATATG
3
NC
NC
NC

521
934
GAGTAGTTTGCATTGGATAT
2
NC
NC
NC

522
935
TGAGTAGTTTGCATTGGATA
3
2
NC
NC

523
936
CTGAGTAGTTTGCATTGGAT
2
2
NC
NC

524
937
ACTGAGTAGTTTGCATTGGA
3
2
NC
NC

525
938
CACTGAGTAGTTTGCATTGG
3
2
NC
NC

526
939
TCACTGAGTAGTTTGCATTG
2
2
NC
NC

527
940
TTCACTGAGTAGTTTGCATT
1
NC
NC
NC

528
941
CTTCACTGAGTAGTTTGCAT
2
2
NC
NC

529
942
TCTTCACTGAGTAGTTTGCA
2
2
NC
NC

530
943
TTCTTCACTGAGTAGTTTGC
2
2
NC
NC

531
944
CTTCTTCACTGAGTAGTTTG
2
NC
NC
NC

532
945
ACTTCTTCACTGAGTAGTTT
2
NC
NC
NC

533
946
CACTTCTTCACTGAGTAGTT
2
NC
NC
NC

534
948
TGCACTTCTTCACTGAGTAG
2
NC
NC
NC

535
949
ATGCACTTCTTCACTGAGTA
2
NC
NC
NC

536
950
GATGCACTTCTTCACTGAGT
2
NC
NC
NC

537
951
AGATGCACTTCTTCACTGAG
2
NC
NC
NC

538
952
AAGATGCACTTCTTCACTGA
2
NC
NC
NC

539
962
GAAGAGTAAGAAGATGCACT
2
NC
NC
NC

540
963
TGAAGAGTAAGAAGATGCAC
2
NC
NC
NC

541
964
ATGAAGAGTAAGAAGATGCA
2
NC
NC
NC

542
965
GATGAAGAGTAAGAAGATGC
2
2
NC
NC

543
966
TGATGAAGAGTAAGAAGATG
1
1
NC
NC

544
967
TTGATGAAGAGTAAGAAGAT
2
NC
NC
NC

545
968
GTTGATGAAGAGTAAGAAGA
2
2
NC
NC

546
969
GGTTGATGAAGAGTAAGAAG
2
2
NC
NC

547
970
TGGTTGATGAAGAGTAAGAA
2
2
NC
NC

548
971
ATGGTTGATGAAGAGTAAGA
2
2
NC
NC

549
972
GATGGTTGATGAAGAGTAAG
2
3
NC
NC

550
973
CGATGGTTGATGAAGAGTAA
2
NC
NC
NC

551
974
ACGATGGTTGATGAAGAGTA
2
2
NC
NC

552
975
GACGATGGTTGATGAAGAGT
3
1
NC
NC

553
976
AGACGATGGTTGATGAAGAG
2
1
NC
NC

554
977
CAGACGATGGTTGATGAAGA
2
2
NC
NC

555
988
GTTGATTCTACCAGACGATG
3
2
NC
NC

556
989
AGTTGATTCTACCAGACGAT
3
3
NC
NC

557
990
AAGTTGATTCTACCAGACGA
3
2
NC
NC

558
991
GAAGTTGATTCTACCAGACG
3
2
NC
NC

559
992
GGAAGTTGATTCTACCAGAC
2
3
NC
NC

560
993
AGGAAGTTGATTCTACCAGA
2
2
NC
NC

561
994
AAGGAAGTTGATTCTACCAG
2
NC
NC
NC

562
995
CAAGGAAGTTGATTCTACCA
2
NC
NC
NC

563
1004
GGCTTTTCTCAAGGAAGTTG
2
1
NC
NC

564
1005
TGGCTTTTCTCAAGGAAGTT
2
1
NC
NC

565
1006
ATGGCTTTTCTCAAGGAAGT
2
2
NC
NC

566
1007
TATGGCTTTTCTCAAGGAAG
2
2
NC
NC

567
1008
CTATGGCTTTTCTCAAGGAA
1
2
NC
NC

568
1009
TCTATGGCTTTTCTCAAGGA
1
3
NC
NC

569
1010
TTCTATGGCTTTTCTCAAGG
1
2
NC
NC

570
1011
TTTCTATGGCTTTTCTCAAG
1
2
NC
NC

571
1012
GTTTCTATGGCTTTTCTCAA
2
2
NC
NC

572
1013
TGTTTCTATGGCTTTTCTCA
1
1
NC
NC

573
1014
CTGTTTCTATGGCTTTTCTC
1
2
NC
NC

574
1015
ACTGTTTCTATGGCTTTTCT
2
1
NC
NC

575
1016
CACTGTTTCTATGGCTTTTC
2
3
NC
NC

576
1017
ACACTGTTTCTATGGCTTTT
2
2
NC
NC

577
1018
TACACTGTTTCTATGGCTTT
2
2
NC
NC

578
1019
ATACACTGTTTCTATGGCTT
1
2
NC
NC

579
1020
CATACACTGTTTCTATGGCT
2
2
NC
NC

580
1021
GCATACACTGTTTCTATGGC
1
NC
NC
NC

581
1022
TGCATACACTGTTTCTATGG
1
1
NC
NC

582
1023
CTGCATACACTGTTTCTATG
2
NC
NC
NC

583
1024
GCTGCATACACTGTTTCTAT
2
1
NC
NC

584
1025
GGCTGCATACACTGTTTCTA
1
2
NC
NC

585
1026
AGGCTGCATACACTGTTTCT
2
2
NC
NC

586
1027
TAGGCTGCATACACTGTTTC
2
2
NC
NC

587
1028
ATAGGCTGCATACACTGTTT
2
NC
NC
NC

588
1029
AATAGGCTGCATACACTGTT
2
NC
NC
NC

589
1030
AAATAGGCTGCATACACTGT
2
NC
NC
NC

590
1031
CAAATAGGCTGCATACACTG
2
NC
NC
NC

591
1043
TGTGTTTTTGGGCAAATAGG
2
NC
NC
NC

592
1044
GTGTGTTTTTGGGCAAATAG
2
NC
NC
NC

593
1045
TGTGTGTTTTTGGGCAAATA
1
NC
NC
NC

594
1046
GTGTGTGTTTTTGGGCAAAT
1
NC
NC
NC

595
1047
GGTGTGTGTTTTTGGGCAAA
2
NC
NC
NC

596
1048
GGGTGTGTGTTTTTGGGCAA
2
2
2
NC

597
1049
TGGGTGTGTGTTTTTGGGCA
2
3
2
NC

598
1050
ATGGGTGTGTGTTTTTGGGC
2
2
2
NC

599
1051
AATGGGTGTGTGTTTTTGGG
1
2
2
NC

600
1052
GAATGGGTGTGTGTTTTTGG
1
2
2
NC

601
1053
GGAATGGGTGTGTGTTTTTG
1
2
2
NC

602
1054
AGGAATGGGTGTGTGTTTTT
2
2
2
NC

603
1055
CAGGAATGGGTGTGTGTTTT
2
2
2
NC

604
1056
ACAGGAATGGGTGTGTGTTT
2
2
1
NC

605
1057
TACAGGAATGGGTGTGTGTT
2
2
1
NC

606
1058
GTACAGGAATGGGTGTGTGT
1
2
1
NC

607
1059
GGTACAGGAATGGGTGTGTG
2
2
1
NC

608
1060
AGGTACAGGAATGGGTGTGT
2
2
2
NC

609
1061
GAGGTACAGGAATGGGTGTG
1
1
1
NC

610
1062
TGAGGTACAGGAATGGGTGT
2
1
2
NC

611
1073
GATTTCTAAACTGAGGTACA
1
2
NC
NC

612
1074
TGATTTCTAAACTGAGGTAC
2
1
NC
NC

613
1075
CTGATTTCTAAACTGAGGTA
2
2
NC
NC

614
1076
ACTGATTTCTAAACTGAGGT
1
1
NC
NC

615
1077
GACTGATTTCTAAACTGAGG
2
1
NC
NC

616
1078
GGACTGATTTCTAAACTGAG
2
2
NC
NC

617
1079
GGGACTGATTTCTAAACTGA
2
2
NC
NC

618
1099
ACATTAACATCCACATTCTG
2
NC
NC
NC

619
1100
CACATTAACATCCACATTCT
2
2
NC
NC

620
1101
GCACATTAACATCCACATTC
2
2
NC
NC

621
1102
TGCACATTAACATCCACATT
2
2
NC
NC

622
1103
GTGCACATTAACATCCACAT
2
2
NC
NC

623
1104
GGTGCACATTAACATCCACA
1
2
NC
NC

624
1105
GGGTGCACATTAACATCCAC
1
2
NC
NC

625
1123
TGAACTTCATGCTTTGTGGG
1
2
NC
NC

626
1124
GTGAACTTCATGCTTTGTGG
2
2
NC
NC

627
1125
AGTGAACTTCATGCTTTGTG
2
2
NC
NC

628
1126
AAGTGAACTTCATGCTTTGT
2
3
NC
NC

629
1177
TTGCTCTCGATGTGCTGCTG
1
NC
NC
NC

630
1178
CTTGCTCTCGATGTGCTGCT
1
NC
NC
NC

631
1180
AGCTTGCTCTCGATGTGCTG
2
2
NC
NC

632
1181
GAGCTTGCTCTCGATGTGCT
2
1
NC
NC

633
1183
AGGAGCTTGCTCTCGATGTG
2
2
NC
NC

634
1184
CAGGAGCTTGCTCTCGATGT
2
2
NC
NC

635
1214
GGTGAAGTACATCCTGGAGG
2
2
NC
NC

636
1221
AAGTCTGGGTGAAGTACATC
2
2
NC
NC

637
1222
AAAGTCTGGGTGAAGTACAT
2
2
NC
NC

638
1223
CAAAGTCTGGGTGAAGTACA
1
1
NC
NC

639
1224
GCAAAGTCTGGGTGAAGTAC
2
2
NC
NC

640
1225
AGCAAAGTCTGGGTGAAGTA
2
2
NC
NC

641
1226
TAGCAAAGTCTGGGTGAAGT
2
2
NC
NC

642
1227
GTAGCAAAGTCTGGGTGAAG
2
2
NC
NC

643
1228
GGTAGCAAAGTCTGGGTGAA
3
1
NC
NC

644
1229
TGGTAGCAAAGTCTGGGTGA
2
2
NC
NC

645
1230
CTGGTAGCAAAGTCTGGGTG
2
2
NC
NC

646
1231
CCTGGTAGCAAAGTCTGGGT
2
2
NC
NC

647
1232
TCCTGGTAGCAAAGTCTGGG
2
NC
NC
NC

648
1233
GTCCTGGTAGCAAAGTCTGG
2
1
NC
NC

649
1234
AGTCCTGGTAGCAAAGTCTG
2
2
NC
NC

650
1235
AAGTCCTGGTAGCAAAGTCT
2
1
NC
NC

651
1236
CAAGTCCTGGTAGCAAAGTC
2
2
NC
NC

652
1237
GCAAGTCCTGGTAGCAAAGT
2
2
NC
NC

653
1238
AGCAAGTCCTGGTAGCAAAG
1
2
NC
NC

654
1239
CAGCAAGTCCTGGTAGCAAA
1
2
NC
NC

655
1264
GATTTAACCATCTCCCCAGA
2
2
NC
NC

656
1265
GGATTTAACCATCTCCCCAG
2
2
NC
NC

657
1266
TGGATTTAACCATCTCCCCA
2
2
NC
NC

658
1275
GACTTGTTGTGGATTTAACC
2
NC
NC
NC

659
1276
AGACTTGTTGTGGATTTAAC
2
NC
NC
NC

660
1277
CAGACTTGTTGTGGATTTAA
2
NC
NC
NC

661
1278
TCAGACTTGTTGTGGATTTA
2
NC
NC
NC

662
1279
GTCAGACTTGTTGTGGATTT
2
NC
NC
NC

663
1280
GGTCAGACTTGTTGTGGATT
2
NC
NC
NC

664
1281
AGGTCAGACTTGTTGTGGAT
3
NC
NC
NC

665
1282
GAGGTCAGACTTGTTGTGGA
3
NC
NC
NC

666
1283
CGAGGTCAGACTTGTTGTGG
2
NC
NC
NC

667
1284
ACGAGGTCAGACTTGTTGTG
3
NC
NC
NC

668
1285
GACGAGGTCAGACTTGTTGT
3
NC
NC
NC

669
1286
AGACGAGGTCAGACTTGTTG
2
NC
NC
NC

670
1287
AAGACGAGGTCAGACTTGTT
3
NC
NC
NC

671
1288
GAAGACGAGGTCAGACTTGT
3
NC
NC
NC

672
1289
AGAAGACGAGGTCAGACTTG
2
NC
NC
NC

673
1290
TAGAAGACGAGGTCAGACTT
2
NC
NC
NC

674
1291
GTAGAAGACGAGGTCAGACT
2
NC
NC
NC

675
1292
AGTAGAAGACGAGGTCAGAC
2
NC
NC
NC

676
1293
AAGTAGAAGACGAGGTCAGA
1
NC
NC
NC

677
1294
GAAGTAGAAGACGAGGTCAG
2
NC
NC
NC

678
1295
AGAAGTAGAAGACGAGGTCA
2
NC
NC
NC

679
1296
CAGAAGTAGAAGACGAGGTC
2
NC
NC
NC

680
1297
CCAGAAGTAGAAGACGAGGT
2
NC
NC
NC

681
1298
TCCAGAAGTAGAAGACGAGG
2
NC
NC
NC

682
1299
TTCCAGAAGTAGAAGACGAG
2
NC
NC
NC

683
1311
CCTTATCACTACTTCCAGAA
2
NC
NC
NC

684
1312
ACCTTATCACTACTTCCAGA
2
NC
NC
NC

685
1313
GACCTTATCACTACTTCCAG
3
NC
NC
NC

686
1314
AGACCTTATCACTACTTCCA
2
NC
NC
NC

687
1315
TAGACCTTATCACTACTTCC
2
NC
NC
NC

688
1316
ATAGACCTTATCACTACTTC
2
NC
NC
NC

689
1317
CATAGACCTTATCACTACTT
2
NC
NC
NC

690
1318
GCATAGACCTTATCACTACT
3
NC
NC
NC

691
1319
GGCATAGACCTTATCACTAC
2
NC
NC
NC

692
1320
GGGCATAGACCTTATCACTA
2
NC
NC
NC

693
1321
TGGGCATAGACCTTATCACT
2
NC
NC
NC

694
1322
GTGGGCATAGACCTTATCAC
3
NC
NC
NC

695
1323
GGTGGGCATAGACCTTATCA
2
NC
NC
NC

696
1324
TGGTGGGCATAGACCTTATC
3
NC
NC
NC

697
1325
CTGGTGGGCATAGACCTTAT
3
NC
NC
NC

698
1326
TCTGGTGGGCATAGACCTTA
2
NC
NC
NC

699
1327
ATCTGGTGGGCATAGACCTT
2
2
NC
NC

700
1328
CATCTGGTGGGCATAGACCT
2
2
NC
NC

701
1342
GAATCTGTACGAACCATCTG
3
2
NC
NC

702
1343
GGAATCTGTACGAACCATCT
3
2
NC
NC

703
1344
GGGAATCTGTACGAACCATC
3
2
NC
NC

704
1345
CGGGAATCTGTACGAACCAT
2
2
NC
NC

705
1346
CCGGGAATCTGTACGAACCA
2
1
NC
NC

706
1348
TCCCGGGAATCTGTACGAAC
3
2
NC
NC

707
1360
TCAAGCTTCTGTTCCCGGGA
2
2
NC
NC

708
1361
ATCAAGCTTCTGTTCCCGGG
3
2
NC
NC

709
1362
CATCAAGCTTCTGTTCCCGG
3
2
NC
NC

710
1381
CTCAGAGGCTGCAGAAATGC
1
2
NC
NC

711
1382
GCTCAGAGGCTGCAGAAATG
1
2
NC
NC

712
1383
TGCTCAGAGGCTGCAGAAAT
1
2
NC
NC

713
1384
TTGCTCAGAGGCTGCAGAAA
1
NC
NC
NC

714
1385
TTTGCTCAGAGGCTGCAGAA
2
2
NC
NC

715
1386
GTTTGCTCAGAGGCTGCAGA
2
2
NC
NC

716
1423
TCCTCTGTGACAATGGCCTG
2
NC
NC
NC

717
1424
ATCCTCTGTGACAATGGCCT
2
NC
NC
NC

718
1425
TATCCTCTGTGACAATGGCC
2
NC
NC
NC

719
1426
TTATCCTCTGTGACAATGGC
2
NC
NC
NC

720
1427
CTTATCCTCTGTGACAATGG
2
NC
NC
NC

721
1428
TCTTATCCTCTGTGACAATG
2
NC
NC
NC

722
1429
GTCTTATCCTCTGTGACAAT
2
NC
NC
NC

723
1430
TGTCTTATCCTCTGTGACAA
2
NC
NC
NC

724
1431
CTGTCTTATCCTCTGTGACA
1
NC
NC
NC

725
1432
TCTGTCTTATCCTCTGTGAC
1
NC
NC
NC

726
1433
ATCTGTCTTATCCTCTGTGA
1
NC
NC
NC

727
1434
TATCTGTCTTATCCTCTGTG
1
NC
NC
NC

728
1435
ATATCTGTCTTATCCTCTGT
2
NC
NC
NC

729
1436
AATATCTGTCTTATCCTCTG
2
2
NC
NC

730
1437
AAATATCTGTCTTATCCTCT
2
2
NC
NC

731
1438
GAAATATCTGTCTTATCCTC
2
1
NC
NC

732
1439
AGAAATATCTGTCTTATCCT
2
NC
NC
NC

733
1440
TAGAAATATCTGTCTTATCC
2
2
NC
NC

734
1441
CTAGAAATATCTGTCTTATC
2
2
NC
NC

735
1442
ACTAGAAATATCTGTCTTAT
2
1
NC
NC

736
1443
CACTAGAAATATCTGTCTTA
2
2
NC
NC

737
1444
CCACTAGAAATATCTGTCTT
2
3
NC
NC

738
1445
GCCACTAGAAATATCTGTCT
2
2
NC
NC

739
1446
TGCCACTAGAAATATCTGTC
2
2
NC
NC

740
1447
CTGCCACTAGAAATATCTGT
2
2
NC
NC

741
1448
CCTGCCACTAGAAATATCTG
2
2
NC
NC

742
1450
GCCCTGCCACTAGAAATATC
2
2
NC
NC

743
1451
AGCCCTGCCACTAGAAATAT
2
NC
NC
NC

744
1452
TAGCCCTGCCACTAGAAATA
2
2
NC
NC

745
1453
CTAGCCCTGCCACTAGAAAT
2
2
NC
NC

746
1454
CCTAGCCCTGCCACTAGAAA
2
1
NC
NC

747
1469
CTCCTCATCTTGCTGCCTAG
2
2
NC
NC

748
1470
TCTCCTCATCTTGCTGCCTA
1
2
NC
NC

749
1471
ATCTCCTCATCTTGCTGCCT
2
2
NC
NC

750
1472
CATCTCCTCATCTTGCTGCC
2
NC
NC
NC

751
1473
GCATCTCCTCATCTTGCTGC
2
1
NC
NC

752
1476
CAAGCATCTCCTCATCTTGC
2
2
NC
NC

753
1477
TCAAGCATCTCCTCATCTTG
2
2
NC
NC

754
1478
TTCAAGCATCTCCTCATCTT
2
2
NC
NC

755
1479
GTTCAAGCATCTCCTCATCT
1
2
NC
NC

756
1480
AGTTCAAGCATCTCCTCATC
1
2
NC
NC

757
1481
GAGTTCAAGCATCTCCTCAT
2
2
NC
NC

758
1486
GCTGGGAGTTCAAGCATCTC
2
NC
NC
NC

759
1487
GGCTGGGAGTTCAAGCATCT
1
NC
NC
NC

760
1508
TTTGGCAGCCACTTCAGCAG
1
1
NC
NC

761
1509
TTTTGGCAGCCACTTCAGCA
1
2
NC
NC

762
1510
TTTTTGGCAGCCACTTCAGC
2
1
NC
NC

763
1511
ATTTTTGGCAGCCACTTCAG
2
2
NC
NC

764
1512
GATTTTTGGCAGCCACTTCA
2
NC
NC
NC

765
1513
TGATTTTTGGCAGCCACTTC
2
2
NC
NC

766
1514
CTGATTTTTGGCAGCCACTT
2
2
NC
NC

767
1515
TCTGATTTTTGGCAGCCACT
1
2
NC
NC

768
1516
CTCTGATTTTTGGCAGCCAC
2
2
NC
NC

769
1517
GCTCTGATTTTTGGCAGCCA
2
2
NC
NC

770
1518
AGCTCTGATTTTTGGCAGCC
2
NC
NC
NC

771
1519
AAGCTCTGATTTTTGGCAGC
2
NC
NC
NC

772
1520
CAAGCTCTGATTTTTGGCAG
2
NC
NC
NC

773
1521
CCAAGCTCTGATTTTTGGCA
2
NC
NC
NC

774
1524
CCTCCAAGCTCTGATTTTTG
2
NC
NC
NC

775
1525
CCCTCCAAGCTCTGATTTTT
1
NC
NC
NC

776
1526
CCCCTCCAAGCTCTGATTTT
1
NC
NC
NC

777
1527
CCCCCTCCAAGCTCTGATTT
2
NC
NC
NC

778
1528
TCCCCCTCCAAGCTCTGATT
2
NC
NC
NC

779
1529
ATCCCCCTCCAAGCTCTGAT
2
NC
NC
NC

780
1530
TATCCCCCTCCAAGCTCTGA
2
NC
NC
NC

781
1532
TGTATCCCCCTCCAAGCTCT
2
NC
NC
NC

782
1533
TTGTATCCCCCTCCAAGCTC
2
NC
NC
NC

783
1534
GTTGTATCCCCCTCCAAGCT
2
NC
NC
NC

784
1535
TGTTGTATCCCCCTCCAAGC
2
NC
NC
NC

785
1536
TTGTTGTATCCCCCTCCAAG
2
NC
NC
NC

786
1537
TTTGTTGTATCCCCCTCCAA
2
NC
NC
NC

787
1538
CTTTGTTGTATCCCCCTCCA
2
NC
NC
NC

788
1539
CCTTTGTTGTATCCCCCTCC
2
2
NC
NC

789
1540
CCCTTTGTTGTATCCCCCTC
2
NC
NC
NC

790
1541
CCCCTTTGTTGTATCCCCCT
2
2
NC
NC

791
1542
TCCCCTTTGTTGTATCCCCC
2
2
NC
NC

792
1543
GTCCCCTTTGTTGTATCCCC
2
1
NC
NC

793
1544
AGTCCCCTTTGTTGTATCCC
2
NC
NC
NC

794
1545
AAGTCCCCTTTGTTGTATCC
2
NC
NC
NC

795
1546
GAAGTCCCCTTTGTTGTATC
2
NC
NC
NC

796
1547
TGAAGTCCCCTTTGTTGTAT
2
NC
NC
NC

797
1548
CTGAAGTCCCCTTTGTTGTA
2
NC
NC
NC

798
1549
TCTGAAGTCCCCTTTGTTGT
2
NC
NC
NC

799
1550
TTCTGAAGTCCCCTTTGTTG
1
NC
NC
NC

800
1551
TTTCTGAAGTCCCCTTTGTT
1
NC
NC
NC

801
1552
ATTTCTGAAGTCCCCTTTGT
1
NC
NC
NC

802
1553
CATTTCTGAAGTCCCCTTTG
1
NC
NC
NC

803
1554
ACATTTCTGAAGTCCCCTTT
1
NC
NC
NC

804
1555
GACATTTCTGAAGTCCCCTT
1
NC
NC
NC

805
1556
TGACATTTCTGAAGTCCCCT
1
NC
NC
NC

806
1557
CTGACATTTCTGAAGTCCCC
2
NC
NC
NC

807
1558
TCTGACATTTCTGAAGTCCC
2
NC
NC
NC

808
1559
CTCTGACATTTCTGAAGTCC
2
NC
NC
NC

809
1560
TCTCTGACATTTCTGAAGTC
2
NC
NC
NC

810
1561
TTCTCTGACATTTCTGAAGT
2
NC
NC
NC

811
1562
CTTCTCTGACATTTCTGAAG
2
NC
NC
NC

812
1563
TCTTCTCTGACATTTCTGAA
2
NC
NC
NC

813
1564
CTCTTCTCTGACATTTCTGA
2
2
NC
NC

814
1565
TCTCTTCTCTGACATTTCTG
1
2
NC
NC

815
1566
CTCTCTTCTCTGACATTTCT
1
2
NC
NC

816
1567
CCTCTCTTCTCTGACATTTC
2
NC
NC
NC

817
1568
TCCTCTCTTCTCTGACATTT
2
2
NC
NC

818
1569
GTCCTCTCTTCTCTGACATT
1
3
NC
NC

819
1570
GGTCCTCTCTTCTCTGACAT
2
2
NC
NC

820
1571
AGGTCCTCTCTTCTCTGACA
2
NC
NC
NC

821
1572
TAGGTCCTCTCTTCTCTGAC
2
NC
NC
NC

822
1573
GTAGGTCCTCTCTTCTCTGA
2
NC
NC
NC

823
1574
AGTAGGTCCTCTCTTCTCTG
2
NC
NC
NC

824
1575
AAGTAGGTCCTCTCTTCTCT
2
NC
NC
NC

825
1576
GAAGTAGGTCCTCTCTTCTC
2
NC
NC
NC

826
1577
GGAAGTAGGTCCTCTCTTCT
2
NC
NC
NC

827
1603
TCCCGATGTCTCTTTCTGGG
2
2
NC
NC

828
1604
TTCCCGATGTCTCTTTCTGG
2
3
NC
NC

829
1605
CTTCCCGATGTCTCTTTCTG
2
2
NC
NC

830
1606
TCTTCCCGATGTCTCTTTCT
2
2
NC
NC

831
1607
ATCTTCCCGATGTCTCTTTC
2
2
NC
NC

832
1608
AATCTTCCCGATGTCTCTTT
2
1
NC
NC

833
1609
GAATCTTCCCGATGTCTCTT
2
2
NC
NC

834
1610
AGAATCTTCCCGATGTCTCT
2
2
NC
NC

835
1611
CAGAATCTTCCCGATGTCTC
3
2
NC
NC

836
1612
TCAGAATCTTCCCGATGTCT
2
NC
NC
NC

837
1613
ATCAGAATCTTCCCGATGTC
2
NC
NC
NC

838
1614
CATCAGAATCTTCCCGATGT
2
NC
NC
NC

839
1616
CACATCAGAATCTTCCCGAT
2
NC
NC
NC

840
1617
CCACATCAGAATCTTCCCGA
2
2
NC
NC

841
1618
TCCACATCAGAATCTTCCCG
2
2
NC
NC

842
1619
TTCCACATCAGAATCTTCCC
2
2
NC
NC

843
1620
TTTCCACATCAGAATCTTCC
1
3
NC
NC

844
1621
ATTTCCACATCAGAATCTTC
1
2
NC
NC

845
1622
CATTTCCACATCAGAATCTT
2
3
NC
NC

846
1623
CCATTTCCACATCAGAATCT
2
NC
NC
NC

847
1624
ACCATTTCCACATCAGAATC
2
2
NC
NC

848
1625
CACCATTTCCACATCAGAAT
2
2
NC
NC

849
1626
CCACCATTTCCACATCAGAA
2
2
NC
NC

850
1627
TCCACCATTTCCACATCAGA
2
2
2
NC

851
1628
TTCCACCATTTCCACATCAG
1
1
2
NC

852
1629
CTTCCACCATTTCCACATCA
2
3
NC
NC

853
1630
TCTTCCACCATTTCCACATC
2
2
NC
NC

854
1631
ATCTTCCACCATTTCCACAT
1
2
NC
NC

855
1632
CATCTTCCACCATTTCCACA
1
2
NC
NC

856
1633
TCATCTTCCACCATTTCCAC
1
2
NC
NC

857
1634
ATCATCTTCCACCATTTCCA
2
3
NC
NC

858
1635
AATCATCTTCCACCATTTCC
1
2
NC
NC

859
1636
GAATCATCTTCCACCATTTC
2
2
NC
NC

860
1644
CCTTTCGGGAATCATCTTCC
2
2
NC
NC

861
1655
TGCAGTCATTTCCTTTCGGG
2
2
NC
NC

862
1656
CTGCAGTCATTTCCTTTCGG
2
1
NC
NC

863
1657
GCTGCAGTCATTTCCTTTCG
2
NC
NC
NC

864
1658
AGCTGCAGTCATTTCCTTTC
1
1
NC
NC

865
1659
AAGCTGCAGTCATTTCCTTT
1
2
NC
NC

866
1660
CAAGCTGCAGTCATTTCCTT
2
2
NC
NC

867
1661
ACAAGCTGCAGTCATTTCCT
2
1
NC
NC

868
1662
TACAAGCTGCAGTCATTTCC
2
2
NC
NC

869
1663
GTACAAGCTGCAGTCATTTC
2
2
NC
NC

870
1664
GGTACAAGCTGCAGTCATTT
2
2
NC
NC

871
1665
GGGTACAAGCTGCAGTCATT
2
2
NC
NC

872
1685
GTTAATGATCCTTCTCCGGG
3
2
NC
NC

873
1686
GGTTAATGATCCTTCTCCGG
3
2
NC
NC

874
1687
AGGTTAATGATCCTTCTCCG
2
2
NC
NC

875
1688
GAGGTTAATGATCCTTCTCC
1
2
NC
NC

876
1689
TGAGGTTAATGATCCTTCTC
2
2
NC
NC

877
1690
GTGAGGTTAATGATCCTTCT
2
1
NC
NC

878
1691
AGTGAGGTTAATGATCCTTC
2
2
NC
NC

879
1692
TAGTGAGGTTAATGATCCTT
2
1
NC
NC

880
1693
CTAGTGAGGTTAATGATCCT
2
1
NC
NC

881
1694
ACTAGTGAGGTTAATGATCC
2
1
NC
NC

882
1695
CACTAGTGAGGTTAATGATC
2
2
NC
NC

883
1707
GGAGACTCAAAACACTAGTG
2
2
NC
NC

884
1708
TGGAGACTCAAAACACTAGT
2
2
NC
NC

885
1709
CTGGAGACTCAAAACACTAG
2
2
NC
NC

886
1710
CCTGGAGACTCAAAACACTA
2
1
NC
NC

887
1711
TCCTGGAGACTCAAAACACT
2
2
NC
NC

888
1712
TTCCTGGAGACTCAAAACAC
1
2
NC
NC

889
1713
CTTCCTGGAGACTCAAAACA
2
2
NC
NC

890
1714
TCTTCCTGGAGACTCAAAAC
2
2
NC
NC

891
1715
TTCTTCCTGGAGACTCAAAA
2
1
NC
NC

892
1716
TTTCTTCCTGGAGACTCAAA
1
2
NC
NC

893
1717
ATTTCTTCCTGGAGACTCAA
2
2
NC
NC

894
1718
AATTTCTTCCTGGAGACTCA
2
2
NC
NC

895
1719
TAATTTCTTCCTGGAGACTC
2
NC
NC
NC

896
1720
TTAATTTCTTCCTGGAGACT
1
NC
NC
NC

897
1721
ATTAATTTCTTCCTGGAGAC
1
2
NC
NC

898
1722
CATTAATTTCTTCCTGGAGA
1
1
NC
NC

899
1723
TCATTAATTTCTTCCTGGAG
1
3
NC
NC

900
1724
CTCATTAATTTCTTCCTGGA
1
2
NC
NC

901
1725
GCTCATTAATTTCTTCCTGG
2
2
NC
NC

902
1726
TGCTCATTAATTTCTTCCTG
1
NC
NC
NC

903
1727
CTGCTCATTAATTTCTTCCT
2
NC
NC
NC

904
1728
CCTGCTCATTAATTTCTTCC
2
NC
NC
NC

905
1729
CCCTGCTCATTAATTTCTTC
2
NC
NC
NC

906
1730
TCCCTGCTCATTAATTTCTT
2
NC
NC
NC

907
1731
GTCCCTGCTCATTAATTTCT
2
NC
NC
NC

908
1732
TGTCCCTGCTCATTAATTTC
2
NC
NC
NC

909
1733
ATGTCCCTGCTCATTAATTT
2
NC
NC
NC

910
1734
CATGTCCCTGCTCATTAATT
2
NC
NC
NC

911
1735
TCATGTCCCTGCTCATTAAT
2
NC
NC
NC

912
1736
CTCATGTCCCTGCTCATTAA
2
NC
NC
NC

913
1737
CCTCATGTCCCTGCTCATTA
2
NC
NC
NC

914
1738
ACCTCATGTCCCTGCTCATT
2
NC
NC
NC

915
1739
AACCTCATGTCCCTGCTCAT
2
NC
NC
NC

916
1740
GAACCTCATGTCCCTGCTCA
2
NC
NC
NC

917
1741
AGAACCTCATGTCCCTGCTC
2
NC
NC
NC

918
1742
GAGAACCTCATGTCCCTGCT
1
NC
NC
NC

919
1749
TCTCCCGGAGAACCTCATGT
3
NC
NC
NC

920
1759
TTATGCAACATCTCCCGGAG
2
NC
NC
NC

921
1760
GTTATGCAACATCTCCCGGA
3
NC
NC
NC

922
1761
GGTTATGCAACATCTCCCGG
3
NC
NC
NC

923
1762
TGGTTATGCAACATCTCCCG
2
1
NC
NC

924
1763
GTGGTTATGCAACATCTCCC
2
2
NC
NC

925
1764
AGTGGTTATGCAACATCTCC
2
2
NC
NC

926
1765
GAGTGGTTATGCAACATCTC
2
2
NC
NC

927
1766
GGAGTGGTTATGCAACATCT
2
1
NC
NC

928
1767
AGGAGTGGTTATGCAACATC
2
1
NC
NC

929
1768
AAGGAGTGGTTATGCAACAT
2
2
NC
NC

930
1769
GAAGGAGTGGTTATGCAACA
2
1
NC
NC

931
1770
CGAAGGAGTGGTTATGCAAC
2
1
NC
NC

932
1771
ACGAAGGAGTGGTTATGCAA
3
NC
NC
NC

933
1785
GATTCACACAGCCCACGAAG
2
2
NC
NC

934
1786
GGATTCACACAGCCCACGAA
2
2
NC
NC

935
1787
AGGATTCACACAGCCCACGA
2
NC
NC
NC

936
1789
TGAGGATTCACACAGCCCAC
2
2
2
2

937
1790
CTGAGGATTCACACAGCCCA
2
2
1
1

938
1791
ACTGAGGATTCACACAGCCC
2
2
2
2

939
1792
CACTGAGGATTCACACAGCC
2
NC
2
2

940
1814
GGTTTGATGCTGTGCCAAGG
3
NC
NC
NC

941
1815
TGGTTTGATGCTGTGCCAAG
2
NC
NC
NC

942
1816
TTGGTTTGATGCTGTGCCAA
2
NC
NC
NC

943
1817
CTTGGTTTGATGCTGTGCCA
1
NC
NC
NC

944
1818
ACTTGGTTTGATGCTGTGCC
2
NC
NC
NC

945
1819
AACTTGGTTTGATGCTGTGC
2
NC
NC
NC

946
1820
TAACTTGGTTTGATGCTGTG
2
NC
NC
NC

947
1821
ATAACTTGGTTTGATGCTGT
1
NC
NC
NC

948
1822
TATAACTTGGTTTGATGCTG
2
NC
NC
NC

949
1823
GTATAACTTGGTTTGATGCT
2
NC
NC
NC

950
1824
GGTATAACTTGGTTTGATGC
3
NC
NC
NC

951
1825
AGGTATAACTTGGTTTGATG
2
NC
NC
NC

952
1826
AAGGTATAACTTGGTTTGAT
2
NC
NC
NC

953
1827
GAAGGTATAACTTGGTTTGA
2
NC
NC
NC

954
1828
AGAAGGTATAACTTGGTTTG
2
NC
NC
NC

955
1829
GAGAAGGTATAACTTGGTTT
2
NC
NC
NC

956
1830
TGAGAAGGTATAACTTGGTT
2
NC
NC
NC

957
1831
TTGAGAAGGTATAACTTGGT
2
NC
NC
NC

958
1832
GTTGAGAAGGTATAACTTGG
2
NC
NC
NC

959
1833
TGTTGAGAAGGTATAACTTG
2
NC
NC
NC

960
1834
GTGTTGAGAAGGTATAACTT
2
NC
NC
NC

961
1835
GGTGTTGAGAAGGTATAACT
2
NC
NC
NC

962
1836
TGGTGTTGAGAAGGTATAAC
2
NC
NC
NC

963
1837
GTGGTGTTGAGAAGGTATAA
2
NC
NC
NC

964
1838
GGTGGTGTTGAGAAGGTATA
2
NC
NC
NC

965
1839
TGGTGGTGTTGAGAAGGTAT
2
NC
NC
NC

966
1840
TTGGTGGTGTTGAGAAGGTA
2
NC
NC
NC

967
1841
CTTGGTGGTGTTGAGAAGGT
2
NC
NC
NC

968
1842
GCTTGGTGGTGTTGAGAAGG
2
NC
NC
NC

969
1843
AGCTTGGTGGTGTTGAGAAG
2
NC
NC
NC

970
1844
AAGCTTGGTGGTGTTGAGAA
2
NC
NC
NC

971
1845
TAAGCTTGGTGGTGTTGAGA
2
NC
NC
NC

972
1846
CTAAGCTTGGTGGTGTTGAG
3
NC
NC
NC

973
1847
ACTAAGCTTGGTGGTGTTGA
2
NC
NC
NC

974
1848
CACTAAGCTTGGTGGTGTTG
2
NC
NC
NC

975
1855
AGTTCTTCACTAAGCTTGGT
2
2
NC
NC

976
1856
CAGTTCTTCACTAAGCTTGG
1
2
NC
NC

977
1857
ACAGTTCTTCACTAAGCTTG
2
2
NC
NC

978
1858
AACAGTTCTTCACTAAGCTT
1
1
NC
NC

979
1859
GAACAGTTCTTCACTAAGCT
2
2
NC
NC

980
1860
AGAACAGTTCTTCACTAAGC
2
2
NC
NC

981
1874
AATGAGTATCTGGTAGAACA
2
2
2
NC

982
1875
AAATGAGTATCTGGTAGAAC
2
2
2
NC

983
1876
TAAATGAGTATCTGGTAGAA
1
1
2
1

984
1877
ATAAATGAGTATCTGGTAGA
1
2
1
NC

985
1878
CATAAATGAGTATCTGGTAG
2
2
1
NC

986
1879
TCATAAATGAGTATCTGGTA
2
2
2
NC

987
1890
AATTGGCAAAATCATAAATG
1
2
NC
NC

988
1893
CAAAATTGGCAAAATCATAA
1
3
NC
NC

989
1905
ACCTGAGAACACCAAAATTG
2
NC
NC
NC

990
1906
AACCTGAGAACACCAAAATT
2
NC
NC
NC

991
1907
TAACCTGAGAACACCAAAAT
2
NC
NC
NC

992
1908
ATAACCTGAGAACACCAAAA
2
NC
NC
NC

993
1909
GATAACCTGAGAACACCAAA
1
NC
NC
NC

994
1910
CGATAACCTGAGAACACCAA
2
NC
NC
NC

995
1911
CCGATAACCTGAGAACACCA
2
NC
NC
NC

996
1912
TCCGATAACCTGAGAACACC
2
NC
NC
NC

997
1913
CTCCGATAACCTGAGAACAC
2
NC
NC
NC

998
1914
GCTCCGATAACCTGAGAACA
3
NC
NC
NC

999
1915
GGCTCCGATAACCTGAGAAC
3
NC
NC
NC

1000
1916
TGGCTCCGATAACCTGAGAA
3
NC
NC
NC

1001
1917
CTGGCTCCGATAACCTGAGA
2
NC
NC
NC

1002
1919
TGCTGGCTCCGATAACCTGA
3
NC
NC
NC

1003
1934
AAGGTCAAAGAGCGGTGCTG
2
NC
NC
NC

1004
1938
TGGCAAGGTCAAAGAGCGGT
2
NC
NC
NC

1005
1939
ATGGCAAGGTCAAAGAGCGG
2
NC
NC
NC

1006
1940
CATGGCAAGGTCAAAGAGCG
2
NC
NC
NC

1007
1941
GCATGGCAAGGTCAAAGAGC
3
NC
NC
NC

1008
1942
AGCATGGCAAGGTCAAAGAG
2
NC
NC
NC

1009
1943
AAGCATGGCAAGGTCAAAGA
2
NC
NC
NC

1010
1944
CAAGCATGGCAAGGTCAAAG
2
NC
NC
NC

1011
1945
GCAAGCATGGCAAGGTCAAA
2
NC
NC
NC

1012
1955
ACTATCTAAGGCAAGCATGG
2
2
NC
NC

1013
1956
GACTATCTAAGGCAAGCATG
2
2
NC
NC

1014
1957
GGACTATCTAAGGCAAGCAT
3
NC
NC
NC

1015
1958
TGGACTATCTAAGGCAAGCA
2
NC
NC
NC

1016
1959
CTGGACTATCTAAGGCAAGC
2
2
NC
NC

1017
1960
TCTGGACTATCTAAGGCAAG
3
NC
NC
NC

1018
1961
CTCTGGACTATCTAAGGCAA
2
1
NC
NC

1019
1962
TCTCTGGACTATCTAAGGCA
2
2
NC
NC

1020
1963
CTCTCTGGACTATCTAAGGC
2
2
NC
NC

1021
1964
ACTCTCTGGACTATCTAAGG
2
2
NC
NC

1022
1965
CACTCTCTGGACTATCTAAG
2
2
NC
NC

1023
1979
TTCCTCTGTCCAGCCACTCT
2
1
NC
NC

1024
1981
TCTTCCTCTGTCCAGCCACT
2
2
NC
NC

1025
1982
ATCTTCCTCTGTCCAGCCAC
2
2
NC
NC

1026
1983
CATCTTCCTCTGTCCAGCCA
2
1
NC
NC

1027
1985
ACCATCTTCCTCTGTCCAGC
2
2
NC
NC

1028
1986
GACCATCTTCCTCTGTCCAG
2
2
NC
NC

1029
1989
TGGGACCATCTTCCTCTGTC
2
2
NC
NC

1030
1990
TTGGGACCATCTTCCTCTGT
2
2
NC
NC

1031
1991
TTTGGGACCATCTTCCTCTG
2
2
NC
NC

1032
1992
CTTTGGGACCATCTTCCTCT
2
1
NC
NC

1033
1993
TCTTTGGGACCATCTTCCTC
1
NC
NC
NC

1034
1994
TTCTTTGGGACCATCTTCCT
2
2
NC
NC

1035
1995
CTTCTTTGGGACCATCTTCC
2
NC
NC
NC

1036
1996
CCTTCTTTGGGACCATCTTC
2
2
NC
NC

1037
1997
TCCTTCTTTGGGACCATCTT
2
1
NC
NC

1038
2004
CAGCAAGTCCTTCTTTGGGA
2
2
NC
NC

1039
2005
TCAGCAAGTCCTTCTTTGGG
2
2
NC
NC

1040
2006
TTCAGCAAGTCCTTCTTTGG
2
1
NC
NC

1041
2007
ATTCAGCAAGTCCTTCTTTG
2
2
NC
NC

1042
2008
TATTCAGCAAGTCCTTCTTT
1
NC
NC
NC

1043
2009
GTATTCAGCAAGTCCTTCTT
2
2
NC
NC

1044
2010
TGTATTCAGCAAGTCCTTCT
2
2
NC
NC

1045
2011
ATGTATTCAGCAAGTCCTTC
2
2
NC
NC

1046
2012
AATGTATTCAGCAAGTCCTT
1
2
NC
NC

1047
2013
CAATGTATTCAGCAAGTCCT
2
2
NC
NC

1048
2014
ACAATGTATTCAGCAAGTCC
2
1
NC
NC

1049
2015
AACAATGTATTCAGCAAGTC
2
2
NC
NC

1050
2016
CAACAATGTATTCAGCAAGT
2
2
NC
NC

1051
2017
TCAACAATGTATTCAGCAAG
2
2
NC
NC

1052
2018
CTCAACAATGTATTCAGCAA
1
2
NC
NC

1053
2019
ACTCAACAATGTATTCAGCA
1
1
NC
NC

1054
2020
AACTCAACAATGTATTCAGC
2
3
NC
NC

1055
2021
AAACTCAACAATGTATTCAG
2
2
NC
NC

1056
2022
GAAACTCAACAATGTATTCA
2
2
NC
NC

1057
2023
AGAAACTCAACAATGTATTC
2
1
NC
NC

1058
2024
CAGAAACTCAACAATGTATT
2
2
NC
NC

1059
2025
TCAGAAACTCAACAATGTAT
1
2
NC
NC

1060
2026
TTCAGAAACTCAACAATGTA
1
2
2
NC

1061
2027
CTTCAGAAACTCAACAATGT
1
2
2
NC

1062
2028
TCTTCAGAAACTCAACAATG
1
2
1
NC

1063
2029
TTCTTCAGAAACTCAACAAT
2
2
2
NC

1064
2030
CTTCTTCAGAAACTCAACAA
2
2
2
NC

1065
2031
TCTTCTTCAGAAACTCAACA
2
2
1
NC

1066
2032
TTCTTCTTCAGAAACTCAAC
1
2
2
NC

1067
2040
TCTCAGCCTTCTTCTTCAGA
2
NC
NC
NC

1068
2041
ATCTCAGCCTTCTTCTTCAG
2
NC
NC
NC

1069
2042
CATCTCAGCCTTCTTCTTCA
1
NC
NC
NC

1070
2043
GCATCTCAGCCTTCTTCTTC
2
NC
NC
NC

1071
2044
AGCATCTCAGCCTTCTTCTT
1
NC
NC
NC

1072
2045
AAGCATCTCAGCCTTCTTCT
1
NC
NC
NC

1073
2046
CAAGCATCTCAGCCTTCTTC
1
NC
NC
NC

1074
2047
GCAAGCATCTCAGCCTTCTT
2
NC
NC
NC

1075
2048
TGCAAGCATCTCAGCCTTCT
2
NC
NC
NC

1076
2049
CTGCAAGCATCTCAGCCTTC
2
NC
NC
NC

1077
2050
TCTGCAAGCATCTCAGCCTT
2
NC
NC
NC

1078
2051
GTCTGCAAGCATCTCAGCCT
2
NC
NC
NC

1079
2052
AGTCTGCAAGCATCTCAGCC
2
NC
NC
NC

1080
2053
TAGTCTGCAAGCATCTCAGC
2
NC
NC
NC

1081
2054
ATAGTCTGCAAGCATCTCAG
2
NC
NC
NC

1082
2055
AATAGTCTGCAAGCATCTCA
2
NC
NC
NC

1083
2056
AAATAGTCTGCAAGCATCTC
1
2
2
NC

1084
2057
GAAATAGTCTGCAAGCATCT
2
2
2
2

1085
2058
AGAAATAGTCTGCAAGCATC
2
2
2
2

1086
2059
GAGAAATAGTCTGCAAGCAT
2
2
2
3

1087
2060
AGAGAAATAGTCTGCAAGCA
1
NC
2
2

1088
2061
AAGAGAAATAGTCTGCAAGC
2
NC
NC
NC

1089
2062
AAAGAGAAATAGTCTGCAAG
2
2
NC
NC

1090
2063
CAAAGAGAAATAGTCTGCAA
1
1
NC
NC

1091
2064
CCAAAGAGAAATAGTCTGCA
1
2
NC
NC

1092
2065
TCCAAAGAGAAATAGTCTGC
1
1
NC
NC

1093
2066
TTCCAAAGAGAAATAGTCTG
2
2
NC
NC

1094
2074
TCATCAATTTCCAAAGAGAA
2
2
NC
NC

1095
2075
CTCATCAATTTCCAAAGAGA
2
2
NC
NC

1096
2076
CCTCATCAATTTCCAAAGAG
2
2
NC
NC

1097
2077
TCCTCATCAATTTCCAAAGA
2
2
NC
NC

1098
2078
TTCCTCATCAATTTCCAAAG
2
2
NC
NC

1099
2079
CTTCCTCATCAATTTCCAAA
2
2
NC
NC

1100
2080
CCTTCCTCATCAATTTCCAA
2
2
NC
NC

1101
2081
CCCTTCCTCATCAATTTCCA
2
1
NC
NC

1102
2082
TCCCTTCCTCATCAATTTCC
1
NC
NC
NC

1103
2083
TTCCCTTCCTCATCAATTTC
1
2
NC
NC

1104
2084
GTTCCCTTCCTCATCAATTT
2
2
NC
NC

1105
2085
GGTTCCCTTCCTCATCAATT
2
2
NC
NC

1106
2086
AGGTTCCCTTCCTCATCAAT
2
NC
NC
NC

1107
2087
CAGGTTCCCTTCCTCATCAA
2
2
NC
NC

1108
2088
TCAGGTTCCCTTCCTCATCA
1
2
NC
NC

1109
2089
ATCAGGTTCCCTTCCTCATC
2
1
2
2

1110
2090
AATCAGGTTCCCTTCCTCAT
2
2
2
1

1111
2091
CAATCAGGTTCCCTTCCTCA
1
2
1
1

1112
2092
CCAATCAGGTTCCCTTCCTC
2
2
1
1

1113
2117
ATAGTTGTCAATCAGAAGGG
2
2
NC
NC

1114
2118
CATAGTTGTCAATCAGAAGG
2
2
NC
NC

1115
2119
ACATAGTTGTCAATCAGAAG
2
2
NC
NC

1116
2120
CACATAGTTGTCAATCAGAA
2
2
NC
NC

1117
2121
GCACATAGTTGTCAATCAGA
2
1
NC
NC

1118
2122
GGCACATAGTTGTCAATCAG
2
1
NC
NC

1119
2123
GGGCACATAGTTGTCAATCA
2
3
NC
NC

1120
2151
GAATGAAGATAGGCAGTCCC
2
NC
2
2

1121
2152
AGAATGAAGATAGGCAGTCC
1
2
2
2

1122
2153
AAGAATGAAGATAGGCAGTC
1
2
2
2

1123
2154
GAAGAATGAAGATAGGCAGT
2
2
2
1

1124
2155
CGAAGAATGAAGATAGGCAG
1
2
2
1

1125
2156
TCGAAGAATGAAGATAGGCA
2
2
2
2

1126
2169
CCTCAGTGGCTAGTCGAAGA
2
2
NC
NC

1127
2182
TCGTCCCAATTCACCTCAGT
2
NC
NC
NC

1128
2183
TTCGTCCCAATTCACCTCAG
3
NC
NC
NC

1129
2184
CTTCGTCCCAATTCACCTCA
3
NC
NC
NC

1130
2185
TCTTCGTCCCAATTCACCTC
2
NC
NC
NC

1131
2186
TTCTTCGTCCCAATTCACCT
2
2
NC
NC

1132
2187
TTTCTTCGTCCCAATTCACC
2
2
NC
NC

1133
2188
TTTTCTTCGTCCCAATTCAC
2
2
NC
NC

1134
2189
CTTTTCTTCGTCCCAATTCA
2
1
NC
NC

1135
2190
CCTTTTCTTCGTCCCAATTC
1
2
NC
NC

1136
2191
TCCTTTTCTTCGTCCCAATT
2
2
NC
NC

1137
2192
TTCCTTTTCTTCGTCCCAAT
1
1
NC
NC

1138
2193
ATTCCTTTTCTTCGTCCCAA
2
2
NC
NC

1139
2194
CATTCCTTTTCTTCGTCCCA
2
2
NC
NC

1140
2195
ACATTCCTTTTCTTCGTCCC
2
2
NC
NC

1141
2196
AACATTCCTTTTCTTCGTCC
2
2
NC
NC

1142
2197
AAACATTCCTTTTCTTCGTC
2
2
NC
NC

1143
2198
AAAACATTCCTTTTCTTCGT
2
2
NC
NC

1144
2199
CAAAACATTCCTTTTCTTCG
2
1
NC
NC

1145
2200
TCAAAACATTCCTTTTCTTC
1
2
NC
NC

1146
2201
TTCAAAACATTCCTTTTCTT
1
2
NC
NC

1147
2202
TTTCAAAACATTCCTTTTCT
1
2
NC
NC

1148
2203
CTTTCAAAACATTCCTTTTC
1
3
NC
NC

1149
2204
GCTTTCAAAACATTCCTTTT
1
2
NC
NC

1150
2205
GGCTTTCAAAACATTCCTTT
1
2
NC
NC

1151
2206
AGGCTTTCAAAACATTCCTT
2
2
NC
NC

1152
2207
GAGGCTTTCAAAACATTCCT
2
2
NC
NC

1153
2208
TGAGGCTTTCAAAACATTCC
1
2
NC
NC

1154
2209
CTGAGGCTTTCAAAACATTC
1
1
NC
NC

1155
2210
ACTGAGGCTTTCAAAACATT
1
2
NC
NC

1156
2211
TACTGAGGCTTTCAAAACAT
2
2
NC
NC

1157
2212
TTACTGAGGCTTTCAAAACA
2
NC
NC
NC

1158
2213
TTTACTGAGGCTTTCAAAAC
2
2
NC
NC

1159
2214
CTTTACTGAGGCTTTCAAAA
2
2
NC
NC

1160
2215
TCTTTACTGAGGCTTTCAAA
2
NC
NC
NC

1161
2216
TTCTTTACTGAGGCTTTCAA
2
2
NC
NC

1162
2217
ATTCTTTACTGAGGCTTTCA
2
3
NC
NC

1163
2218
CATTCTTTACTGAGGCTTTC
2
2
NC
NC

1164
2219
GCATTCTTTACTGAGGCTTT
2
NC
NC
NC

1165
2220
CGCATTCTTTACTGAGGCTT
2
NC
NC
NC

1166
2221
GCGCATTCTTTACTGAGGCT
2
NC
NC
NC

1167
2222
AGCGCATTCTTTACTGAGGC
2
NC
NC
NC

1168
2223
TAGCGCATTCTTTACTGAGG
3
NC
NC
NC

1169
2224
ATAGCGCATTCTTTACTGAG
2
NC
NC
NC

1170
2225
CATAGCGCATTCTTTACTGA
2
NC
NC
NC

1171
2226
ACATAGCGCATTCTTTACTG
2
NC
NC
NC

1172
2227
AACATAGCGCATTCTTTACT
2
NC
NC
NC

1173
2228
GAACATAGCGCATTCTTTAC
3
NC
NC
NC

1174
2229
AGAACATAGCGCATTCTTTA
2
NC
NC
NC

1175
2230
TAGAACATAGCGCATTCTTT
3
NC
NC
NC

1176
2231
ATAGAACATAGCGCATTCTT
2
NC
NC
NC

1177
2232
AATAGAACATAGCGCATTCT
2
NC
NC
NC

1178
2233
GAATAGAACATAGCGCATTC
2
NC
NC
NC

1179
2234
GGAATAGAACATAGCGCATT
3
NC
NC
NC

1180
2235
TGGAATAGAACATAGCGCAT
2
NC
NC
NC

1181
2236
ATGGAATAGAACATAGCGCA
2
NC
NC
NC

1182
2237
GATGGAATAGAACATAGCGC
2
NC
NC
NC

1183
2238
GGATGGAATAGAACATAGCG
3
NC
NC
NC

1184
2239
CGGATGGAATAGAACATAGC
2
NC
NC
NC

1185
2240
CCGGATGGAATAGAACATAG
3
NC
NC
NC

1186
2252
TATGTACTGCTTCCGGATGG
2
2
NC
NC

1187
2253
ATATGTACTGCTTCCGGATG
3
2
NC
NC

1188
2254
GATATGTACTGCTTCCGGAT
3
2
NC
NC

1189
2255
AGATATGTACTGCTTCCGGA
3
2
NC
NC

1190
2256
CAGATATGTACTGCTTCCGG
3
3
NC
NC

1191
2257
TCAGATATGTACTGCTTCCG
2
1
NC
NC

1192
2258
CTCAGATATGTACTGCTTCC
2
2
NC
NC

1193
2259
CCTCAGATATGTACTGCTTC
2
2
NC
NC

1194
2260
TCCTCAGATATGTACTGCTT
2
2
NC
NC

1195
2261
CTCCTCAGATATGTACTGCT
2
2
NC
NC

1196
2262
ACTCCTCAGATATGTACTGC
2
NC
NC
NC

1197
2264
CGACTCCTCAGATATGTACT
2
1
NC
NC

1198
2265
TCGACTCCTCAGATATGTAC
2
NC
NC
NC

1199
2266
GTCGACTCCTCAGATATGTA
3
2
NC
NC

1200
2267
GGTCGACTCCTCAGATATGT
3
2
NC
NC

1201
2268
GGGTCGACTCCTCAGATATG
3
2
NC
NC

1202
2269
AGGGTCGACTCCTCAGATAT
2
NC
NC
NC

1203
2290
ACTTCACTCTGCTGGCCTGA
2
1
NC
NC

1204
2302
ATGGAGCCAGGCACTTCACT
2
1
NC
NC

1205
2303
AATGGAGCCAGGCACTTCAC
2
2
NC
NC

1206
2304
GAATGGAGCCAGGCACTTCA
2
2
NC
NC

1207
2306
TGGAATGGAGCCAGGCACTT
2
3
NC
NC

1208
2308
TTTGGAATGGAGCCAGGCAC
2
2
NC
NC

1209
2309
GTTTGGAATGGAGCCAGGCA
2
2
NC
NC

1210
2310
AGTTTGGAATGGAGCCAGGC
1
2
NC
NC

1211
2311
GAGTTTGGAATGGAGCCAGG
1
2
NC
NC

1212
2312
GGAGTTTGGAATGGAGCCAG
1
2
NC
NC

1213
2313
AGGAGTTTGGAATGGAGCCA
1
NC
NC
NC

1214
2314
CAGGAGTTTGGAATGGAGCC
2
3
NC
NC

1215
2324
AGTCCACTTCCAGGAGTTTG
2
2
NC
NC

1216
2325
CAGTCCACTTCCAGGAGTTT
2
2
NC
NC

1217
2326
ACAGTCCACTTCCAGGAGTT
1
2
NC
NC

1218
2329
TCCACAGTCCACTTCCAGGA
2
2
NC
NC

1219
2330
TTCCACAGTCCACTTCCAGG
1
NC
NC
NC

1220
2331
GTTCCACAGTCCACTTCCAG
2
3
NC
NC

1221
2332
TGTTCCACAGTCCACTTCCA
2
2
NC
NC

1222
2333
GTGTTCCACAGTCCACTTCC
2
2
NC
NC

1223
2334
TGTGTTCCACAGTCCACTTC
2
1
NC
NC

1224
2344
TTATAGACAATGTGTTCCAC
2
NC
NC
NC

1225
2345
TTTATAGACAATGTGTTCCA
1
NC
NC
NC

1226
2346
CTTTATAGACAATGTGTTCC
2
NC
NC
NC

1227
2347
GCTTTATAGACAATGTGTTC
1
NC
NC
NC

1228
2348
GGCTTTATAGACAATGTGTT
1
NC
NC
NC

1229
2349
AGGCTTTATAGACAATGTGT
1
NC
NC
NC

1230
2350
AAGGCTTTATAGACAATGTG
1
NC
NC
NC

1231
2351
CAAGGCTTTATAGACAATGT
1
NC
NC
NC

1232
2352
GCAAGGCTTTATAGACAATG
1
NC
NC
NC

1233
2353
CGCAAGGCTTTATAGACAAT
2
NC
NC
NC

1234
2380
AAATGTTTAGGAGGCAGAAT
2
1
NC
NC

1235
2381
GAAATGTTTAGGAGGCAGAA
2
2
NC
NC

1236
2382
TGAAATGTTTAGGAGGCAGA
2
2
NC
NC

1237
2383
GTGAAATGTTTAGGAGGCAG
3
2
NC
NC

1238
2384
TGTGAAATGTTTAGGAGGCA
2
2
NC
NC

1239
2385
CTGTGAAATGTTTAGGAGGC
2
2
NC
NC

1240
2386
TCTGTGAAATGTTTAGGAGG
2
2
NC
NC

1241
2387
TTCTGTGAAATGTTTAGGAG
2
2
NC
NC

1242
2388
CTTCTGTGAAATGTTTAGGA
1
1
NC
NC

1243
2389
TCTTCTGTGAAATGTTTAGG
1
2
NC
NC

1244
2390
ATCTTCTGTGAAATGTTTAG
2
2
NC
NC

1245
2391
CATCTTCTGTGAAATGTTTA
2
2
NC
NC

1246
2392
CCATCTTCTGTGAAATGTTT
2
2
NC
NC

1247
2393
TCCATCTTCTGTGAAATGTT
2
2
NC
NC

1248
2394
TTCCATCTTCTGTGAAATGT
1
NC
NC
NC

1249
2395
TTTCCATCTTCTGTGAAATG
1
2
NC
NC

1250
2396
ATTTCCATCTTCTGTGAAAT
1
2
NC
NC

1251
2397
TATTTCCATCTTCTGTGAAA
1
2
NC
NC

1252
2398
ATATTTCCATCTTCTGTGAA
2
2
NC
NC

1253
2399
GATATTTCCATCTTCTGTGA
2
1
NC
NC

1254
2424
GATCAGGCAGGTTAGCAAGC
2
NC
NC
NC

1255
2425
AGATCAGGCAGGTTAGCAAG
1
1
NC
NC

1256
2426
TAGATCAGGCAGGTTAGCAA
2
NC
NC
NC

1257
2427
ATAGATCAGGCAGGTTAGCA
2
1
NC
NC

1258
2428
TATAGATCAGGCAGGTTAGC
2
3
NC
NC

1259
2429
GTATAGATCAGGCAGGTTAG
2
2
NC
NC

1260
2430
TGTATAGATCAGGCAGGTTA
2
2
NC
NC

1261
2431
TTGTATAGATCAGGCAGGTT
2
NC
NC
NC

1262
2432
TTTGTATAGATCAGGCAGGT
2
2
NC
NC

1263
2433
CTTTGTATAGATCAGGCAGG
2
2
NC
NC

1264
2434
ACTTTGTATAGATCAGGCAG
2
2
NC
NC

1265
2435
GACTTTGTATAGATCAGGCA
2
2
NC
NC

1266
2436
AGACTTTGTATAGATCAGGC
3
2
NC
NC

1267
2437
AAGACTTTGTATAGATCAGG
2
2
NC
NC

1268
2438
AAAGACTTTGTATAGATCAG
2
1
NC
NC

1269
2439
CAAAGACTTTGTATAGATCA
2
2
NC
NC

1270
2449
TAACACCTCTCAAAGACTTT
2
1
NC
NC

1271
2450
TTAACACCTCTCAAAGACTT
1
2
NC
NC

1272
2451
TTTAACACCTCTCAAAGACT
1
2
NC
NC

1273
2452
ATTTAACACCTCTCAAAGAC
2
3
NC
NC

1274
2453
TATTTAACACCTCTCAAAGA
2
2
NC
NC

1275
2454
ATATTTAACACCTCTCAAAG
2
3
NC
NC

1276
2455
CATATTTAACACCTCTCAAA
2
NC
NC
NC

1277
2456
CCATATTTAACACCTCTCAA
2
2
NC
NC

1278
2457
ACCATATTTAACACCTCTCA
2
1
NC
NC

1279
2458
AACCATATTTAACACCTCTC
2
NC
NC
NC

1280
2459
TAACCATATTTAACACCTCT
2
NC
NC
NC

1281
2460
ATAACCATATTTAACACCTC
2
NC
NC
NC

1282
2461
AATAACCATATTTAACACCT
2
NC
NC
NC

1283
2462
AAATAACCATATTTAACACC
2
NC
NC
NC

1284
2469
AGTGCATAAATAACCATATT
2
NC
NC
NC

1285
2470
CAGTGCATAAATAACCATAT
2
NC
NC
NC

1286
2471
ACAGTGCATAAATAACCATA
2
NC
NC
NC

1287
2472
CACAGTGCATAAATAACCAT
2
NC
NC
NC

1288
2473
CCACAGTGCATAAATAACCA
2
NC
NC
NC

1289
2474
CCCACAGTGCATAAATAACC
2
NC
NC
NC

1290
2475
TCCCACAGTGCATAAATAAC
2
NC
NC
NC

1291
2476
ATCCCACAGTGCATAAATAA
2
NC
NC
NC

1292
2477
CATCCCACAGTGCATAAATA
2
NC
NC
NC

1293
2478
ACATCCCACAGTGCATAAAT
2
2
NC
NC

1294
2479
CACATCCCACAGTGCATAAA
2
2
NC
NC

1295
2486
AGAAGAACACATCCCACAGT
1
NC
NC
NC

1296
2487
AAGAAGAACACATCCCACAG
1
NC
NC
NC

1297
2488
AAAGAAGAACACATCCCACA
2
NC
NC
NC

1298
2489
GAAAGAAGAACACATCCCAC
2
NC
NC
NC

1299
2490
AGAAAGAAGAACACATCCCA
2
NC
NC
NC

1300
2491
GAGAAAGAAGAACACATCCC
2
NC
NC
NC

1301
2492
AGAGAAAGAAGAACACATCC
1
NC
NC
NC

1302
2493
CAGAGAAAGAAGAACACATC
2
NC
NC
NC

1303
2494
ACAGAGAAAGAAGAACACAT
1
NC
NC
NC

1304
2495
TACAGAGAAAGAAGAACACA
2
NC
NC
NC

1305
2496
ATACAGAGAAAGAAGAACAC
2
NC
NC
NC

1306
2497
AATACAGAGAAAGAAGAACA
1
NC
NC
NC

1307
2498
GAATACAGAGAAAGAAGAAC
1
NC
NC
NC

1308
2499
GGAATACAGAGAAAGAAGAA
1
NC
NC
NC

1309
2500
CGGAATACAGAGAAAGAAGA
2
NC
NC
NC

1310
2501
TCGGAATACAGAGAAAGAAG
2
NC
NC
NC

1311
2502
ATCGGAATACAGAGAAAGAA
2
2
NC
NC

1312
2503
TATCGGAATACAGAGAAAGA
2
2
NC
NC

1313
2504
GTATCGGAATACAGAGAAAG
2
2
NC
NC

1314
2513
CAACACTTTGTATCGGAATA
3
1
NC
NC

1315
2524
ACACTTTGATACAACACTTT
2
2
NC
NC

1316
2525
CACACTTTGATACAACACTT
2
2
NC
NC

1317
2526
TCACACTTTGATACAACACT
2
NC
NC
NC

1318
2535
CTTTGTATATCACACTTTGA
2
NC
NC
NC

1319
2536
ACTTTGTATATCACACTTTG
1
NC
NC
NC

1320
2537
CACTTTGTATATCACACTTT
2
NC
NC
NC

1321
2538
ACACTTTGTATATCACACTT
2
NC
NC
NC

1322
2539
TACACTTTGTATATCACACT
2
NC
NC
NC

1323
2540
GTACACTTTGTATATCACAC
2
NC
NC
NC

1324
2541
GGTACACTTTGTATATCACA
3
NC
NC
NC

1325
2542
TGGTACACTTTGTATATCAC
2
NC
NC
NC

1326
2543
TTGGTACACTTTGTATATCA
2
NC
NC
NC

1327
2544
GTTGGTACACTTTGTATATC
2
NC
NC
NC

1328
2545
TGTTGGTACACTTTGTATAT
2
NC
NC
NC

1329
2546
ATGTTGGTACACTTTGTATA
2
NC
NC
NC

1330
2547
TATGTTGGTACACTTTGTAT
2
NC
NC
NC

1331
2548
TTATGTTGGTACACTTTGTA
2
NC
NC
NC

1332
2549
CTTATGTTGGTACACTTTGT
2
NC
NC
NC

1333
2550
ACTTATGTTGGTACACTTTG
2
NC
NC
NC

1334
2551
CACTTATGTTGGTACACTTT
3
NC
NC
NC

1335
2552
ACACTTATGTTGGTACACTT
2
NC
NC
NC

1336
2569
AGTCTTAAGTGCTACCAACA
1
1
NC
NC

1337
2570
AAGTCTTAAGTGCTACCAAC
2
2
NC
NC

1338
2571
TAAGTCTTAAGTGCTACCAA
2
2
NC
NC

1339
2572
ATAAGTCTTAAGTGCTACCA
2
2
NC
NC

1340
2573
TATAAGTCTTAAGTGCTACC
2
1
NC
NC

1341
2574
GTATAAGTCTTAAGTGCTAC
2
NC
NC
NC

1342
2575
AGTATAAGTCTTAAGTGCTA
2
2
NC
NC

1343
2576
AAGTATAAGTCTTAAGTGCT
2
2
NC
NC

1344
2577
CAAGTATAAGTCTTAAGTGC
2
2
NC
NC

1345
2578
GCAAGTATAAGTCTTAAGTG
2
2
NC
NC

1346
2579
GGCAAGTATAAGTCTTAAGT
2
2
NC
NC

1347
2580
AGGCAAGTATAAGTCTTAAG
2
2
NC
NC

1348
2581
AAGGCAAGTATAAGTCTTAA
2
2
NC
NC

1349
2582
GAAGGCAAGTATAAGTCTTA
2
2
NC
NC

1350
2583
AGAAGGCAAGTATAAGTCTT
2
2
NC
NC

1351
2584
CAGAAGGCAAGTATAAGTCT
2
NC
NC
NC

1352
2585
TCAGAAGGCAAGTATAAGTC
2
NC
NC
NC

1353
2586
ATCAGAAGGCAAGTATAAGT
2
NC
NC
NC

1354
2587
TATCAGAAGGCAAGTATAAG
2
NC
NC
NC

1355
2588
CTATCAGAAGGCAAGTATAA
1
NC
NC
NC

1356
2589
ACTATCAGAAGGCAAGTATA
2
NC
NC
NC

1357
2590
TACTATCAGAAGGCAAGTAT
2
NC
NC
NC

1358
2591
ATACTATCAGAAGGCAAGTA
2
NC
NC
NC

1359
2592
AATACTATCAGAAGGCAAGT
2
NC
NC
NC

1360
2593
GAATACTATCAGAAGGCAAG
1
NC
NC
NC

1361
2594
GGAATACTATCAGAAGGCAA
1
NC
NC
NC

1362
2595
AGGAATACTATCAGAAGGCA
2
NC
NC
NC

1363
2596
AAGGAATACTATCAGAAGGC
2
NC
NC
NC

1364
2597
AAAGGAATACTATCAGAAGG
2
NC
NC
NC

1365
2598
TAAAGGAATACTATCAGAAG
2
NC
NC
NC

1366
2599
ATAAAGGAATACTATCAGAA
1
NC
NC
NC

1367
2600
TATAAAGGAATACTATCAGA
1
NC
NC
NC

1368
2601
GTATAAAGGAATACTATCAG
1
NC
NC
NC

1369
2602
TGTATAAAGGAATACTATCA
2
NC
NC
NC

1370
2603
GTGTATAAAGGAATACTATC
2
NC
NC
NC

1371
2604
TGTGTATAAAGGAATACTAT
1
NC
NC
NC

1372
2605
CTGTGTATAAAGGAATACTA
2
NC
NC
NC

1373
2606
ACTGTGTATAAAGGAATACT
2
NC
NC
NC

1374
2607
CACTGTGTATAAAGGAATAC
2
NC
NC
NC

1375
2608
CCACTGTGTATAAAGGAATA
2
NC
NC
NC

1376
2609
TCCACTGTGTATAAAGGAAT
2
NC
NC
NC

1377
2610
ATCCACTGTGTATAAAGGAA
2
NC
NC
NC

1378
2611
AATCCACTGTGTATAAAGGA
2
NC
NC
NC

1379
2612
CAATCCACTGTGTATAAAGG
2
NC
NC
NC

1380
2613
TCAATCCACTGTGTATAAAG
2
NC
NC
NC

1381
2614
ATCAATCCACTGTGTATAAA
2
NC
NC
NC

1382
2615
AATCAATCCACTGTGTATAA
2
NC
NC
NC

1383
2616
TAATCAATCCACTGTGTATA
2
NC
NC
NC

1384
2617
ATAATCAATCCACTGTGTAT
2
NC
NC
NC

1385
2618
TATAATCAATCCACTGTGTA
2
NC
NC
NC

1386
2619
TTATAATCAATCCACTGTGT
2
NC
NC
NC

1387
2620
TTTATAATCAATCCACTGTG
2
NC
NC
NC

1388
2621
ATTTATAATCAATCCACTGT
2
NC
NC
NC

1389
2622
TATTTATAATCAATCCACTG
1
NC
NC
NC

1390
2639
GTTAAGACACATCTATTTAT
1
NC
NC
NC

1391
2640
TGTTAAGACACATCTATTTA
1
NC
NC
NC

1392
2641
ATGTTAAGACACATCTATTT
1
NC
NC
NC

1393
2642
TATGTTAAGACACATCTATT
2
NC
NC
NC

dsRNAs

Selection of 19mer Oligonucleotide Sequences

All sense 18mer sub-sequences and complementary antisense sequences per transcript were generated. An A nucleotide was added to the 3′ end of the sense strand, with a complementary U at the 5′ end of the antisense strand, to yield a 19mer duplex. This nucleotide pair was chosen because the antisense (“guide”) strand's first (5′) nucleotide is not exposed and does not bind to target mRNAs when loaded in the RISC complex, and the core AGO protein subunit shows preference for 5′ U nucleotides (Noland and Doudna (2013), RNA, 19: 639-648, Nakanishi (2016), WIREs RNA, 7: 637-660). Candidate 19mer duplexes were selected that met the following thermodynamic and physical characteristics: predicted melting temperature of <60° C., no homopolymers of 5 or longer, and at least 4 U or A nucleotides in the seed region (antisense strand positions 2-9). These selected duplexes were further evaluated for specificity (off-target scoring, below).

Off-Target Scoring

The specificity of the selected duplexes was evaluated via alignment of both strands to all unspliced RefSeq transcripts (“NM” models for human, mouse, and rat; “NM” and “XM” models for cynomolgus monkey), using the FASTA algorithm with an E value cutoff of 1000. The number of mismatches between each strand and each transcript (per species) was tallied. Duplexes were selected with at least one 8mer seed (positions 2-9) mismatch on each strand to any transcript other than those encoded by the MLH1 gene, since seed mismatches govern specificity of dsRNA activity (Boudreau et al., (2011), Mol. Therapy 19: 2169-2177).

The sequences, positions in human transcript, conservation in other species and species-specific seed mismatch counts of each duplex are given in Table 4. In some embodiments, the 3′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T). Furthermore, duplexes with sequence conservation in cynologous monkey, mouse, and rat are provided in Tables 5-11.

TABLE 4

Exemplary dsRNAs

SEQ ID

SEQ ID

NO
Sense
Antisense
NO
Pos
Cyno
Mouse
Rat

1394
GAGACCCAGCAACCCACAA
UUGUGGGUUGCUGGGUCUC
1395
4
Yes
No
No

1396
CCCAGCAACCCACAGAGUA
UACUCUGUGGGUUGCUGGG
1397
8
Yes
No
No

1398
CCAGCAACCCACAGAGUUA
UAACUCUGUGGGUUGCUGG
1399
9
Yes
No
No

1400
CAGCAACCCACAGAGUUGA
UCAACUCUGUGGGUUGCUG
1401
10
Yes
No
No

1402
CAACCCACAGAGUUGAGAA
UUCUCAACUCUGUGGGUUG
1403
13
Yes
No
No

1404
AACCCACAGAGUUGAGAAA
UUUCUCAACUCUGUGGGUU
1405
14
Yes
No
No

1406
ACCCACAGAGUUGAGAAAA
UUUUCUCAACUCUGUGGGU
1407
15
Yes
No
No

1408
CCCACAGAGUUGAGAAAUA
UAUUUCUCAACUCUGUGGG
1409
16
Yes
No
No

1410
CACAGAGUUGAGAAAUUUA
UAAAUUUCUCAACUCUGUG
1411
18
Yes
No
No

1412
ACAGAGUUGAGAAAUUUGA
UCAAAUUUCUCAACUCUGU
1413
19
Yes
No
No

1414
GAGUUGAGAAAUUUGACUA
UAGUCAAAUUUCUCAACUC
1415
22
Yes
No
No

1416
AGUUGAGAAAUUUGACUGA
UCAGUCAAAUUUCUCAACU
1417
23
Yes
No
No

1418
GUUGAGAAAUUUGACUGGA
UCCAGUCAAAUUUCUCAAC
1419
24
Yes
No
No

1420
GAGAAAUUUGACUGGCAUA
UAUGCCAGUCAAAUUUCUC
1421
27
Yes
No
No

1422
AAAUUUGACUGGCAUUCAA
UUGAAUGCCAGUCAAAUUU
1423
30
Yes
No
No

1424
UUGACUGGCAUUCAAGCUA
UAGCUUGAAUGCCAGUCAA
1425
34
Yes
No
No

1426
ACUGGCAUUCAAGCUGUCA
UGACAGCUUGAAUGCCAGU
1427
37
No
No
No

1428
GGCAUUCAAGCUGUCCAAA
UUUGGACAGCUUGAAUGCC
1429
40
No
No
No

1430
GCAUUCAAGCUGUCCAAUA
UAUUGGACAGCUUGAAUGC
1431
41
No
No
No

1432
CAUUCAAGCUGUCCAAUCA
UGAUUGGACAGCUUGAAUG
1433
42
No
No
No

1434
UUCAAGCUGUCCAAUCAAA
UUUGAUUGGACAGCUUGAA
1435
44
No
No
No

1436
UCAAGCUGUCCAAUCAAUA
UAUUGAUUGGACAGCUUGA
1437
45
No
No
No

1438
AGCUGUCCAAUCAAUAGCA
UGCUAUUGAUUGGACAGCU
1439
48
No
No
No

1440
GCUGUCCAAUCAAUAGCUA
UAGCUAUUGAUUGGACAGC
1441
49
No
No
No

1442
CUGUCCAAUCAAUAGCUGA
UCAGCUAUUGAUUGGACAG
1443
50
No
No
No

1444
UGUCCAAUCAAUAGCUGCA
UGCAGCUAUUGAUUGGACA
1445
51
No
No
No

1446
UAGCUGCCGCUGAAGGGUA
UACCCUUCAGCGGCAGCUA
1447
62
No
No
No

1448
UGGGGCUGGAUGGCGUAAA
UUUACGCCAUCCAGCCCCA
1449
79
No
No
No

1450
UGGAUGGCGUAAGCUACAA
UUGUAGCUUACGCCAUCCA
1451
85
No
No
No

1452
GGAUGGCGUAAGCUACAGA
UCUGUAGCUUACGCCAUCC
1453
86
No
No
No

1454
AUGGCGUAAGCUACAGCUA
UAGCUGUAGCUUACGCCAU
1455
88
No
No
No

1456
GGCGUAAGCUACAGCUGAA
UUCAGCUGUAGCUUACGCC
1457
90
No
No
No

1458
GCGUAAGCUACAGCUGAAA
UUUCAGCUGUAGCUUACGC
1459
91
No
No
No

1460
CGUAAGCUACAGCUGAAGA
UCUUCAGCUGUAGCUUACG
1461
92
No
No
No

1462
AAGCUACAGCUGAAGGAAA
UUUCCUUCAGCUGUAGCUU
1463
95
No
No
No

1464
CUACAGCUGAAGGAAGAAA
UUUCUUCCUUCAGCUGUAG
1465
98
No
No
No

1466
AGCUGAAGGAAGAACGUGA
UCACGUUCUUCCUUCAGCU
1467
102
No
No
No

1468
GCUGAAGGAAGAACGUGAA
UUCACGUUCUUCCUUCAGC
1469
103
No
No
No

1470
ACGAGGCACUGAGGUGAUA
UAUCACCUCAGUGCCUCGU
1471
123
No
No
No

1472
CGAGGCACUGAGGUGAUUA
UAAUCACCUCAGUGCCUCG
1473
124
No
No
No

1474
AGGCACUGAGGUGAUUGGA
UCCAAUCACCUCAGUGCCU
1475
126
No
No
No

1476
GGCACUGAGGUGAUUGGCA
UGCCAAUCACCUCAGUGCC
1477
127
No
No
No

1478
ACUGAGGUGAUUGGCUGAA
UUCAGCCAAUCACCUCAGU
1479
130
No
No
No

1480
CUGAGGUGAUUGGCUGAAA
UUUCAGCCAAUCACCUCAG
1481
131
No
No
No

1482
UGAAGGCACUUCCGUUGAA
UUCAACGGAAGUGCCUUCA
1483
145
Yes
No
No

1484
AGGCACUUCCGUUGAGCAA
UUGCUCAACGGAAGUGCCU
1485
148
Yes
No
No

1486
GGCACUUCCGUUGAGCAUA
UAUGCUCAACGGAAGUGCC
1487
149
Yes
No
No

1488
GCACUUCCGUUGAGCAUCA
UGAUGCUCAACGGAAGUGC
1489
150
Yes
No
No

1490
CUUCCGUUGAGCAUCUAGA
UCUAGAUGCUCAACGGAAG
1491
153
Yes
No
No

1492
UUCCGUUGAGCAUCUAGAA
UUCUAGAUGCUCAACGGAA
1493
154
Yes
No
No

1494
CCGUUGAGCAUCUAGACGA
UCGUCUAGAUGCUCAACGG
1495
156
Yes
No
No

1496
CGUUGAGCAUCUAGACGUA
UACGUCUAGAUGCUCAACG
1497
157
Yes
No
No

1498
AGCAUCUAGACGUUUCCUA
UAGGAAACGUCUAGAUGCU
1499
162
Yes
No
No

1500
AUCUAGACGUUUCCUUGGA
UCCAAGGAAACGUCUAGAU
1501
165
No
No
No

1502
AGACGUUUCCUUGGCUCUA
UAGAGCCAAGGAAACGUCU
1503
169
No
No
No

1504
GACGUUUCCUUGGCUCUUA
UAAGAGCCAAGGAAACGUC
1505
170
No
No
No

1506
CUUCUGGCGCCAAAAUGUA
UACAUUUUGGCGCCAGAAG
1507
185
No
No
No

1508
UUCUGGCGCCAAAAUGUCA
UGACAUUUUGGCGCCAGAA
1509
186
No
No
No

1510
UCUGGCGCCAAAAUGUCGA
UCGACAUUUUGGCGCCAGA
1511
187
Yes
No
No

1512
UGGCGCCAAAAUGUCGUUA
UAACGACAUUUUGGCGCCA
1513
189
Yes
No
No

1514
GGCGCCAAAAUGUCGUUCA
UGAACGACAUUUUGGCGCC
1515
190
Yes
No
No

1516
CGCCAAAAUGUCGUUCGUA
UACGAACGACAUUUUGGCG
1517
192
Yes
No
No

1518
CGUUCGUGGCAGGGGUUAA
UUAACCCCUGCCACGAACG
1519
203
Yes
No
No

1520
GUUCGUGGCAGGGGUUAUA
UAUAACCCCUGCCACGAAC
1521
204
Yes
No
No

1522
UUCGUGGCAGGGGUUAUUA
UAAUAACCCCUGCCACGAA
1523
205
Yes
No
No

1524
UCGUGGCAGGGGUUAUUCA
UGAAUAACCCCUGCCACGA
1525
206
Yes
No
No

1526
CGUGGCAGGGGUUAUUCGA
UCGAAUAACCCCUGCCACG
1527
207
Yes
No
No

1528
GUGGCAGGGGUUAUUCGGA
UCCGAAUAACCCCUGCCAC
1529
208
Yes
No
No

1530
UGGACGAGACAGUGGUGAA
UUCACCACUGUCUCGUCCA
1531
230
Yes
No
No

1532
GGACGAGACAGUGGUGAAA
UUUCACCACUGUCUCGUCC
1533
231
Yes
No
No

1534
GACGAGACAGUGGUGAACA
UGUUCACCACUGUCUCGUC
1535
232
Yes
No
No

1536
UAUCCAGCGGCCAGCUAAA
UUUAGCUGGCCGCUGGAUA
1537
270
Yes
No
No

1538
AUCCAGCGGCCAGCUAAUA
UAUUAGCUGGCCGCUGGAU
1539
271
Yes
No
No

1540
UCCAGCGGCCAGCUAAUGA
UCAUUAGCUGGCCGCUGGA
1541
272
Yes
No
No

1542
AGCGGCCAGCUAAUGCUAA
UUAGCAUUAGCUGGCCGCU
1543
275
Yes
No
No

1544
GCCAGCUAAUGCUAUCAAA
UUUGAUAGCAUUAGCUGGC
1545
279
Yes
No
No

1546
CCAGCUAAUGCUAUCAAAA
UUUUGAUAGCAUUAGCUGG
1547
280
Yes
No
No

1548
CAGCUAAUGCUAUCAAAGA
UCUUUGAUAGCAUUAGCUG
1549
281
Yes
No
No

1550
AGCUAAUGCUAUCAAAGAA
UUCUUUGAUAGCAUUAGCU
1551
282
Yes
No
No

1552
GCUAAUGCUAUCAAAGAGA
UCUCUUUGAUAGCAUUAGC
1553
283
Yes
No
No

1554
CUAAUGCUAUCAAAGAGAA
UUCUCUUUGAUAGCAUUAG
1555
284
Yes
No
No

1556
UAAUGCUAUCAAAGAGAUA
UAUCUCUUUGAUAGCAUUA
1557
285
Yes
No
No

1558
AUGCUAUCAAAGAGAUGAA
UUCAUCUCUUUGAUAGCAU
1559
287
Yes
Yes
Yes

1560
GCUAUCAAAGAGAUGAUUA
UAAUCAUCUCUUUGAUAGC
1561
289
Yes
No
No

1562
CUAUCAAAGAGAUGAUUGA
UCAAUCAUCUCUUUGAUAG
1563
290
Yes
No
No

1564
GAGAUGAUUGAGAACUGUA
UACAGUUCUCAAUCAUCUC
1565
298
Yes
No
No

1566
AGAUGAUUGAGAACUGUUA
UAACAGUUCUCAAUCAUCU
1567
299
Yes
No
No

1568
GAUGAUUGAGAACUGUUUA
UAAACAGUUCUCAAUCAUC
1569
300
Yes
No
No

1570
AUGAUUGAGAACUGUUUAA
UUAAACAGUUCUCAAUCAU
1571
301
Yes
No
No

1572
AUUGAGAACUGUUUAGAUA
UAUCUAAACAGUUCUCAAU
1573
304
Yes
No
No

1574
AGAACUGUUUAGAUGCAAA
UUUGCAUCUAAACAGUUCU
1575
308
Yes
No
No

1576
GAACUGUUUAGAUGCAAAA
UUUUGCAUCUAAACAGUUC
1577
309
Yes
No
No

1578
ACUGUUUAGAUGCAAAAUA
UAUUUUGCAUCUAAACAGU
1579
311
Yes
Yes
Yes

1580
UGUUUAGAUGCAAAAUCCA
UGGAUUUUGCAUCUAAACA
1581
313
Yes
No
No

1582
GUUUAGAUGCAAAAUCCAA
UUGGAUUUUGCAUCUAAAC
1583
314
Yes
No
No

1584
UUUAGAUGCAAAAUCCACA
UGUGGAUUUUGCAUCUAAA
1585
315
Yes
No
No

1586
UUAGAUGCAAAAUCCACAA
UUGUGGAUUUUGCAUCUAA
1587
316
Yes
No
No

1588
AGAUGCAAAAUCCACAAGA
UCUUGUGGAUUUUGCAUCU
1589
318
Yes
No
No

1590
GCAAAAUCCACAAGUAUUA
UAAUACUUGUGGAUUUUGC
1591
322
Yes
No
No

1592
AAAAUCCACAAGUAUUCAA
UUGAAUACUUGUGGAUUUU
1593
324
Yes
No
No

1594
AAUCCACAAGUAUUCAAGA
UCUUGAAUACUUGUGGAUU
1595
326
Yes
No
No

1596
UCCACAAGUAUUCAAGUGA
UCACUUGAAUACUUGUGGA
1597
328
Yes
No
No

1598
CCACAAGUAUUCAAGUGAA
UUCACUUGAAUACUUGUGG
1599
329
Yes
No
No

1600
CACAAGUAUUCAAGUGAUA
UAUCACUUGAAUACUUGUG
1601
330
Yes
No
No

1602
ACAAGUAUUCAAGUGAUUA
UAAUCACUUGAAUACUUGU
1603
331
Yes
No
No

1604
AAGUAUUCAAGUGAUUGUA
UACAAUCACUUGAAUACUU
1605
333
Yes
No
No

1606
AGUAUUCAAGUGAUUGUUA
UAACAAUCACUUGAAUACU
1607
334
Yes
No
No

1608
GUAUUCAAGUGAUUGUUAA
UUAACAAUCACUUGAAUAC
1609
335
Yes
No
No

1610
UCAAGUGAUUGUUAAAGAA
UUCUUUAACAAUCACUUGA
1611
339
Yes
No
No

1612
CAAGUGAUUGUUAAAGAGA
UCUCUUUAACAAUCACUUG
1613
340
Yes
No
No

1614
AGUGAUUGUUAAAGAGGGA
UCCCUCUUUAACAAUCACU
1615
342
Yes
No
No

1616
GAGGGAGGCCUGAAGUUGA
UCAACUUCAGGCCUCCCUC
1617
355
Yes
No
No

1618
AGGGAGGCCUGAAGUUGAA
UUCAACUUCAGGCCUCCCU
1619
356
Yes
No
No

1620
GGGAGGCCUGAAGUUGAUA
UAUCAACUUCAGGCCUCCC
1621
357
Yes
No
No

1622
GAGGCCUGAAGUUGAUUCA
UGAAUCAACUUCAGGCCUC
1623
359
Yes
No
No

1624
CUGAAGUUGAUUCAGAUCA
UGAUCUGAAUCAACUUCAG
1625
364
Yes
No
No

1626
GAAGUUGAUUCAGAUCCAA
UUGGAUCUGAAUCAACUUC
1627
366
Yes
No
No

1628
AAGUUGAUUCAGAUCCAAA
UUUGGAUCUGAAUCAACUU
1629
367
Yes
No
No

1630
AGUUGAUUCAGAUCCAAGA
UCUUGGAUCUGAAUCAACU
1631
368
Yes
No
No

1632
GUUGAUUCAGAUCCAAGAA
UUCUUGGAUCUGAAUCAAC
1633
369
Yes
No
No

1634
UGAUUCAGAUCCAAGACAA
UUGUCUUGGAUCUGAAUCA
1635
371
Yes
No
No

1636
AUUCAGAUCCAAGACAAUA
UAUUGUCUUGGAUCUGAAU
1637
373
Yes
Yes
Yes

1638
GCACCGGGAUCAGGAAAGA
UCUUUCCUGAUCCCGGUGC
1639
392
No
No
No

1640
CACCGGGAUCAGGAAAGAA
UUCUUUCCUGAUCCCGGUG
1641
393
No
No
No

1642
GAUCAGGAAAGAAGAUCUA
UAGAUCUUCUUUCCUGAUC
1643
399
Yes
No
No

1644
GAAAGAAGAUCUGGAUAUA
UAUAUCCAGAUCUUCUUUC
1645
405
Yes
No
No

1646
AAAGAAGAUCUGGAUAUUA
UAAUAUCCAGAUCUUCUUU
1647
406
Yes
No
No

1648
AAGAAGAUCUGGAUAUUGA
UCAAUAUCCAGAUCUUCUU
1649
407
Yes
No
No

1650
AGAUCUGGAUAUUGUAUGA
UCAUACAAUAUCCAGAUCU
1651
411
Yes
No
No

1652
GAUCUGGAUAUUGUAUGUA
UACAUACAAUAUCCAGAUC
1653
412
Yes
No
No

1654
AUCUGGAUAUUGUAUGUGA
UCACAUACAAUAUCCAGAU
1655
413
Yes
No
No

1656
UCUGGAUAUUGUAUGUGAA
UUCACAUACAAUAUCCAGA
1657
414
Yes
No
No

1658
GGAUAUUGUAUGUGAAAGA
UCUUUCACAUACAAUAUCC
1659
417
Yes
No
No

1660
UAUUGUAUGUGAAAGGUUA
UAACCUUUCACAUACAAUA
1661
420
Yes
No
No

1662
AUUGUAUGUGAAAGGUUCA
UGAACCUUUCACAUACAAU
1663
421
Yes
No
No

1664
UUGUAUGUGAAAGGUUCAA
UUGAACCUUUCACAUACAA
1665
422
Yes
No
No

1666
UGUAUGUGAAAGGUUCACA
UGUGAACCUUUCACAUACA
1667
423
Yes
No
No

1668
GUAUGUGAAAGGUUCACUA
UAGUGAACCUUUCACAUAC
1669
424
Yes
No
No

1670
AUGUGAAAGGUUCACUACA
UGUAGUGAACCUUUCACAU
1671
426
Yes
No
No

1672
UGUGAAAGGUUCACUACUA
UAGUAGUGAACCUUUCACA
1673
427
No
No
No

1674
GUGAAAGGUUCACUACUAA
UUAGUAGUGAACCUUUCAC
1675
428
No
No
No

1676
UGAAAGGUUCACUACUAGA
UCUAGUAGUGAACCUUUCA
1677
429
No
No
No

1678
GAAAGGUUCACUACUAGUA
UACUAGUAGUGAACCUUUC
1679
430
No
No
No

1680
AAGGUUCACUACUAGUAAA
UUUACUAGUAGUGAACCUU
1681
432
No
No
No

1682
AGGUUCACUACUAGUAAAA
UUUUACUAGUAGUGAACCU
1683
433
No
No
No

1684
GUUCACUACUAGUAAACUA
UAGUUUACUAGUAGUGAAC
1685
435
No
No
No

1686
UUCACUACUAGUAAACUGA
UCAGUUUACUAGUAGUGAA
1687
436
No
No
No

1688
UCACUACUAGUAAACUGCA
UGCAGUUUACUAGUAGUGA
1689
437
No
No
No

1690
CUACUAGUAAACUGCAGUA
UACUGCAGUUUACUAGUAG
1691
440
No
No
No

1692
UAGUAAACUGCAGUCCUUA
UAAGGACUGCAGUUUACUA
1693
444
No
No
No

1694
AGUAAACUGCAGUCCUUUA
UAAAGGACUGCAGUUUACU
1695
445
Yes
No
No

1696
GUAAACUGCAGUCCUUUGA
UCAAAGGACUGCAGUUUAC
1697
446
Yes
No
No

1698
UAAACUGCAGUCCUUUGAA
UUCAAAGGACUGCAGUUUA
1699
447
Yes
No
No

1700
AAACUGCAGUCCUUUGAGA
UCUCAAAGGACUGCAGUUU
1701
448
Yes
No
No

1702
UGCAGUCCUUUGAGGAUUA
UAAUCCUCAAAGGACUGCA
1703
452
Yes
No
No

1704
GCAGUCCUUUGAGGAUUUA
UAAAUCCUCAAAGGACUGC
1705
453
Yes
No
No

1706
CAGUCCUUUGAGGAUUUAA
UUAAAUCCUCAAAGGACUG
1707
454
Yes
No
No

1708
AGUCCUUUGAGGAUUUAGA
UCUAAAUCCUCAAAGGACU
1709
455
Yes
No
No

1710
GUCCUUUGAGGAUUUAGCA
UGCUAAAUCCUCAAAGGAC
1711
456
Yes
No
No

1712
UCCUUUGAGGAUUUAGCCA
UGGCUAAAUCCUCAAAGGA
1713
457
Yes
No
No

1714
CUUUGAGGAUUUAGCCAGA
UCUGGCUAAAUCCUCAAAG
1715
459
Yes
No
No

1716
UUUGAGGAUUUAGCCAGUA
UACUGGCUAAAUCCUCAAA
1717
460
Yes
Yes
No

1718
UUGAGGAUUUAGCCAGUAA
UUACUGGCUAAAUCCUCAA
1719
461
Yes
Yes
No

1720
UGAGGAUUUAGCCAGUAUA
UAUACUGGCUAAAUCCUCA
1721
462
Yes
Yes
No

1722
GAGGAUUUAGCCAGUAUUA
UAAUACUGGCUAAAUCCUC
1723
463
Yes
Yes
No

1724
AGGAUUUAGCCAGUAUUUA
UAAAUACUGGCUAAAUCCU
1725
464
Yes
Yes
No

1726
GGAUUUAGCCAGUAUUUCA
UGAAAUACUGGCUAAAUCC
1727
465
Yes
Yes
No

1728
GAUUUAGCCAGUAUUUCUA
UAGAAAUACUGGCUAAAUC
1729
466
Yes
Yes
No

1730
UUAGCCAGUAUUUCUACCA
UGGUAGAAAUACUGGCUAA
1731
469
Yes
Yes
No

1732
UAGCCAGUAUUUCUACCUA
UAGGUAGAAAUACUGGCUA
1733
470
Yes
Yes
No

1734
AGCCAGUAUUUCUACCUAA
UUAGGUAGAAAUACUGGCU
1735
471
Yes
Yes
No

1736
GCCAGUAUUUCUACCUAUA
UAUAGGUAGAAAUACUGGC
1737
472
Yes
Yes
No

1738
CAGUAUUUCUACCUAUGGA
UCCAUAGGUAGAAAUACUG
1739
474
Yes
Yes
No

1740
GUAUUUCUACCUAUGGCUA
UAGCCAUAGGUAGAAAUAC
1741
476
Yes
Yes
No

1742
UAUUUCUACCUAUGGCUUA
UAAGCCAUAGGUAGAAAUA
1743
477
Yes
Yes
No

1744
AUUUCUACCUAUGGCUUUA
UAAAGCCAUAGGUAGAAAU
1745
478
Yes
Yes
No

1746
UUUCUACCUAUGGCUUUCA
UGAAAGCCAUAGGUAGAAA
1747
479
Yes
Yes
No

1748
UCUACCUAUGGCUUUCGAA
UUCGAAAGCCAUAGGUAGA
1749
481
Yes
No
No

1750
CUACCUAUGGCUUUCGAGA
UCUCGAAAGCCAUAGGUAG
1751
482
Yes
No
No

1752
UACCUAUGGCUUUCGAGGA
UCCUCGAAAGCCAUAGGUA
1753
483
Yes
No
No

1754
ACCUAUGGCUUUCGAGGUA
UACCUCGAAAGCCAUAGGU
1755
484
Yes
No
No

1756
GCUUUCGAGGUGAGGCUUA
UAAGCCUCACCUCGAAAGC
1757
491
Yes
No
No

1758
UUUCGAGGUGAGGCUUUGA
UCAAAGCCUCACCUCGAAA
1759
493
Yes
No
No

1760
GAGGUGAGGCUUUGGCCAA
UUGGCCAAAGCCUCACCUC
1761
497
Yes
No
No

1762
GCUUUGGCCAGCAUAAGCA
UGCUUAUGCUGGCCAAAGC
1763
505
Yes
No
No

1764
AGCAUAAGCCAUGUGGCUA
UAGCCACAUGGCUUAUGCU
1765
514
Yes
No
No

1766
AGCCAUGUGGCUCAUGUUA
UAACAUGAGCCACAUGGCU
1767
520
Yes
No
No

1768
CAUGUGGCUCAUGUUACUA
UAGUAACAUGAGCCACAUG
1769
523
Yes
No
No

1770
AUGUGGCUCAUGUUACUAA
UUAGUAACAUGAGCCACAU
1771
524
Yes
No
No

1772
UGUGGCUCAUGUUACUAUA
UAUAGUAACAUGAGCCACA
1773
525
Yes
No
No

1774
GUGGCUCAUGUUACUAUUA
UAAUAGUAACAUGAGCCAC
1775
526
Yes
No
No

1776
UGGCUCAUGUUACUAUUAA
UUAAUAGUAACAUGAGCCA
1777
527
Yes
No
No

1778
GGCUCAUGUUACUAUUACA
UGUAAUAGUAACAUGAGCC
1779
528
Yes
No
No

1780
CUCAUGUUACUAUUACAAA
UUUGUAAUAGUAACAUGAG
1781
530
Yes
No
No

1782
UCAUGUUACUAUUACAACA
UGUUGUAAUAGUAACAUGA
1783
531
Yes
No
No

1784
CAUGUUACUAUUACAACGA
UCGUUGUAAUAGUAACAUG
1785
532
No
No
No

1786
AUGUUACUAUUACAACGAA
UUCGUUGUAAUAGUAACAU
1787
533
No
No
No

1788
UGUUACUAUUACAACGAAA
UUUCGUUGUAAUAGUAACA
1789
534
No
No
No

1790
UACUAUUACAACGAAAACA
UGUUUUCGUUGUAAUAGUA
1791
537
No
No
No

1792
ACUAUUACAACGAAAACAA
UUGUUUUCGUUGUAAUAGU
1793
538
No
No
No

1794
AUUACAACGAAAACAGCUA
UAGCUGUUUUCGUUGUAAU
1795
541
No
No
No

1796
UACAACGAAAACAGCUGAA
UUCAGCUGUUUUCGUUGUA
1797
543
No
No
No

1798
ACAACGAAAACAGCUGAUA
UAUCAGCUGUUUUCGUUGU
1799
544
No
No
No

1800
CAACGAAAACAGCUGAUGA
UCAUCAGCUGUUUUCGUUG
1801
545
No
No
No

1802
ACGAAAACAGCUGAUGGAA
UUCCAUCAGCUGUUUUCGU
1803
547
No
No
No

1804
CGAAAACAGCUGAUGGAAA
UUUCCAUCAGCUGUUUUCG
1805
548
No
No
No

1806
AAACAGCUGAUGGAAAGUA
UACUUUCCAUCAGCUGUUU
1807
551
Yes
No
No

1808
ACAGCUGAUGGAAAGUGUA
UACACUUUCCAUCAGCUGU
1809
553
Yes
No
No

1810
CAGCUGAUGGAAAGUGUGA
UCACACUUUCCAUCAGCUG
1811
554
Yes
No
No

1812
AGCUGAUGGAAAGUGUGCA
UGCACACUUUCCAUCAGCU
1813
555
Yes
No
No

1814
CUGAUGGAAAGUGUGCAUA
UAUGCACACUUUCCAUCAG
1815
557
Yes
No
No

1816
UGAUGGAAAGUGUGCAUAA
UUAUGCACACUUUCCAUCA
1817
558
Yes
No
No

1818
AUGGAAAGUGUGCAUACAA
UUGUAUGCACACUUUCCAU
1819
560
Yes
No
No

1820
UGGAAAGUGUGCAUACAGA
UCUGUAUGCACACUUUCCA
1821
561
Yes
No
No

1822
GGAAAGUGUGCAUACAGAA
UUCUGUAUGCACACUUUCC
1823
562
Yes
No
No

1824
GAAAGUGUGCAUACAGAGA
UCUCUGUAUGCACACUUUC
1825
563
Yes
No
No

1826
AAAGUGUGCAUACAGAGCA
UGCUCUGUAUGCACACUUU
1827
564
Yes
No
No

1828
AAGUGUGCAUACAGAGCAA
UUGCUCUGUAUGCACACUU
1829
565
Yes
No
No

1830
GUGUGCAUACAGAGCAAGA
UCUUGCUCUGUAUGCACAC
1831
567
Yes
No
No

1832
GCAUACAGAGCAAGUUACA
UGUAACUUGCUCUGUAUGC
1833
571
Yes
No
Yes

1834
CAUACAGAGCAAGUUACUA
UAGUAACUUGCUCUGUAUG
1835
572
Yes
No
Yes

1836
AUACAGAGCAAGUUACUCA
UGAGUAACUUGCUCUGUAU
1837
573
Yes
No
Yes

1838
UACAGAGCAAGUUACUCAA
UUGAGUAACUUGCUCUGUA
1839
574
Yes
Yes
Yes

1840
CAGAGCAAGUUACUCAGAA
UUCUGAGUAACUUGCUCUG
1841
576
Yes
Yes
Yes

1842
GAGCAAGUUACUCAGAUGA
UCAUCUGAGUAACUUGCUC
1843
578
Yes
Yes
Yes

1844
CAAGUUACUCAGAUGGAAA
UUUCCAUCUGAGUAACUUG
1845
581
Yes
Yes
Yes

1846
AAGUUACUCAGAUGGAAAA
UUUUCCAUCUGAGUAACUU
1847
582
Yes
Yes
Yes

1848
ACUGAAAGCCCCUCCUAAA
UUUAGGAGGGGCUUUCAGU
1849
600
No
No
No

1850
CUGAAAGCCCCUCCUAAAA
UUUUAGGAGGGGCUUUCAG
1851
601
No
No
No

1852
GAAAGCCCCUCCUAAACCA
UGGUUUAGGAGGGGCUUUC
1853
603
No
No
No

1854
AAAGCCCCUCCUAAACCAA
UUGGUUUAGGAGGGGCUUU
1855
604
No
No
No

1856
AAGCCCCUCCUAAACCAUA
UAUGGUUUAGGAGGGGCUU
1857
605
No
No
No

1858
GCCCCUCCUAAACCAUGUA
UACAUGGUUUAGGAGGGGC
1859
607
No
No
No

1860
CCCCUCCUAAACCAUGUGA
UCACAUGGUUUAGGAGGGG
1861
608
No
No
No

1862
CCUCCUAAACCAUGUGCUA
UAGCACAUGGUUUAGGAGG
1863
610
Yes
No
No

1864
CUCCUAAACCAUGUGCUGA
UCAGCACAUGGUUUAGGAG
1865
611
Yes
No
No

1866
AAACCAUGUGCUGGCAAUA
UAUUGCCAGCACAUGGUUU
1867
616
Yes
No
No

1868
AACCAUGUGCUGGCAAUCA
UGAUUGCCAGCACAUGGUU
1869
617
Yes
No
No

1870
ACCAUGUGCUGGCAAUCAA
UUGAUUGCCAGCACAUGGU
1871
618
Yes
No
No

1872
CAUGUGCUGGCAAUCAAGA
UCUUGAUUGCCAGCACAUG
1873
620
Yes
No
No

1874
AUGUGCUGGCAAUCAAGGA
UCCUUGAUUGCCAGCACAU
1875
621
Yes
No
No

1876
UGUGCUGGCAAUCAAGGGA
UCCCUUGAUUGCCAGCACA
1877
622
Yes
No
No

1878
CAAUCAAGGGACCCAGAUA
UAUCUGGGUCCCUUGAUUG
1879
630
Yes
No
No

1880
CAAGGGACCCAGAUCACGA
UCGUGAUCUGGGUCCCUUG
1881
634
Yes
No
No

1882
AUCACGGUGGAGGACCUUA
UAAGGUCCUCCACCGUGAU
1883
646
Yes
No
No

1884
UCACGGUGGAGGACCUUUA
UAAAGGUCCUCCACCGUGA
1885
647
Yes
No
No

1886
ACAUAGCCACGAGGAGAAA
UUUCUCCUCGUGGCUAUGU
1887
671
Yes
No
No

1888
CAUAGCCACGAGGAGAAAA
UUUUCUCCUCGUGGCUAUG
1889
672
Yes
No
No

1890
AGCCACGAGGAGAAAAGCA
UGCUUUUCUCCUCGUGGCU
1891
675
Yes
No
No

1892
GCCACGAGGAGAAAAGCUA
UAGCUUUUCUCCUCGUGGC
1893
676
Yes
No
No

1894
CCACGAGGAGAAAAGCUUA
UAAGCUUUUCUCCUCGUGG
1895
677
Yes
No
No

1896
CGAGGAGAAAAGCUUUAAA
UUUAAAGCUUUUCUCCUCG
1897
680
Yes
No
No

1898
UGGGAAAAUUUUGGAAGUA
UACUUCCAAAAUUUUCCCA
1899
717
No
No
No

1900
GGAAAAUUUUGGAAGUUGA
UCAACUUCCAAAAUUUUCC
1901
719
No
No
No

1902
AAAUUUUGGAAGUUGUUGA
UCAACAACUUCCAAAAUUU
1903
722
No
Yes
No

1904
AAUUUUGGAAGUUGUUGGA
UCCAACAACUUCCAAAAUU
1905
723
No
Yes
No

1906
AUUUUGGAAGUUGUUGGCA
UGCCAACAACUUCCAAAAU
1907
724
No
Yes
Yes

1908
GAAGUUGUUGGCAGGUAUA
UAUACCUGCCAACAACUUC
1909
730
No
Yes
No

1910
AGUUGUUGGCAGGUAUUCA
UGAAUACCUGCCAACAACU
1911
732
No
Yes
No

1912
GGCAGGUAUUCAGUACACA
UGUGUACUGAAUACCUGCC
1913
739
No
No
No

1914
GCAGGUAUUCAGUACACAA
UUGUGUACUGAAUACCUGC
1915
740
No
No
No

1916
CAGGUAUUCAGUACACAAA
UUUGUGUACUGAAUACCUG
1917
741
No
No
No

1918
AGGUAUUCAGUACACAAUA
UAUUGUGUACUGAAUACCU
1919
742
No
No
No

1920
GGUAUUCAGUACACAAUGA
UCAUUGUGUACUGAAUACC
1921
743
No
No
No

1922
GUAUUCAGUACACAAUGCA
UGCAUUGUGUACUGAAUAC
1923
744
No
No
No

1924
UUCAGUACACAAUGCAGGA
UCCUGCAUUGUGUACUGAA
1925
747
No
No
No

1926
UACACAAUGCAGGCAUUAA
UUAAUGCCUGCAUUGUGUA
1927
752
Yes
No
No

1928
CACAAUGCAGGCAUUAGUA
UACUAAUGCCUGCAUUGUG
1929
754
Yes
No
No

1930
ACAAUGCAGGCAUUAGUUA
UAACUAAUGCCUGCAUUGU
1931
755
Yes
No
No

1932
CAAUGCAGGCAUUAGUUUA
UAAACUAAUGCCUGCAUUG
1933
756
Yes
No
No

1934
AAUGCAGGCAUUAGUUUCA
UGAAACUAAUGCCUGCAUU
1935
757
Yes
No
No

1936
UGCAGGCAUUAGUUUCUCA
UGAGAAACUAAUGCCUGCA
1937
759
Yes
No
No

1938
GCAGGCAUUAGUUUCUCAA
UUGAGAAACUAAUGCCUGC
1939
760
Yes
No
No

1940
GCAUUAGUUUCUCAGUUAA
UUAACUGAGAAACUAAUGC
1941
764
Yes
Yes
No

1942
AAAACAAGGAGAGACAGUA
UACUGUCUCUCCUUGUUUU
1943
783
No
No
No

1944
AACAAGGAGAGACAGUAGA
UCUACUGUCUCUCCUUGUU
1945
785
No
No
No

1946
ACAAGGAGAGACAGUAGCA
UGCUACUGUCUCUCCUUGU
1947
786
No
No
No

1948
CAAGGAGAGACAGUAGCUA
UAGCUACUGUCUCUCCUUG
1949
787
No
No
No

1950
GAGACAGUAGCUGAUGUUA
UAACAUCAGCUACUGUCUC
1951
793
Yes
No
No

1952
AGACAGUAGCUGAUGUUAA
UUAACAUCAGCUACUGUCU
1953
794
Yes
No
No

1954
GACAGUAGCUGAUGUUAGA
UCUAACAUCAGCUACUGUC
1955
795
Yes
No
No

1956
ACAGUAGCUGAUGUUAGGA
UCCUAACAUCAGCUACUGU
1957
796
Yes
No
No

1958
CAGUAGCUGAUGUUAGGAA
UUCCUAACAUCAGCUACUG
1959
797
Yes
No
No

1960
AGUAGCUGAUGUUAGGACA
UGUCCUAACAUCAGCUACU
1961
798
Yes
No
No

1962
GUAGCUGAUGUUAGGACAA
UUGUCCUAACAUCAGCUAC
1963
799
Yes
No
No

1964
UAGCUGAUGUUAGGACACA
UGUGUCCUAACAUCAGCUA
1965
800
Yes
No
No

1966
AGCUGAUGUUAGGACACUA
UAGUGUCCUAACAUCAGCU
1967
801
Yes
No
No

1968
GCUGAUGUUAGGACACUAA
UUAGUGUCCUAACAUCAGC
1969
802
Yes
No
No

1970
UGAUGUUAGGACACUACCA
UGGUAGUGUCCUAACAUCA
1971
804
Yes
No
No

1972
GUUAGGACACUACCCAAUA
UAUUGGGUAGUGUCCUAAC
1973
808
Yes
No
No

1974
UUAGGACACUACCCAAUGA
UCAUUGGGUAGUGUCCUAA
1975
809
Yes
No
No

1976
GACACUACCCAAUGCCUCA
UGAGGCAUUGGGUAGUGUC
1977
813
Yes
No
No

1978
ACACUACCCAAUGCCUCAA
UUGAGGCAUUGGGUAGUGU
1979
814
Yes
No
No

1980
CACUACCCAAUGCCUCAAA
UUUGAGGCAUUGGGUAGUG
1981
815
Yes
No
No

1982
UGCCUCAACCGUGGACAAA
UUUGUCCACGGUUGAGGCA
1983
825
No
No
No

1984
GCCUCAACCGUGGACAAUA
UAUUGUCCACGGUUGAGGC
1985
826
No
No
No

1986
CUCAACCGUGGACAAUAUA
UAUAUUGUCCACGGUUGAG
1987
828
No
No
No

1988
UCAACCGUGGACAAUAUUA
UAAUAUUGUCCACGGUUGA
1989
829
No
No
No

1990
CAACCGUGGACAAUAUUCA
UGAAUAUUGUCCACGGUUG
1991
830
No
No
No

1992
AACCGUGGACAAUAUUCGA
UCGAAUAUUGUCCACGGUU
1993
831
No
No
No

1994
ACCGUGGACAAUAUUCGCA
UGCGAAUAUUGUCCACGGU
1995
832
No
No
No

1996
CCGUGGACAAUAUUCGCUA
UAGCGAAUAUUGUCCACGG
1997
833
No
No
No

1998
ACAAUAUUCGCUCCAUCUA
UAGAUGGAGCGAAUAUUGU
1999
839
Yes
No
No

2000
CAAUAUUCGCUCCAUCUUA
UAAGAUGGAGCGAAUAUUG
2001
840
Yes
No
No

2002
AAUAUUCGCUCCAUCUUUA
UAAAGAUGGAGCGAAUAUU
2003
841
Yes
No
No

2004
UAUUCGCUCCAUCUUUGGA
UCCAAAGAUGGAGCGAAUA
2005
843
Yes
No
No

2006
AUUCGCUCCAUCUUUGGAA
UUCCAAAGAUGGAGCGAAU
2007
844
Yes
Yes
Yes

2008
UUCGCUCCAUCUUUGGAAA
UUUCCAAAGAUGGAGCGAA
2009
845
Yes
Yes
Yes

2010
UCGCUCCAUCUUUGGAAAA
UUUUCCAAAGAUGGAGCGA
2011
846
Yes
Yes
Yes

2012
CGCUCCAUCUUUGGAAAUA
UAUUUCCAAAGAUGGAGCG
2013
847
Yes
Yes
Yes

2014
UCCAUCUUUGGAAAUGCUA
UAGCAUUUCCAAAGAUGGA
2015
850
Yes
No
Yes

2016
AUCUUUGGAAAUGCUGUUA
UAACAGCAUUUCCAAAGAU
2017
853
Yes
No
Yes

2018
UCUUUGGAAAUGCUGUUAA
UUAACAGCAUUUCCAAAGA
2019
854
Yes
No
Yes

2020
CUUUGGAAAUGCUGUUAGA
UCUAACAGCAUUUCCAAAG
2021
855
Yes
No
Yes

2022
UUGGAAAUGCUGUUAGUCA
UGACUAACAGCAUUUCCAA
2023
857
Yes
No
Yes

2024
UGGAAAUGCUGUUAGUCGA
UCGACUAACAGCAUUUCCA
2025
858
Yes
No
Yes

2026
GGAAAUGCUGUUAGUCGAA
UUCGACUAACAGCAUUUCC
2027
859
Yes
No
Yes

2028
GAAAUGCUGUUAGUCGAGA
UCUCGACUAACAGCAUUUC
2029
860
Yes
No
Yes

2030
AAAUGCUGUUAGUCGAGAA
UUCUCGACUAACAGCAUUU
2031
861
Yes
No
Yes

2032
AAUGCUGUUAGUCGAGAAA
UUUCUCGACUAACAGCAUU
2033
862
Yes
No
Yes

2034
AUGCUGUUAGUCGAGAACA
UGUUCUCGACUAACAGCAU
2035
863
Yes
No
Yes

2036
UGCUGUUAGUCGAGAACUA
UAGUUCUCGACUAACAGCA
2037
864
Yes
No
Yes

2038
GCUGUUAGUCGAGAACUGA
UCAGUUCUCGACUAACAGC
2039
865
Yes
No
Yes

2040
UGUUAGUCGAGAACUGAUA
UAUCAGUUCUCGACUAACA
2041
867
Yes
No
Yes

2042
GUUAGUCGAGAACUGAUAA
UUAUCAGUUCUCGACUAAC
2043
868
Yes
Yes
No

2044
UUAGUCGAGAACUGAUAGA
UCUAUCAGUUCUCGACUAA
2045
869
Yes
Yes
No

2046
UAGUCGAGAACUGAUAGAA
UUCUAUCAGUUCUCGACUA
2047
870
Yes
Yes
No

2048
AGUCGAGAACUGAUAGAAA
UUUCUAUCAGUUCUCGACU
2049
871
Yes
Yes
No

2050
GUCGAGAACUGAUAGAAAA
UUUUCUAUCAGUUCUCGAC
2051
872
Yes
No
No

2052
UCGAGAACUGAUAGAAAUA
UAUUUCUAUCAGUUCUCGA
2053
873
Yes
No
No

2054
CGAGAACUGAUAGAAAUUA
UAAUUUCUAUCAGUUCUCG
2055
874
Yes
No
No

2056
AGAACUGAUAGAAAUUGGA
UCCAAUUUCUAUCAGUUCU
2057
876
Yes
No
No

2058
GAACUGAUAGAAAUUGGAA
UUCCAAUUUCUAUCAGUUC
2059
877
Yes
No
No

2060
ACUGAUAGAAAUUGGAUGA
UCAUCCAAUUUCUAUCAGU
2061
879
Yes
No
No

2062
UGAUAGAAAUUGGAUGUGA
UCACAUCCAAUUUCUAUCA
2063
881
Yes
No
No

2064
GAUAGAAAUUGGAUGUGAA
UUCACAUCCAAUUUCUAUC
2065
882
Yes
No
No

2066
UAGAAAUUGGAUGUGAGGA
UCCUCACAUCCAAUUUCUA
2067
884
Yes
No
No

2068
AAUUGGAUGUGAGGAUAAA
UUUAUCCUCACAUCCAAUU
2069
888
Yes
No
No

2070
AUUGGAUGUGAGGAUAAAA
UUUUAUCCUCACAUCCAAU
2071
889
Yes
No
No

2072
UGGAUGUGAGGAUAAAACA
UGUUUUAUCCUCACAUCCA
2073
891
Yes
No
No

2074
GAUGUGAGGAUAAAACCCA
UGGGUUUUAUCCUCACAUC
2075
893
Yes
No
No

2076
AUGUGAGGAUAAAACCCUA
UAGGGUUUUAUCCUCACAU
2077
894
Yes
No
No

2078
UGUGAGGAUAAAACCCUAA
UUAGGGUUUUAUCCUCACA
2079
895
Yes
Yes
Yes

2080
GUGAGGAUAAAACCCUAGA
UCUAGGGUUUUAUCCUCAC
2081
896
Yes
Yes
Yes

2082
GGAUAAAACCCUAGCCUUA
UAAGGCUAGGGUUUUAUCC
2083
900
Yes
No
No

2084
UAAAACCCUAGCCUUCAAA
UUUGAAGGCUAGGGUUUUA
2085
903
Yes
No
No

2086
AACCCUAGCCUUCAAAAUA
UAUUUUGAAGGCUAGGGUU
2087
906
Yes
No
No

2088
ACCCUAGCCUUCAAAAUGA
UCAUUUUGAAGGCUAGGGU
2089
907
Yes
No
No

2090
CUAGCCUUCAAAAUGAAUA
UAUUCAUUUUGAAGGCUAG
2091
910
Yes
No
No

2092
AGCCUUCAAAAUGAAUGGA
UCCAUUCAUUUUGAAGGCU
2093
912
Yes
No
No

2094
UCAAAAUGAAUGGUUACAA
UUGUAACCAUUCAUUUUGA
2095
917
No
No
No

2096
CAAAAUGAAUGGUUACAUA
UAUGUAACCAUUCAUUUUG
2097
918
No
No
No

2098
AAAUGAAUGGUUACAUAUA
UAUAUGUAACCAUUCAUUU
2099
920
No
No
No

2100
AAUGAAUGGUUACAUAUCA
UGAUAUGUAACCAUUCAUU
2101
921
No
No
No

2102
AUGAAUGGUUACAUAUCCA
UGGAUAUGUAACCAUUCAU
2103
922
No
No
No

2104
UGAAUGGUUACAUAUCCAA
UUGGAUAUGUAACCAUUCA
2105
923
No
No
No

2106
GAAUGGUUACAUAUCCAAA
UUUGGAUAUGUAACCAUUC
2107
924
No
No
No

2108
GGUUACAUAUCCAAUGCAA
UUGCAUUGGAUAUGUAACC
2109
928
No
No
No

2110
UUACAUAUCCAAUGCAAAA
UUUUGCAUUGGAUAUGUAA
2111
930
No
No
No

2112
UACAUAUCCAAUGCAAACA
UGUUUGCAUUGGAUAUGUA
2113
931
No
No
No

2114
UAUCCAAUGCAAACUACUA
UAGUAGUUUGCAUUGGAUA
2115
935
Yes
No
No

2116
AUCCAAUGCAAACUACUCA
UGAGUAGUUUGCAUUGGAU
2117
936
Yes
No
No

2118
UCCAAUGCAAACUACUCAA
UUGAGUAGUUUGCAUUGGA
2119
937
Yes
No
No

2120
CCAAUGCAAACUACUCAGA
UCUGAGUAGUUUGCAUUGG
2121
938
Yes
No
No

2122
CAAUGCAAACUACUCAGUA
UACUGAGUAGUUUGCAUUG
2123
939
Yes
No
No

2124
AUGCAAACUACUCAGUGAA
UUCACUGAGUAGUUUGCAU
2125
941
Yes
No
Yes

2126
CAAACUACUCAGUGAAGAA
UUCUUCACUGAGUAGUUUG
2127
944
Yes
No
Yes

2128
AAACUACUCAGUGAAGAAA
UUUCUUCACUGAGUAGUUU
2129
945
Yes
No
Yes

2130
AACUACUCAGUGAAGAAGA
UCUUCUUCACUGAGUAGUU
2131
946
No
No
Yes

2132
ACUCAGUGAAGAAGUGCAA
UUGCACUUCUUCACUGAGU
2133
950
No
No
Yes

2134
UCAGUGAAGAAGUGCAUCA
UGAUGCACUUCUUCACUGA
2135
952
No
No
No

2136
CAGUGAAGAAGUGCAUCUA
UAGAUGCACUUCUUCACUG
2137
953
No
No
No

2138
GUGAAGAAGUGCAUCUUCA
UGAAGAUGCACUUCUUCAC
2139
955
No
No
No

2140
UGAAGAAGUGCAUCUUCUA
UAGAAGAUGCACUUCUUCA
2141
956
No
No
No

2142
AGAAGUGCAUCUUCUUACA
UGUAAGAAGAUGCACUUCU
2143
959
No
No
No

2144
AAGUGCAUCUUCUUACUCA
UGAGUAAGAAGAUGCACUU
2145
961
No
No
No

2146
AGUGCAUCUUCUUACUCUA
UAGAGUAAGAAGAUGCACU
2147
962
No
No
No

2148
GUGCAUCUUCUUACUCUUA
UAAGAGUAAGAAGAUGCAC
2149
963
No
No
No

2150
UCUUCUUACUCUUCAUCAA
UUGAUGAAGAGUAAGAAGA
2151
968
Yes
No
No

2152
CUUCUUACUCUUCAUCAAA
UUUGAUGAAGAGUAAGAAG
2153
969
Yes
No
No

2154
UCUUACUCUUCAUCAACCA
UGGUUGAUGAAGAGUAAGA
2155
971
Yes
No
No

2156
UUACUCUUCAUCAACCAUA
UAUGGUUGAUGAAGAGUAA
2157
973
Yes
No
No

2158
ACUCUUCAUCAACCAUCGA
UCGAUGGUUGAUGAAGAGU
2159
975
Yes
No
No

2160
CUCUUCAUCAACCAUCGUA
UACGAUGGUUGAUGAAGAG
2161
976
Yes
No
No

2162
CUUCAUCAACCAUCGUCUA
UAGACGAUGGUUGAUGAAG
2163
978
Yes
No
No

2164
UUCAUCAACCAUCGUCUGA
UCAGACGAUGGUUGAUGAA
2165
979
Yes
No
No

2166
AUCAACCAUCGUCUGGUAA
UUACCAGACGAUGGUUGAU
2167
982
Yes
No
No

2168
UCAACCAUCGUCUGGUAGA
UCUACCAGACGAUGGUUGA
2169
983
Yes
No
No

2170
CAACCAUCGUCUGGUAGAA
UUCUACCAGACGAUGGUUG
2171
984
Yes
No
No

2172
AACCAUCGUCUGGUAGAAA
UUUCUACCAGACGAUGGUU
2173
985
Yes
No
No

2174
CCAUCGUCUGGUAGAAUCA
UGAUUCUACCAGACGAUGG
2175
987
Yes
No
No

2176
CAUCGUCUGGUAGAAUCAA
UUGAUUCUACCAGACGAUG
2177
988
Yes
No
No

2178
AUCGUCUGGUAGAAUCAAA
UUUGAUUCUACCAGACGAU
2179
989
Yes
No
No

2180
CGUCUGGUAGAAUCAACUA
UAGUUGAUUCUACCAGACG
2181
991
Yes
No
No

2182
UCUGGUAGAAUCAACUUCA
UGAAGUUGAUUCUACCAGA
2183
993
Yes
No
No

2184
CUGGUAGAAUCAACUUCCA
UGGAAGUUGAUUCUACCAG
2185
994
Yes
No
No

2186
UGGUAGAAUCAACUUCCUA
UAGGAAGUUGAUUCUACCA
2187
995
Yes
No
No

2188
GAAUCAACUUCCUUGAGAA
UUCUCAAGGAAGUUGAUUC
2189
1000
Yes
No
No

2190
AAUCAACUUCCUUGAGAAA
UUUCUCAAGGAAGUUGAUU
2191
1001
Yes
No
No

2192
CAACUUCCUUGAGAAAAGA
UCUUUUCUCAAGGAAGUUG
2193
1004
Yes
No
No

2194
AACUUCCUUGAGAAAAGCA
UGCUUUUCUCAAGGAAGUU
2195
1005
Yes
No
No

2196
ACUUCCUUGAGAAAAGCCA
UGGCUUUUCUCAAGGAAGU
2197
1006
Yes
No
No

2198
UUCCUUGAGAAAAGCCAUA
UAUGGCUUUUCUCAAGGAA
2199
1008
Yes
No
No

2200
CCUUGAGAAAAGCCAUAGA
UCUAUGGCUUUUCUCAAGG
2201
1010
Yes
No
No

2202
UGAGAAAAGCCAUAGAAAA
UUUUCUAUGGCUUUUCUCA
2203
1013
Yes
No
No

2204
AGCCAUAGAAACAGUGUAA
UUACACUGUUUCUAUGGCU
2205
1020
Yes
No
No

2206
CAUAGAAACAGUGUAUGCA
UGCAUACACUGUUUCUAUG
2207
1023
Yes
No
No

2208
UAGAAACAGUGUAUGCAGA
UCUGCAUACACUGUUUCUA
2209
1025
Yes
No
No

2210
AGAAACAGUGUAUGCAGCA
UGCUGCAUACACUGUUUCU
2211
1026
Yes
No
No

2212
ACAGUGUAUGCAGCCUAUA
UAUAGGCUGCAUACACUGU
2213
1030
No
No
No

2214
CAGUGUAUGCAGCCUAUUA
UAAUAGGCUGCAUACACUG
2215
1031
No
No
No

2216
GUGUAUGCAGCCUAUUUGA
UCAAAUAGGCUGCAUACAC
2217
1033
No
No
No

2218
UGUAUGCAGCCUAUUUGCA
UGCAAAUAGGCUGCAUACA
2219
1034
No
No
No

2220
GUAUGCAGCCUAUUUGCCA
UGGCAAAUAGGCUGCAUAC
2221
1035
No
No
No

2222
GCAGCCUAUUUGCCCAAAA
UUUUGGGCAAAUAGGCUGC
2223
1039
No
No
No

2224
AAACACACACCCAUUCCUA
UAGGAAUGGGUGUGUGUUU
2225
1056
Yes
Yes
No

2226
AACACACACCCAUUCCUGA
UCAGGAAUGGGUGUGUGUU
2227
1057
Yes
Yes
No

2228
ACACACACCCAUUCCUGUA
UACAGGAAUGGGUGUGUGU
2229
1058
Yes
Yes
No

2230
CACACACCCAUUCCUGUAA
UUACAGGAAUGGGUGUGUG
2231
1059
Yes
Yes
No

2232
ACACACCCAUUCCUGUACA
UGUACAGGAAUGGGUGUGU
2233
1060
Yes
Yes
No

2234
ACACCCAUUCCUGUACCUA
UAGGUACAGGAAUGGGUGU
2235
1062
Yes
Yes
No

2236
ACCCAUUCCUGUACCUCAA
UUGAGGUACAGGAAUGGGU
2237
1064
Yes
Yes
Yes

2238
CCCAUUCCUGUACCUCAGA
UCUGAGGUACAGGAAUGGG
2239
1065
Yes
Yes
No

2240
CAUUCCUGUACCUCAGUUA
UAACUGAGGUACAGGAAUG
2241
1067
Yes
Yes
No

2242
UUCCUGUACCUCAGUUUAA
UUAAACUGAGGUACAGGAA
2243
1069
Yes
No
No

2244
UCCUGUACCUCAGUUUAGA
UCUAAACUGAGGUACAGGA
2245
1070
Yes
No
No

2246
CCUGUACCUCAGUUUAGAA
UUCUAAACUGAGGUACAGG
2247
1071
Yes
No
No

2248
GUACCUCAGUUUAGAAAUA
UAUUUCUAAACUGAGGUAC
2249
1074
Yes
No
No

2250
CUCAGUUUAGAAAUCAGUA
UACUGAUUUCUAAACUGAG
2251
1078
Yes
No
No

2252
UCAGUUUAGAAAUCAGUCA
UGACUGAUUUCUAAACUGA
2253
1079
Yes
No
No

2254
CAGUUUAGAAAUCAGUCCA
UGGACUGAUUUCUAAACUG
2255
1080
Yes
No
No

2256
AAUCAGUCCCCAGAAUGUA
UACAUUCUGGGGACUGAUU
2257
1089
Yes
No
No

2258
UCAGUCCCCAGAAUGUGGA
UCCACAUUCUGGGGACUGA
2259
1091
Yes
No
No

2260
CAGUCCCCAGAAUGUGGAA
UUCCACAUUCUGGGGACUG
2261
1092
Yes
No
No

2262
AGUCCCCAGAAUGUGGAUA
UAUCCACAUUCUGGGGACU
2263
1093
Yes
No
No

2264
UCCCCAGAAUGUGGAUGUA
UACAUCCACAUUCUGGGGA
2265
1095
Yes
No
No

2266
CCCCAGAAUGUGGAUGUUA
UAACAUCCACAUUCUGGGG
2267
1096
Yes
No
No

2268
CCCAGAAUGUGGAUGUUAA
UUAACAUCCACAUUCUGGG
2269
1097
Yes
No
No

2270
CCAGAAUGUGGAUGUUAAA
UUUAACAUCCACAUUCUGG
2271
1098
Yes
No
No

2272
CAGAAUGUGGAUGUUAAUA
UAUUAACAUCCACAUUCUG
2273
1099
Yes
No
No

2274
AUGUGGAUGUUAAUGUGCA
UGCACAUUAACAUCCACAU
2275
1103
Yes
No
No

2276
UGUGGAUGUUAAUGUGCAA
UUGCACAUUAACAUCCACA
2277
1104
Yes
No
No

2278
GUGGAUGUUAAUGUGCACA
UGUGCACAUUAACAUCCAC
2279
1105
Yes
No
No

2280
UAAUGUGCACCCCACAAAA
UUUUGUGGGGUGCACAUUA
2281
1113
Yes
No
No

2282
AAUGUGCACCCCACAAAGA
UCUUUGUGGGGUGCACAUU
2283
1114
Yes
No
No

2284
UGCACCCCACAAAGCAUGA
UCAUGCUUUGUGGGGUGCA
2285
1118
Yes
No
No

2286
GCACCCCACAAAGCAUGAA
UUCAUGCUUUGUGGGGUGC
2287
1119
Yes
No
No

2288
CCCACAAAGCAUGAAGUUA
UAACUUCAUGCUUUGUGGG
2289
1123
Yes
No
No

2290
CCACAAAGCAUGAAGUUCA
UGAACUUCAUGCUUUGUGG
2291
1124
Yes
No
No

2292
CAAAGCAUGAAGUUCACUA
UAGUGAACUUCAUGCUUUG
2293
1127
Yes
No
No

2294
AAAGCAUGAAGUUCACUUA
UAAGUGAACUUCAUGCUUU
2295
1128
Yes
No
No

2296
AAGCAUGAAGUUCACUUCA
UGAAGUGAACUUCAUGCUU
2297
1129
Yes
No
No

2298
GCAUGAAGUUCACUUCCUA
UAGGAAGUGAACUUCAUGC
2299
1131
Yes
No
No

2300
CCUGCACGAGGAGAGCAUA
UAUGCUCUCCUCGUGCAGG
2301
1146
Yes
No
No

2302
CUGCACGAGGAGAGCAUCA
UGAUGCUCUCCUCGUGCAG
2303
1147
Yes
No
No

2304
CGAGGAGAGCAUCCUGGAA
UUCCAGGAUGCUCUCCUCG
2305
1152
Yes
No
No

2306
GGUGCAGCAGCACAUCGAA
UUCGAUGUGCUGCUGCACC
2307
1173
No
No
No

2308
CAGCACAUCGAGAGCAAGA
UCUUGCUCUCGAUGUGCUG
2309
1180
Yes
No
Yes

2310
GCACAUCGAGAGCAAGCUA
UAGCUUGCUCUCGAUGUGC
2311
1182
Yes
No
Yes

2312
CAUCGAGAGCAAGCUCCUA
UAGGAGCUUGCUCUCGAUG
2313
1185
Yes
No
No

2314
AAGCUCCUGGGCUCCAAUA
UAUUGGAGCCCAGGAGCUU
2315
1195
Yes
No
No

2316
AGCUCCUGGGCUCCAAUUA
UAAUUGGAGCCCAGGAGCU
2317
1196
Yes
No
No

2318
CUCCUGGGCUCCAAUUCCA
UGGAAUUGGAGCCCAGGAG
2319
1198
Yes
No
No

2320
UCCUGGGCUCCAAUUCCUA
UAGGAAUUGGAGCCCAGGA
2321
1199
Yes
No
No

2322
CCUGGGCUCCAAUUCCUCA
UGAGGAAUUGGAGCCCAGG
2323
1200
Yes
No
No

2324
CUCCAAUUCCUCCAGGAUA
UAUCCUGGAGGAAUUGGAG
2325
1206
Yes
Yes
No

2326
CCAAUUCCUCCAGGAUGUA
UACAUCCUGGAGGAAUUGG
2327
1208
Yes
Yes
No

2328
AAUUCCUCCAGGAUGUACA
UGUACAUCCUGGAGGAAUU
2329
1210
Yes
No
No

2330
AUUCCUCCAGGAUGUACUA
UAGUACAUCCUGGAGGAAU
2331
1211
Yes
No
No

2332
UUCCUCCAGGAUGUACUUA
UAAGUACAUCCUGGAGGAA
2333
1212
Yes
No
No

2334
CCUCCAGGAUGUACUUCAA
UUGAAGUACAUCCUGGAGG
2335
1214
Yes
No
No

2336
CUCCAGGAUGUACUUCACA
UGUGAAGUACAUCCUGGAG
2337
1215
Yes
No
No

2338
CAGGAUGUACUUCACCCAA
UUGGGUGAAGUACAUCCUG
2339
1218
Yes
No
No

2340
UGUACUUCACCCAGACUUA
UAAGUCUGGGUGAAGUACA
2341
1223
Yes
No
No

2342
UACUUCACCCAGACUUUGA
UCAAAGUCUGGGUGAAGUA
2343
1225
Yes
No
No

2344
CUUCACCCAGACUUUGCUA
UAGCAAAGUCUGGGUGAAG
2345
1227
Yes
No
No

2346
UUCACCCAGACUUUGCUAA
UUAGCAAAGUCUGGGUGAA
2347
1228
Yes
No
No

2348
UCACCCAGACUUUGCUACA
UGUAGCAAAGUCUGGGUGA
2349
1229
Yes
No
No

2350
ACCCAGACUUUGCUACCAA
UUGGUAGCAAAGUCUGGGU
2351
1231
Yes
No
No

2352
CAGACUUUGCUACCAGGAA
UUCCUGGUAGCAAAGUCUG
2353
1234
Yes
No
No

2354
ACUUUGCUACCAGGACUUA
UAAGUCCUGGUAGCAAAGU
2355
1237
Yes
No
No

2356
CUUUGCUACCAGGACUUGA
UCAAGUCCUGGUAGCAAAG
2357
1238
Yes
No
No

2358
UUGCUACCAGGACUUGCUA
UAGCAAGUCCUGGUAGCAA
2359
1240
Yes
No
No

2360
UGCUACCAGGACUUGCUGA
UCAGCAAGUCCUGGUAGCA
2361
1241
Yes
No
No

2362
CUCUGGGGAGAUGGUUAAA
UUUAACCAUCUCCCCAGAG
2363
1263
Yes
No
No

2364
CUGGGGAGAUGGUUAAAUA
UAUUUAACCAUCUCCCCAG
2365
1265
Yes
No
No

2366
UGGGGAGAUGGUUAAAUCA
UGAUUUAACCAUCUCCCCA
2367
1266
Yes
No
No

2368
GGGGAGAUGGUUAAAUCCA
UGGAUUUAACCAUCUCCCC
2369
1267
Yes
No
No

2370
GGGAGAUGGUUAAAUCCAA
UUGGAUUUAACCAUCUCCC
2371
1268
Yes
No
No

2372
GGAGAUGGUUAAAUCCACA
UGUGGAUUUAACCAUCUCC
2373
1269
Yes
No
No

2374
UGGUUAAAUCCACAACAAA
UUUGUUGUGGAUUUAACCA
2375
1274
No
No
No

2376
GGUUAAAUCCACAACAAGA
UCUUGUUGUGGAUUUAACC
2377
1275
No
No
No

2378
GUUAAAUCCACAACAAGUA
UACUUGUUGUGGAUUUAAC
2379
1276
No
No
No

2380
UAAAUCCACAACAAGUCUA
UAGACUUGUUGUGGAUUUA
2381
1278
No
No
No

2382
AUCCACAACAAGUCUGACA
UGUCAGACUUGUUGUGGAU
2383
1281
No
No
No

2384
AAGUCUGACCUCGUCUUCA
UGAAGACGAGGUCAGACUU
2385
1290
No
No
No

2386
AGUCUGACCUCGUCUUCUA
UAGAAGACGAGGUCAGACU
2387
1291
No
No
No

2388
UCUGACCUCGUCUUCUACA
UGUAGAAGACGAGGUCAGA
2389
1293
No
No
No

2390
CUGACCUCGUCUUCUACUA
UAGUAGAAGACGAGGUCAG
2391
1294
No
No
No

2392
UGACCUCGUCUUCUACUUA
UAAGUAGAAGACGAGGUCA
2393
1295
No
No
No

2394
UCUACUUCUGGAAGUAGUA
UACUACUUCCAGAAGUAGA
2395
1306
Yes
No
No

2396
CUACUUCUGGAAGUAGUGA
UCACUACUUCCAGAAGUAG
2397
1307
Yes
No
No

2398
UACUUCUGGAAGUAGUGAA
UUCACUACUUCCAGAAGUA
2399
1308
Yes
No
No

2400
UCUGGAAGUAGUGAUAAGA
UCUUAUCACUACUUCCAGA
2401
1312
No
No
No

2402
CUGGAAGUAGUGAUAAGGA
UCCUUAUCACUACUUCCAG
2403
1313
No
No
No

2404
UGGAAGUAGUGAUAAGGUA
UACCUUAUCACUACUUCCA
2405
1314
No
No
No

2406
GGAAGUAGUGAUAAGGUCA
UGACCUUAUCACUACUUCC
2407
1315
No
No
No

2408
GAAGUAGUGAUAAGGUCUA
UAGACCUUAUCACUACUUC
2409
1316
No
No
No

2410
AAGUAGUGAUAAGGUCUAA
UUAGACCUUAUCACUACUU
2411
1317
No
No
No

2412
AGUAGUGAUAAGGUCUAUA
UAUAGACCUUAUCACUACU
2413
1318
No
No
No

2414
GUAGUGAUAAGGUCUAUGA
UCAUAGACCUUAUCACUAC
2415
1319
No
No
No

2416
UAGUGAUAAGGUCUAUGCA
UGCAUAGACCUUAUCACUA
2417
1320
No
No
No

2418
UGAUAAGGUCUAUGCCCAA
UUGGGCAUAGACCUUAUCA
2419
1323
No
No
No

2420
GGUCUAUGCCCACCAGAUA
UAUCUGGUGGGCAUAGACC
2421
1329
Yes
No
No

2422
GUCUAUGCCCACCAGAUGA
UCAUCUGGUGGGCAUAGAC
2423
1330
Yes
No
No

2424
CUAUGCCCACCAGAUGGUA
UACCAUCUGGUGGGCAUAG
2425
1332
Yes
No
No

2426
UAUGCCCACCAGAUGGUUA
UAACCAUCUGGUGGGCAUA
2427
1333
Yes
No
No

2428
UGCCCACCAGAUGGUUCGA
UCGAACCAUCUGGUGGGCA
2429
1335
Yes
No
No

2430
GCCCACCAGAUGGUUCGUA
UACGAACCAUCUGGUGGGC
2431
1336
Yes
No
No

2432
CCCACCAGAUGGUUCGUAA
UUACGAACCAUCUGGUGGG
2433
1337
Yes
No
No

2434
CCACCAGAUGGUUCGUACA
UGUACGAACCAUCUGGUGG
2435
1338
Yes
No
No

2436
CCAGAUGGUUCGUACAGAA
UUCUGUACGAACCAUCUGG
2437
1341
Yes
No
No

2438
UGGUUCGUACAGAUUCCCA
UGGGAAUCUGUACGAACCA
2439
1346
Yes
No
No

2440
CAGAUUCCCGGGAACAGAA
UUCUGUUCCCGGGAAUCUG
2441
1355
Yes
No
No

2442
AGAUUCCCGGGAACAGAAA
UUUCUGUUCCCGGGAAUCU
2443
1356
Yes
No
No

2444
AUUCCCGGGAACAGAAGCA
UGCUUCUGUUCCCGGGAAU
2445
1358
Yes
No
No

2446
UUCCCGGGAACAGAAGCUA
UAGCUUCUGUUCCCGGGAA
2447
1359
Yes
No
No

2448
CCCGGGAACAGAAGCUUGA
UCAAGCUUCUGUUCCCGGG
2449
1361
Yes
No
No

2450
CCGGGAACAGAAGCUUGAA
UUCAAGCUUCUGUUCCCGG
2451
1362
Yes
No
No

2452
CGGGAACAGAAGCUUGAUA
UAUCAAGCUUCUGUUCCCG
2453
1363
Yes
No
No

2454
GGAACAGAAGCUUGAUGCA
UGCAUCAAGCUUCUGUUCC
2455
1365
Yes
No
No

2456
ACAGAAGCUUGAUGCAUUA
UAAUGCAUCAAGCUUCUGU
2457
1368
Yes
No
No

2458
AGAAGCUUGAUGCAUUUCA
UGAAAUGCAUCAAGCUUCU
2459
1370
Yes
No
No

2460
GAAGCUUGAUGCAUUUCUA
UAGAAAUGCAUCAAGCUUC
2461
1371
Yes
No
No

2462
GCUUGAUGCAUUUCUGCAA
UUGCAGAAAUGCAUCAAGC
2463
1374
Yes
No
No

2464
GCAGCCUCUGAGCAAACCA
UGGUUUGCUCAGAGGCUGC
2465
1389
Yes
No
No

2466
AGCAAACCCCUGUCCAGUA
UACUGGACAGGGGUUUGCU
2467
1399
Yes
No
No

2468
AGCCCCAGGCCAUUGUCAA
UUGACAAUGGCCUGGGGCU
2469
1418
No
No
No

2470
CCCCAGGCCAUUGUCACAA
UUGUGACAAUGGCCUGGGG
2471
1420
No
No
No

2472
CCCAGGCCAUUGUCACAGA
UCUGUGACAAUGGCCUGGG
2473
1421
No
No
No

2474
GGCCAUUGUCACAGAGGAA
UUCCUCUGUGACAAUGGCC
2475
1425
No
No
No

2476
CCAUUGUCACAGAGGAUAA
UUAUCCUCUGUGACAAUGG
2477
1427
No
No
No

2478
CAUUGUCACAGAGGAUAAA
UUUAUCCUCUGUGACAAUG
2479
1428
No
No
No

2480
UUGUCACAGAGGAUAAGAA
UUCUUAUCCUCUGUGACAA
2481
1430
No
No
No

2482
UGUCACAGAGGAUAAGACA
UGUCUUAUCCUCUGUGACA
2483
1431
No
No
No

2484
CACAGAGGAUAAGACAGAA
UUCUGUCUUAUCCUCUGUG
2485
1434
No
No
No

2486
AGAGGAUAAGACAGAUAUA
UAUAUCUGUCUUAUCCUCU
2487
1437
Yes
No
No

2488
GAGGAUAAGACAGAUAUUA
UAAUAUCUGUCUUAUCCUC
2489
1438
Yes
No
No

2490
GAUAAGACAGAUAUUUCUA
UAGAAAUAUCUGUCUUAUC
2491
1441
Yes
No
No

2492
AUAAGACAGAUAUUUCUAA
UUAGAAAUAUCUGUCUUAU
2493
1442
Yes
No
No

2494
GACAGAUAUUUCUAGUGGA
UCCACUAGAAAUAUCUGUC
2495
1446
Yes
No
No

2496
AGGGCUAGGCAGCAAGAUA
UAUCUUGCUGCCUAGCCCU
2497
1465
Yes
No
No

2498
GGGCUAGGCAGCAAGAUGA
UCAUCUUGCUGCCUAGCCC
2499
1466
Yes
No
No

2500
GGCUAGGCAGCAAGAUGAA
UUCAUCUUGCUGCCUAGCC
2501
1467
Yes
No
No

2502
CUAGGCAGCAAGAUGAGGA
UCCUCAUCUUGCUGCCUAG
2503
1469
Yes
No
No

2504
UAGGCAGCAAGAUGAGGAA
UUCCUCAUCUUGCUGCCUA
2505
1470
Yes
No
No

2506
GCAAGAUGAGGAGAUGCUA
UAGCAUCUCCUCAUCUUGC
2507
1476
Yes
No
No

2508
CAAGAUGAGGAGAUGCUUA
UAAGCAUCUCCUCAUCUUG
2509
1477
Yes
No
No

2510
UGAGGAGAUGCUUGAACUA
UAGUUCAAGCAUCUCCUCA
2511
1482
Yes
No
No

2512
GGGAUACAACAAAGGGGAA
UUCCCCUUUGUUGUAUCCC
2513
1544
Yes
No
No

2514
ACAACAAAGGGGACUUCAA
UUGAAGUCCCCUUUGUUGU
2515
1549
No
No
No

2516
ACAAAGGGGACUUCAGAAA
UUUCUGAAGUCCCCUUUGU
2517
1552
No
No
No

2518
CAAAGGGGACUUCAGAAAA
UUUUCUGAAGUCCCCUUUG
2519
1553
No
No
No

2520
AAAGGGGACUUCAGAAAUA
UAUUUCUGAAGUCCCCUUU
2521
1554
No
No
No

2522
AAGGGGACUUCAGAAAUGA
UCAUUUCUGAAGUCCCCUU
2523
1555
No
No
No

2524
GGGGACUUCAGAAAUGUCA
UGACAUUUCUGAAGUCCCC
2525
1557
No
No
No

2526
GAAGAGAGGACCUACUUCA
UGAAGUAGGUCCUCUCUUC
2527
1578
No
No
No

2528
AAGAGAGGACCUACUUCCA
UGGAAGUAGGUCCUCUCUU
2529
1579
No
No
No

2530
AGAGAGGACCUACUUCCAA
UUGGAAGUAGGUCCUCUCU
2531
1580
No
No
No

2532
GAGAGGACCUACUUCCAGA
UCUGGAAGUAGGUCCUCUC
2533
1581
No
No
No

2534
AGGACCUACUUCCAGCAAA
UUUGCUGGAAGUAGGUCCU
2535
1584
No
No
No

2536
CAACCCCAGAAAGAGACAA
UUGUCUCUUUCUGGGGUUG
2537
1599
Yes
No
No

2538
ACCCCAGAAAGAGACAUCA
UGAUGUCUCUUUCUGGGGU
2539
1601
Yes
No
No

2540
CCCCAGAAAGAGACAUCGA
UCGAUGUCUCUUUCUGGGG
2541
1602
Yes
No
No

2542
AGAAAGAGACAUCGGGAAA
UUUCCCGAUGUCUCUUUCU
2543
1606
Yes
No
No

2544
ACAUCGGGAAGAUUCUGAA
UUCAGAAUCUUCCCGAUGU
2545
1614
Yes
No
No

2546
CAUCGGGAAGAUUCUGAUA
UAUCAGAAUCUUCCCGAUG
2547
1615
Yes
No
No

2548
AUCGGGAAGAUUCUGAUGA
UCAUCAGAAUCUUCCCGAU
2549
1616
Yes
No
No

2550
CGGGAAGAUUCUGAUGUGA
UCACAUCAGAAUCUUCCCG
2551
1618
Yes
No
No

2552
GGGAAGAUUCUGAUGUGGA
UCCACAUCAGAAUCUUCCC
2553
1619
Yes
No
No

2554
GGAAGAUUCUGAUGUGGAA
UUCCACAUCAGAAUCUUCC
2555
1620
Yes
No
No

2556
GAAGAUUCUGAUGUGGAAA
UUUCCACAUCAGAAUCUUC
2557
1621
Yes
No
No

2558
AGAUUCUGAUGUGGAAAUA
UAUUUCCACAUCAGAAUCU
2559
1623
Yes
No
No

2560
UGAUGUGGAAAUGGUGGAA
UUCCACCAUUUCCACAUCA
2561
1629
Yes
Yes
No

2562
GAAAUGGUGGAAGAUGAUA
UAUCAUCUUCCACCAUUUC
2563
1636
Yes
No
No

2564
AAAUGGUGGAAGAUGAUUA
UAAUCAUCUUCCACCAUUU
2565
1637
Yes
No
No

2566
AAUGGUGGAAGAUGAUUCA
UGAAUCAUCUUCCACCAUU
2567
1638
Yes
No
No

2568
AUGGUGGAAGAUGAUUCCA
UGGAAUCAUCUUCCACCAU
2569
1639
Yes
No
No

2570
GUGGAAGAUGAUUCCCGAA
UUCGGGAAUCAUCUUCCAC
2571
1642
Yes
No
No

2572
GGAAGAUGAUUCCCGAAAA
UUUUCGGGAAUCAUCUUCC
2573
1644
Yes
No
No

2574
AGAUGAUUCCCGAAAGGAA
UUCCUUUCGGGAAUCAUCU
2575
1647
Yes
No
No

2576
AUGAUUCCCGAAAGGAAAA
UUUUCCUUUCGGGAAUCAU
2577
1649
Yes
No
No

2578
UCCCGAAAGGAAAUGACUA
UAGUCAUUUCCUUUCGGGA
2579
1654
Yes
No
No

2580
CCCGAAAGGAAAUGACUGA
UCAGUCAUUUCCUUUCGGG
2581
1655
Yes
No
No

2582
CCGAAAGGAAAUGACUGCA
UGCAGUCAUUUCCUUUCGG
2583
1656
Yes
No
No

2584
CGAAAGGAAAUGACUGCAA
UUGCAGUCAUUUCCUUUCG
2585
1657
Yes
No
No

2586
AAUGACUGCAGCUUGUACA
UGUACAAGCUGCAGUCAUU
2587
1665
Yes
No
No

2588
AUGACUGCAGCUUGUACCA
UGGUACAAGCUGCAGUCAU
2589
1666
Yes
No
No

2590
UGACUGCAGCUUGUACCCA
UGGGUACAAGCUGCAGUCA
2591
1667
Yes
No
No

2592
CCCCGGAGAAGGAUCAUUA
UAAUGAUCCUUCUCCGGGG
2593
1684
Yes
No
No

2594
CCGGAGAAGGAUCAUUAAA
UUUAAUGAUCCUUCUCCGG
2595
1686
Yes
No
No

2596
GGAGAAGGAUCAUUAACCA
UGGUUAAUGAUCCUUCUCC
2597
1688
Yes
No
No

2598
GAGAAGGAUCAUUAACCUA
UAGGUUAAUGAUCCUUCUC
2599
1689
Yes
No
No

2600
AGAAGGAUCAUUAACCUCA
UGAGGUUAAUGAUCCUUCU
2601
1690
Yes
No
No

2602
GAAGGAUCAUUAACCUCAA
UUGAGGUUAAUGAUCCUUC
2603
1691
Yes
No
No

2604
AAGGAUCAUUAACCUCACA
UGUGAGGUUAAUGAUCCUU
2605
1692
Yes
No
No

2606
AGGAUCAUUAACCUCACUA
UAGUGAGGUUAAUGAUCCU
2607
1693
Yes
No
No

2608
AUCAUUAACCUCACUAGUA
UACUAGUGAGGUUAAUGAU
2609
1696
Yes
No
No

2610
UCAUUAACCUCACUAGUGA
UCACUAGUGAGGUUAAUGA
2611
1697
Yes
No
No

2612
CAUUAACCUCACUAGUGUA
UACACUAGUGAGGUUAAUG
2613
1698
Yes
No
No

2614
AUUAACCUCACUAGUGUUA
UAACACUAGUGAGGUUAAU
2615
1699
Yes
No
No

2616
UUAACCUCACUAGUGUUUA
UAAACACUAGUGAGGUUAA
2617
1700
Yes
No
No

2618
AACCUCACUAGUGUUUUGA
UCAAAACACUAGUGAGGUU
2619
1702
Yes
No
No

2620
ACCUCACUAGUGUUUUGAA
UUCAAAACACUAGUGAGGU
2621
1703
Yes
No
No

2622
CCUCACUAGUGUUUUGAGA
UCUCAAAACACUAGUGAGG
2623
1704
Yes
No
No

2624
CUCACUAGUGUUUUGAGUA
UACUCAAAACACUAGUGAG
2625
1705
Yes
No
No

2626
UCACUAGUGUUUUGAGUCA
UGACUCAAAACACUAGUGA
2627
1706
Yes
No
No

2628
CACUAGUGUUUUGAGUCUA
UAGACUCAAAACACUAGUG
2629
1707
Yes
No
No

2630
ACUAGUGUUUUGAGUCUCA
UGAGACUCAAAACACUAGU
2631
1708
Yes
No
No

2632
UAGUGUUUUGAGUCUCCAA
UUGGAGACUCAAAACACUA
2633
1710
Yes
No
No

2634
GUUUUGAGUCUCCAGGAAA
UUUCCUGGAGACUCAAAAC
2635
1714
Yes
No
Yes

2636
AUUAAUGAGCAGGGACAUA
UAUGUCCCUGCUCAUUAAU
2637
1735
No
No
No

2638
UAAUGAGCAGGGACAUGAA
UUCAUGUCCCUGCUCAUUA
2639
1737
No
No
No

2640
AAUGAGCAGGGACAUGAGA
UCUCAUGUCCCUGCUCAUU
2641
1738
No
No
No

2642
UGAGCAGGGACAUGAGGUA
UACCUCAUGUCCCUGCUCA
2643
1740
No
No
No

2644
GCAGGGACAUGAGGUUCUA
UAGAACCUCAUGUCCCUGC
2645
1743
No
No
No

2646
CAGGGACAUGAGGUUCUCA
UGAGAACCUCAUGUCCCUG
2647
1744
No
No
No

2648
AGGUUCUCCGGGAGAUGUA
UACAUCUCCCGGAGAACCU
2649
1754
No
No
No

2650
GGUUCUCCGGGAGAUGUUA
UAACAUCUCCCGGAGAACC
2651
1755
No
No
No

2652
GUUCUCCGGGAGAUGUUGA
UCAACAUCUCCCGGAGAAC
2653
1756
No
No
No

2654
UUCUCCGGGAGAUGUUGCA
UGCAACAUCUCCCGGAGAA
2655
1757
No
No
No

2656
UCUCCGGGAGAUGUUGCAA
UUGCAACAUCUCCCGGAGA
2657
1758
No
No
No

2658
CUCCGGGAGAUGUUGCAUA
UAUGCAACAUCUCCCGGAG
2659
1759
No
No
No

2660
UCCGGGAGAUGUUGCAUAA
UUAUGCAACAUCUCCCGGA
2661
1760
No
No
No

2662
CCGGGAGAUGUUGCAUAAA
UUUAUGCAACAUCUCCCGG
2663
1761
No
No
No

2664
GGAGAUGUUGCAUAACCAA
UUGGUUAUGCAACAUCUCC
2665
1764
Yes
No
No

2666
CCUUCGUGGGCUGUGUGAA
UUCACACAGCCCACGAAGG
2667
1784
Yes
No
No

2668
CUUCGUGGGCUGUGUGAAA
UUUCACACAGCCCACGAAG
2669
1785
Yes
No
No

2670
UUCGUGGGCUGUGUGAAUA
UAUUCACACAGCCCACGAA
2671
1786
Yes
No
No

2672
UCGUGGGCUGUGUGAAUCA
UGAUUCACACAGCCCACGA
2673
1787
Yes
No
No

2674
GUGGGCUGUGUGAAUCCUA
UAGGAUUCACACAGCCCAC
2675
1789
Yes
Yes
Yes

2676
UGGGCUGUGUGAAUCCUCA
UGAGGAUUCACACAGCCCA
2677
1790
Yes
Yes
Yes

2678
GGGCUGUGUGAAUCCUCAA
UUGAGGAUUCACACAGCCC
2679
1791
Yes
Yes
Yes

2680
UCAGUGGGCCUUGGCACAA
UUGUGCCAAGGCCCACUGA
2681
1806
Yes
Yes
Yes

2682
GCCUUGGCACAGCAUCAAA
UUUGAUGCUGUGCCAAGGC
2683
1813
No
No
No

2684
CCUUGGCACAGCAUCAAAA
UUUUGAUGCUGUGCCAAGG
2685
1814
No
No
No

2686
CUUGGCACAGCAUCAAACA
UGUUUGAUGCUGUGCCAAG
2687
1815
No
No
No

2688
UUGGCACAGCAUCAAACCA
UGGUUUGAUGCUGUGCCAA
2689
1816
No
No
No

2690
GCACAGCAUCAAACCAAGA
UCUUGGUUUGAUGCUGUGC
2691
1819
No
No
No

2692
AGCAUCAAACCAAGUUAUA
UAUAACUUGGUUUGAUGCU
2693
1823
No
No
No

2694
GCAUCAAACCAAGUUAUAA
UUAUAACUUGGUUUGAUGC
2695
1824
No
No
No

2696
AUCAAACCAAGUUAUACCA
UGGUAUAACUUGGUUUGAU
2697
1826
No
No
No

2698
UCAAACCAAGUUAUACCUA
UAGGUAUAACUUGGUUUGA
2699
1827
No
No
No

2700
AAACCAAGUUAUACCUUCA
UGAAGGUAUAACUUGGUUU
2701
1829
No
No
No

2702
AACCAAGUUAUACCUUCUA
UAGAAGGUAUAACUUGGUU
2703
1830
No
No
No

2704
AGUUAUACCUUCUCAACAA
UUGUUGAGAAGGUAUAACU
2705
1835
No
No
No

2706
GUUAUACCUUCUCAACACA
UGUGUUGAGAAGGUAUAAC
2707
1836
No
No
No

2708
UAUACCUUCUCAACACCAA
UUGGUGUUGAGAAGGUAUA
2709
1838
No
No
No

2710
UCUCAACACCACCAAGCUA
UAGCUUGGUGGUGUUGAGA
2711
1845
No
No
No

2712
UCAACACCACCAAGCUUAA
UUAAGCUUGGUGGUGUUGA
2713
1847
No
No
No

2714
CAACACCACCAAGCUUAGA
UCUAAGCUUGGUGGUGUUG
2715
1848
No
No
No

2716
AACACCACCAAGCUUAGUA
UACUAAGCUUGGUGGUGUU
2717
1849
No
No
No

2718
ACACCACCAAGCUUAGUGA
UCACUAAGCUUGGUGGUGU
2719
1850
No
No
No

2720
ACCACCAAGCUUAGUGAAA
UUUCACUAAGCUUGGUGGU
2721
1852
Yes
No
No

2722
CCACCAAGCUUAGUGAAGA
UCUUCACUAAGCUUGGUGG
2723
1853
Yes
No
No

2724
ACCAAGCUUAGUGAAGAAA
UUUCUUCACUAAGCUUGGU
2725
1855
Yes
No
No

2726
CCAAGCUUAGUGAAGAACA
UGUUCUUCACUAAGCUUGG
2727
1856
Yes
No
No

2728
AAGCUUAGUGAAGAACUGA
UCAGUUCUUCACUAAGCUU
2729
1858
Yes
No
No

2730
AGCUUAGUGAAGAACUGUA
UACAGUUCUUCACUAAGCU
2731
1859
Yes
No
No

2732
CUUAGUGAAGAACUGUUCA
UGAACAGUUCUUCACUAAG
2733
1861
Yes
No
No

2734
AGUGAAGAACUGUUCUACA
UGUAGAACAGUUCUUCACU
2735
1864
Yes
No
No

2736
GAAGAACUGUUCUACCAGA
UCUGGUAGAACAGUUCUUC
2737
1867
Yes
No
No

2738
AAGAACUGUUCUACCAGAA
UUCUGGUAGAACAGUUCUU
2739
1868
Yes
No
No

2740
AGAACUGUUCUACCAGAUA
UAUCUGGUAGAACAGUUCU
2741
1869
Yes
No
No

2742
AACUGUUCUACCAGAUACA
UGUAUCUGGUAGAACAGUU
2743
1871
Yes
No
No

2744
ACUGUUCUACCAGAUACUA
UAGUAUCUGGUAGAACAGU
2745
1872
Yes
No
No

2746
UGUUCUACCAGAUACUCAA
UUGAGUAUCUGGUAGAACA
2747
1874
Yes
Yes
No

2748
GUUCUACCAGAUACUCAUA
UAUGAGUAUCUGGUAGAAC
2749
1875
Yes
Yes
No

2750
UCUACCAGAUACUCAUUUA
UAAAUGAGUAUCUGGUAGA
2751
1877
Yes
Yes
Yes

2752
ACCAGAUACUCAUUUAUGA
UCAUAAAUGAGUAUCUGGU
2753
1880
Yes
Yes
No

2754
CCAGAUACUCAUUUAUGAA
UUCAUAAAUGAGUAUCUGG
2755
1881
Yes
Yes
No

2756
AGAUACUCAUUUAUGAUUA
UAAUCAUAAAUGAGUAUCU
2757
1883
Yes
Yes
No

2758
GAUACUCAUUUAUGAUUUA
UAAAUCAUAAAUGAGUAUC
2759
1884
Yes
Yes
No

2760
UACUCAUUUAUGAUUUUGA
UCAAAAUCAUAAAUGAGUA
2761
1886
Yes
Yes
No

2762
CUCAUUUAUGAUUUUGCCA
UGGCAAAAUCAUAAAUGAG
2763
1888
Yes
Yes
No

2764
UCAUUUAUGAUUUUGCCAA
UUGGCAAAAUCAUAAAUGA
2765
1889
Yes
Yes
No

2766
UUAUGAUUUUGCCAAUUUA
UAAAUUGGCAAAAUCAUAA
2767
1893
Yes
No
No

2768
UGAUUUUGCCAAUUUUGGA
UCCAAAAUUGGCAAAAUCA
2769
1896
Yes
No
No

2770
AUUUUGCCAAUUUUGGUGA
UCACCAAAAUUGGCAAAAU
2771
1898
Yes
No
No

2772
UUUGCCAAUUUUGGUGUUA
UAACACCAAAAUUGGCAAA
2773
1900
Yes
No
No

2774
GCCAAUUUUGGUGUUCUCA
UGAGAACACCAAAAUUGGC
2775
1903
No
No
No

2776
CAAUUUUGGUGUUCUCAGA
UCUGAGAACACCAAAAUUG
2777
1905
No
No
No

2778
AUUUUGGUGUUCUCAGGUA
UACCUGAGAACACCAAAAU
2779
1907
No
No
No

2780
UUUUGGUGUUCUCAGGUUA
UAACCUGAGAACACCAAAA
2781
1908
No
No
No

2782
UUUGGUGUUCUCAGGUUAA
UUAACCUGAGAACACCAAA
2783
1909
No
No
No

2784
GGUGUUCUCAGGUUAUCGA
UCGAUAACCUGAGAACACC
2785
1912
No
No
No

2786
GUUCUCAGGUUAUCGGAGA
UCUCCGAUAACCUGAGAAC
2787
1915
No
No
No

2788
GAGCCAGCACCGCUCUUUA
UAAAGAGCGGUGCUGGCUC
2789
1930
No
No
No

2790
GCCAGCACCGCUCUUUGAA
UUCAAAGAGCGGUGCUGGC
2791
1932
No
No
No

2792
CCAGCACCGCUCUUUGACA
UGUCAAAGAGCGGUGCUGG
2793
1933
No
No
No

2794
CACCGCUCUUUGACCUUGA
UCAAGGUCAAAGAGCGGUG
2795
1937
No
No
No

2796
UUUGACCUUGCCAUGCUUA
UAAGCAUGGCAAGGUCAAA
2797
1945
No
No
No

2798
CUUGCCAUGCUUGCCUUAA
UUAAGGCAAGCAUGGCAAG
2799
1951
No
No
No

2800
UGCCAUGCUUGCCUUAGAA
UUCUAAGGCAAGCAUGGCA
2801
1953
No
No
No

2802
CCAUGCUUGCCUUAGAUAA
UUAUCUAAGGCAAGCAUGG
2803
1955
Yes
No
No

2804
CAUGCUUGCCUUAGAUAGA
UCUAUCUAAGGCAAGCAUG
2805
1956
Yes
No
No

2806
GCUUGCCUUAGAUAGUCCA
UGGACUAUCUAAGGCAAGC
2807
1959
Yes
No
No

2808
UUGCCUUAGAUAGUCCAGA
UCUGGACUAUCUAAGGCAA
2809
1961
Yes
No
No

2810
UGCCUUAGAUAGUCCAGAA
UUCUGGACUAUCUAAGGCA
2811
1962
Yes
No
No

2812
UUAGAUAGUCCAGAGAGUA
UACUCUCUGGACUAUCUAA
2813
1966
Yes
No
No

2814
GAGGAAGAUGGUCCCAAAA
UUUUGGGACCAUCUUCCUC
2815
1993
Yes
No
No

2816
AAGAUGGUCCCAAAGAAGA
UCUUCUUUGGGACCAUCUU
2817
1997
Yes
No
No

2818
AGAUGGUCCCAAAGAAGGA
UCCUUCUUUGGGACCAUCU
2819
1998
Yes
No
No

2820
GGUCCCAAAGAAGGACUUA
UAAGUCCUUCUUUGGGACC
2821
2002
Yes
No
No

2822
CCCAAAGAAGGACUUGCUA
UAGCAAGUCCUUCUUUGGG
2823
2005
Yes
No
No

2824
AAAGAAGGACUUGCUGAAA
UUUCAGCAAGUCCUUCUUU
2825
2008
Yes
No
No

2826
AGAAGGACUUGCUGAAUAA
UUAUUCAGCAAGUCCUUCU
2827
2010
Yes
No
No

2828
AAGGACUUGCUGAAUACAA
UUGUAUUCAGCAAGUCCUU
2829
2012
Yes
No
No

2830
AGGACUUGCUGAAUACAUA
UAUGUAUUCAGCAAGUCCU
2831
2013
Yes
No
No

2832
GGACUUGCUGAAUACAUUA
UAAUGUAUUCAGCAAGUCC
2833
2014
Yes
No
No

2834
GACUUGCUGAAUACAUUGA
UCAAUGUAUUCAGCAAGUC
2835
2015
Yes
No
No

2836
ACUUGCUGAAUACAUUGUA
UACAAUGUAUUCAGCAAGU
2837
2016
Yes
No
No

2838
CUUGCUGAAUACAUUGUUA
UAACAAUGUAUUCAGCAAG
2839
2017
Yes
No
No

2840
GCUGAAUACAUUGUUGAGA
UCUCAACAAUGUAUUCAGC
2841
2020
Yes
No
No

2842
CUGAAUACAUUGUUGAGUA
UACUCAACAAUGUAUUCAG
2843
2021
Yes
No
No

2844
UGAAUACAUUGUUGAGUUA
UAACUCAACAAUGUAUUCA
2845
2022
Yes
No
No

2846
AAUACAUUGUUGAGUUUCA
UGAAACUCAACAAUGUAUU
2847
2024
Yes
No
No

2848
UACAUUGUUGAGUUUCUGA
UCAGAAACUCAACAAUGUA
2849
2026
Yes
Yes
No

2850
UUGAGUUUCUGAAGAAGAA
UUCUUCUUCAGAAACUCAA
2851
2033
Yes
Yes
No

2852
GAGUUUCUGAAGAAGAAGA
UCUUCUUCUUCAGAAACUC
2853
2035
Yes
No
No

2854
GUUUCUGAAGAAGAAGGCA
UGCCUUCUUCUUCAGAAAC
2855
2037
Yes
No
No

2856
AGAAGGCUGAGAUGCUUGA
UCAAGCAUCUCAGCCUUCU
2857
2048
No
No
No

2858
GAAGGCUGAGAUGCUUGCA
UGCAAGCAUCUCAGCCUUC
2859
2049
No
No
No

2860
AAGGCUGAGAUGCUUGCAA
UUGCAAGCAUCUCAGCCUU
2861
2050
No
No
No

2862
GGCUGAGAUGCUUGCAGAA
UUCUGCAAGCAUCUCAGCC
2863
2052
No
No
No

2864
GCUGAGAUGCUUGCAGACA
UGUCUGCAAGCAUCUCAGC
2865
2053
No
No
No

2866
CUGAGAUGCUUGCAGACUA
UAGUCUGCAAGCAUCUCAG
2867
2054
No
No
No

2868
UGAGAUGCUUGCAGACUAA
UUAGUCUGCAAGCAUCUCA
2869
2055
No
No
No

2870
GAGAUGCUUGCAGACUAUA
UAUAGUCUGCAAGCAUCUC
2871
2056
Yes
Yes
No

2872
AUGCUUGCAGACUAUUUCA
UGAAAUAGUCUGCAAGCAU
2873
2059
Yes
Yes
Yes

2874
GCUUGCAGACUAUUUCUCA
UGAGAAAUAGUCUGCAAGC
2875
2061
Yes
Yes
Yes

2876
CUUGCAGACUAUUUCUCUA
UAGAGAAAUAGUCUGCAAG
2877
2062
Yes
Yes
Yes

2878
UUGCAGACUAUUUCUCUUA
UAAGAGAAAUAGUCUGCAA
2879
2063
Yes
No
No

2880
UGCAGACUAUUUCUCUUUA
UAAAGAGAAAUAGUCUGCA
2881
2064
Yes
No
No

2882
CAGACUAUUUCUCUUUGGA
UCCAAAGAGAAAUAGUCUG
2883
2066
Yes
No
No

2884
AUUUCUCUUUGGAAAUUGA
UCAAUUUCCAAAGAGAAAU
2885
2072
Yes
No
No

2886
AAUUGAUGAGGAAGGGAAA
UUUCCCUUCCUCAUCAAUU
2887
2085
Yes
No
Yes

2888
AUGAGGAAGGGAACCUGAA
UUCAGGUUCCCUUCCUCAU
2889
2090
Yes
Yes
Yes

2890
GAAGGGAACCUGAUUGGAA
UUCCAAUCAGGUUCCCUUC
2891
2095
Yes
Yes
Yes

2892
AAGGGAACCUGAUUGGAUA
UAUCCAAUCAGGUUCCCUU
2893
2096
Yes
Yes
Yes

2894
GGGAACCUGAUUGGAUUAA
UUAAUCCAAUCAGGUUCCC
2895
2098
Yes
Yes
Yes

2896
CCUGAUUGGAUUACCCCUA
UAGGGGUAAUCCAAUCAGG
2897
2103
Yes
No
No

2898
UUGGAUUACCCCUUCUGAA
UUCAGAAGGGGUAAUCCAA
2899
2108
Yes
No
No

2900
UGGAUUACCCCUUCUGAUA
UAUCAGAAGGGGUAAUCCA
2901
2109
Yes
No
No

2902
GGAUUACCCCUUCUGAUUA
UAAUCAGAAGGGGUAAUCC
2903
2110
Yes
No
No

2904
AUUACCCCUUCUGAUUGAA
UUCAAUCAGAAGGGGUAAU
2905
2112
Yes
No
No

2906
UACCCCUUCUGAUUGACAA
UUGUCAAUCAGAAGGGGUA
2907
2114
Yes
No
No

2908
CCUUCUGAUUGACAACUAA
UUAGUUGUCAAUCAGAAGG
2909
2118
Yes
No
No

2910
UUCUGAUUGACAACUAUGA
UCAUAGUUGUCAAUCAGAA
2911
2120
Yes
No
No

2912
UGAUUGACAACUAUGUGCA
UGCACAUAGUUGUCAAUCA
2913
2123
Yes
No
No

2914
GAUUGACAACUAUGUGCCA
UGGCACAUAGUUGUCAAUC
2915
2124
Yes
No
No

2916
CUUUGGAGGGACUGCCUAA
UUAGGCAGUCCCUCCAAAG
2917
2144
Yes
Yes
Yes

2918
UUUGGAGGGACUGCCUAUA
UAUAGGCAGUCCCUCCAAA
2919
2145
Yes
Yes
Yes

2920
UUGGAGGGACUGCCUAUCA
UGAUAGGCAGUCCCUCCAA
2921
2146
Yes
Yes
Yes

2922
UGGAGGGACUGCCUAUCUA
UAGAUAGGCAGUCCCUCCA
2923
2147
Yes
Yes
Yes

2924
GGAGGGACUGCCUAUCUUA
UAAGAUAGGCAGUCCCUCC
2925
2148
Yes
Yes
Yes

2926
GAGGGACUGCCUAUCUUCA
UGAAGAUAGGCAGUCCCUC
2927
2149
Yes
Yes
Yes

2928
GGGACUGCCUAUCUUCAUA
UAUGAAGAUAGGCAGUCCC
2929
2151
Yes
Yes
Yes

2930
GGACUGCCUAUCUUCAUUA
UAAUGAAGAUAGGCAGUCC
2931
2152
Yes
Yes
Yes

2932
ACUGCCUAUCUUCAUUCUA
UAGAAUGAAGAUAGGCAGU
2933
2154
Yes
Yes
Yes

2934
CUGCCUAUCUUCAUUCUUA
UAAGAAUGAAGAUAGGCAG
2935
2155
Yes
Yes
Yes

2936
GCCUAUCUUCAUUCUUCGA
UCGAAGAAUGAAGAUAGGC
2937
2157
Yes
Yes
Yes

2938
CCUAUCUUCAUUCUUCGAA
UUCGAAGAAUGAAGAUAGG
2939
2158
Yes
Yes
Yes

2940
CUAUCUUCAUUCUUCGACA
UGUCGAAGAAUGAAGAUAG
2941
2159
Yes
Yes
Yes

2942
UAUCUUCAUUCUUCGACUA
UAGUCGAAGAAUGAAGAUA
2943
2160
Yes
Yes
Yes

2944
AUCUUCAUUCUUCGACUAA
UUAGUCGAAGAAUGAAGAU
2945
2161
Yes
No
No

2946
AUUCUUCGACUAGCCACUA
UAGUGGCUAGUCGAAGAAU
2947
2167
Yes
No
No

2948
UCGACUAGCCACUGAGGUA
UACCUCAGUGGCUAGUCGA
2949
2172
Yes
No
No

2950
GACUAGCCACUGAGGUGAA
UUCACCUCAGUGGCUAGUC
2951
2174
No
No
No

2952
ACUAGCCACUGAGGUGAAA
UUUCACCUCAGUGGCUAGU
2953
2175
No
No
No

2954
CUAGCCACUGAGGUGAAUA
UAUUCACCUCAGUGGCUAG
2955
2176
No
No
No

2956
AGCCACUGAGGUGAAUUGA
UCAAUUCACCUCAGUGGCU
2957
2178
No
No
No

2958
GCCACUGAGGUGAAUUGGA
UCCAAUUCACCUCAGUGGC
2959
2179
No
Yes
No

2960
CCACUGAGGUGAAUUGGGA
UCCCAAUUCACCUCAGUGG
2961
2180
No
Yes
No

2962
CACUGAGGUGAAUUGGGAA
UUCCCAAUUCACCUCAGUG
2963
2181
No
Yes
No

2964
ACUGAGGUGAAUUGGGACA
UGUCCCAAUUCACCUCAGU
2965
2182
No
No
No

2966
GGUGAAUUGGGACGAAGAA
UUCUUCGUCCCAAUUCACC
2967
2187
Yes
No
No

2968
UGAAUUGGGACGAAGAAAA
UUUUCUUCGUCCCAAUUCA
2969
2189
Yes
No
No

2970
AAUUGGGACGAAGAAAAGA
UCUUUUCUUCGUCCCAAUU
2971
2191
Yes
No
No

2972
AUUGGGACGAAGAAAAGGA
UCCUUUUCUUCGUCCCAAU
2973
2192
Yes
No
No

2974
UUGGGACGAAGAAAAGGAA
UUCCUUUUCUUCGUCCCAA
2975
2193
Yes
No
No

2976
ACGAAGAAAAGGAAUGUUA
UAACAUUCCUUUUCUUCGU
2977
2198
Yes
No
No

2978
AGAAAAGGAAUGUUUUGAA
UUCAAAACAUUCCUUUUCU
2979
2202
Yes
No
No

2980
GGAAUGUUUUGAAAGCCUA
UAGGCUUUCAAAACAUUCC
2981
2208
Yes
No
No

2982
GAAUGUUUUGAAAGCCUCA
UGAGGCUUUCAAAACAUUC
2983
2209
Yes
No
No

2984
AAUGUUUUGAAAGCCUCAA
UUGAGGCUUUCAAAACAUU
2985
2210
Yes
No
No

2986
GUUUUGAAAGCCUCAGUAA
UUACUGAGGCUUUCAAAAC
2987
2213
Yes
No
No

2988
UUUUGAAAGCCUCAGUAAA
UUUACUGAGGCUUUCAAAA
2989
2214
Yes
No
No

2990
UUGAAAGCCUCAGUAAAGA
UCUUUACUGAGGCUUUCAA
2991
2216
Yes
No
No

2992
GCCUCAGUAAAGAAUGCGA
UCGCAUUCUUUACUGAGGC
2993
2222
No
No
No

2994
CUCAGUAAAGAAUGCGCUA
UAGCGCAUUCUUUACUGAG
2995
2224
No
No
No

2996
UCAGUAAAGAAUGCGCUAA
UUAGCGCAUUCUUUACUGA
2997
2225
No
No
No

2998
GUAAAGAAUGCGCUAUGUA
UACAUAGCGCAUUCUUUAC
2999
2228
No
No
No

3000
AAGAAUGCGCUAUGUUCUA
UAGAACAUAGCGCAUUCUU
3001
2231
No
No
No

3002
AGAAUGCGCUAUGUUCUAA
UUAGAACAUAGCGCAUUCU
3003
2232
No
No
No

3004
GAAUGCGCUAUGUUCUAUA
UAUAGAACAUAGCGCAUUC
3005
2233
No
No
No

3006
AAUGCGCUAUGUUCUAUUA
UAAUAGAACAUAGCGCAUU
3007
2234
No
No
No

3008
UGCGCUAUGUUCUAUUCCA
UGGAAUAGAACAUAGCGCA
3009
2236
No
No
No

3010
GCGCUAUGUUCUAUUCCAA
UUGGAAUAGAACAUAGCGC
3011
2237
No
No
No

3012
CUAUGUUCUAUUCCAUCCA
UGGAUGGAAUAGAACAUAG
3013
2240
No
No
No

3014
GUUCUAUUCCAUCCGGAAA
UUUCCGGAUGGAAUAGAAC
3015
2244
No
No
No

3016
UUCCAUCCGGAAGCAGUAA
UUACUGCUUCCGGAUGGAA
3017
2250
No
No
No

3018
UCCAUCCGGAAGCAGUACA
UGUACUGCUUCCGGAUGGA
3019
2251
Yes
No
No

3020
CAUCCGGAAGCAGUACAUA
UAUGUACUGCUUCCGGAUG
3021
2253
Yes
No
No

3022
UCCGGAAGCAGUACAUAUA
UAUAUGUACUGCUUCCGGA
3023
2255
Yes
No
No

3024
CCGGAAGCAGUACAUAUCA
UGAUAUGUACUGCUUCCGG
3025
2256
Yes
No
No

3026
CGGAAGCAGUACAUAUCUA
UAGAUAUGUACUGCUUCCG
3027
2257
Yes
No
No

3028
GGAAGCAGUACAUAUCUGA
UCAGAUAUGUACUGCUUCC
3029
2258
Yes
No
No

3030
GAAGCAGUACAUAUCUGAA
UUCAGAUAUGUACUGCUUC
3031
2259
Yes
No
No

3032
AAGCAGUACAUAUCUGAGA
UCUCAGAUAUGUACUGCUU
3033
2260
Yes
No
No

3034
AGCAGUACAUAUCUGAGGA
UCCUCAGAUAUGUACUGCU
3035
2261
Yes
No
No

3036
AGUACAUAUCUGAGGAGUA
UACUCCUCAGAUAUGUACU
3037
2264
Yes
No
No

3038
UCAGGCCAGCAGAGUGAAA
UUUCACUCUGCUGGCCUGA
3039
2290
Yes
No
No

3040
AGGCCAGCAGAGUGAAGUA
UACUUCACUCUGCUGGCCU
3041
2292
Yes
No
No

3042
GGCCAGCAGAGUGAAGUGA
UCACUUCACUCUGCUGGCC
3043
2293
Yes
No
No

3044
GCCAGCAGAGUGAAGUGCA
UGCACUUCACUCUGCUGGC
3045
2294
Yes
No
No

3046
GAAGUGCCUGGCUCCAUUA
UAAUGGAGCCAGGCACUUC
3047
2305
Yes
No
No

3048
AAGUGCCUGGCUCCAUUCA
UGAAUGGAGCCAGGCACUU
3049
2306
Yes
No
No

3050
UGCCUGGCUCCAUUCCAAA
UUUGGAAUGGAGCCAGGCA
3051
2309
Yes
No
No

3052
UCCAAACUCCUGGAAGUGA
UCACUUCCAGGAGUUUGGA
3053
2322
Yes
No
No

3054
CUGGAAGUGGACUGUGGAA
UUCCACAGUCCACUUCCAG
3055
2331
Yes
Yes
Yes

3056
GGAAGUGGACUGUGGAACA
UGUUCCACAGUCCACUUCC
3057
2333
Yes
No
Yes

3058
GAAGUGGACUGUGGAACAA
UUGUUCCACAGUCCACUUC
3059
2334
Yes
No
Yes

3060
AAGUGGACUGUGGAACACA
UGUGUUCCACAGUCCACUU
3061
2335
Yes
No
Yes

3062
AGUGGACUGUGGAACACAA
UUGUGUUCCACAGUCCACU
3063
2336
Yes
No
Yes

3064
GGACUGUGGAACACAUUGA
UCAAUGUGUUCCACAGUCC
3065
2339
Yes
No
No

3066
GACUGUGGAACACAUUGUA
UACAAUGUGUUCCACAGUC
3067
2340
Yes
No
No

3068
ACUGUGGAACACAUUGUCA
UGACAAUGUGUUCCACAGU
3069
2341
No
No
No

3070
CUGUGGAACACAUUGUCUA
UAGACAAUGUGUUCCACAG
3071
2342
No
No
No

3072
GUGGAACACAUUGUCUAUA
UAUAGACAAUGUGUUCCAC
3073
2344
No
No
No

3074
UGGAACACAUUGUCUAUAA
UUAUAGACAAUGUGUUCCA
3075
2345
No
No
No

3076
AACACAUUGUCUAUAAAGA
UCUUUAUAGACAAUGUGUU
3077
2348
No
No
No

3078
ACACAUUGUCUAUAAAGCA
UGCUUUAUAGACAAUGUGU
3079
2349
No
No
No

3080
CACAUUGUCUAUAAAGCCA
UGGCUUUAUAGACAAUGUG
3081
2350
No
No
No

3082
ACAUUGUCUAUAAAGCCUA
UAGGCUUUAUAGACAAUGU
3083
2351
No
No
No

3084
GCCUUGCGCUCACACAUUA
UAAUGUGUGAGCGCAAGGC
3085
2365
Yes
No
No

3086
CCUUGCGCUCACACAUUCA
UGAAUGUGUGAGCGCAAGG
3087
2366
Yes
No
No

3088
CUUGCGCUCACACAUUCUA
UAGAAUGUGUGAGCGCAAG
3089
2367
Yes
No
No

3090
UGCGCUCACACAUUCUGCA
UGCAGAAUGUGUGAGCGCA
3091
2369
Yes
No
No

3092
CGCUCACACAUUCUGCCUA
UAGGCAGAAUGUGUGAGCG
3093
2371
Yes
No
No

3094
CACAUUCUGCCUCCUAAAA
UUUUAGGAGGCAGAAUGUG
3095
2377
Yes
No
No

3096
CAUUCUGCCUCCUAAACAA
UUGUUUAGGAGGCAGAAUG
3097
2379
Yes
No
No

3098
UUCUGCCUCCUAAACAUUA
UAAUGUUUAGGAGGCAGAA
3099
2381
Yes
No
No

3100
UCUGCCUCCUAAACAUUUA
UAAAUGUUUAGGAGGCAGA
3101
2382
Yes
No
No

3102
CAGAAGAUGGAAAUAUCCA
UGGAUAUUUCCAUCUUCUG
3103
2402
Yes
No
No

3104
AAGAUGGAAAUAUCCUGCA
UGCAGGAUAUUUCCAUCUU
3105
2405
Yes
No
No

3106
AGAUGGAAAUAUCCUGCAA
UUGCAGGAUAUUUCCAUCU
3107
2406
Yes
No
No

3108
GGAAAUAUCCUGCAGCUUA
UAAGCUGCAGGAUAUUUCC
3109
2410
No
No
No

3110
AAUAUCCUGCAGCUUGCUA
UAGCAAGCUGCAGGAUAUU
3111
2413
No
No
No

3112
AUAUCCUGCAGCUUGCUAA
UUAGCAAGCUGCAGGAUAU
3113
2414
No
No
No

3114
UAUCCUGCAGCUUGCUAAA
UUUAGCAAGCUGCAGGAUA
3115
2415
No
No
No

3116
CCUGCAGCUUGCUAACCUA
UAGGUUAGCAAGCUGCAGG
3117
2418
No
No
No

3118
GCUAACCUGCCUGAUCUAA
UUAGAUCAGGCAGGUUAGC
3119
2428
Yes
No
No

3120
CUAACCUGCCUGAUCUAUA
UAUAGAUCAGGCAGGUUAG
3121
2429
Yes
No
No

3122
UAACCUGCCUGAUCUAUAA
UUAUAGAUCAGGCAGGUUA
3123
2430
Yes
No
No

3124
CCUGCCUGAUCUAUACAAA
UUUGUAUAGAUCAGGCAGG
3125
2433
Yes
No
No

3126
CUGCCUGAUCUAUACAAAA
UUUUGUAUAGAUCAGGCAG
3127
2434
Yes
No
No

3128
UGCCUGAUCUAUACAAAGA
UCUUUGUAUAGAUCAGGCA
3129
2435
Yes
No
No

3130
GCCUGAUCUAUACAAAGUA
UACUUUGUAUAGAUCAGGC
3131
2436
Yes
No
No

3132
CCUGAUCUAUACAAAGUCA
UGACUUUGUAUAGAUCAGG
3133
2437
Yes
No
No

3134
UGAUCUAUACAAAGUCUUA
UAAGACUUUGUAUAGAUCA
3135
2439
Yes
No
No

3136
AUCUAUACAAAGUCUUUGA
UCAAAGACUUUGUAUAGAU
3137
2441
Yes
Yes
No

3138
UCUAUACAAAGUCUUUGAA
UUCAAAGACUUUGUAUAGA
3139
2442
Yes
Yes
No

3140
CUAUACAAAGUCUUUGAGA
UCUCAAAGACUUUGUAUAG
3141
2443
Yes
Yes
No

3142
UAUACAAAGUCUUUGAGAA
UUCUCAAAGACUUUGUAUA
3143
2444
Yes
No
No

3144
UACAAAGUCUUUGAGAGGA
UCCUCUCAAAGACUUUGUA
3145
2446
Yes
No
No

3146
ACAAAGUCUUUGAGAGGUA
UACCUCUCAAAGACUUUGU
3147
2447
Yes
No
No

3148
AAAGUCUUUGAGAGGUGUA
UACACCUCUCAAAGACUUU
3149
2449
Yes
No
No

3150
AAGUCUUUGAGAGGUGUUA
UAACACCUCUCAAAGACUU
3151
2450
Yes
No
No

3152
AGUCUUUGAGAGGUGUUAA
UUAACACCUCUCAAAGACU
3153
2451
Yes
No
No

3154
GUCUUUGAGAGGUGUUAAA
UUUAACACCUCUCAAAGAC
3155
2452
Yes
No
No

3156
UCUUUGAGAGGUGUUAAAA
UUUUAACACCUCUCAAAGA
3157
2453
Yes
No
No

3158
UUGAGAGGUGUUAAAUAUA
UAUAUUUAACACCUCUCAA
3159
2456
Yes
No
No

3160
UGAGAGGUGUUAAAUAUGA
UCAUAUUUAACACCUCUCA
3161
2457
Yes
No
No

3162
GAGAGGUGUUAAAUAUGGA
UCCAUAUUUAACACCUCUC
3163
2458
Yes
No
No

3164
AGAGGUGUUAAAUAUGGUA
UACCAUAUUUAACACCUCU
3165
2459
Yes
No
No

3166
GAGGUGUUAAAUAUGGUUA
UAACCAUAUUUAACACCUC
3167
2460
No
No
No

3168
AGGUGUUAAAUAUGGUUAA
UUAACCAUAUUUAACACCU
3169
2461
No
No
No

3170
GGUGUUAAAUAUGGUUAUA
UAUAACCAUAUUUAACACC
3171
2462
No
No
No

3172
UGUUAAAUAUGGUUAUUUA
UAAAUAACCAUAUUUAACA
3173
2464
No
No
No

3174
UAUGGUUAUUUAUGCACUA
UAGUGCAUAAAUAACCAUA
3175
2471
No
No
No

3176
AUGGUUAUUUAUGCACUGA
UCAGUGCAUAAAUAACCAU
3177
2472
No
No
No

3178
UGGUUAUUUAUGCACUGUA
UACAGUGCAUAAAUAACCA
3179
2473
No
No
No

3180
UUAUGCACUGUGGGAUGUA
UACAUCCCACAGUGCAUAA
3181
2480
Yes
No
No

3182
CACUGUGGGAUGUGUUCUA
UAGAACACAUCCCACAGUG
3183
2485
No
No
No

3184
CUGUGGGAUGUGUUCUUCA
UGAAGAACACAUCCCACAG
3185
2487
No
No
No

3186
UGUGGGAUGUGUUCUUCUA
UAGAAGAACACAUCCCACA
3187
2488
No
No
No

3188
GUGGGAUGUGUUCUUCUUA
UAAGAAGAACACAUCCCAC
3189
2489
No
No
No

3190
UGGGAUGUGUUCUUCUUUA
UAAAGAAGAACACAUCCCA
3191
2490
No
No
No

3192
GGGAUGUGUUCUUCUUUCA
UGAAAGAAGAACACAUCCC
3193
2491
No
No
No

3194
GGAUGUGUUCUUCUUUCUA
UAGAAAGAAGAACACAUCC
3195
2492
No
No
No

3196
GAUGUGUUCUUCUUUCUCA
UGAGAAAGAAGAACACAUC
3197
2493
No
No
No

3198
UGUGUUCUUCUUUCUCUGA
UCAGAGAAAGAAGAACACA
3199
2495
No
No
No

3200
GUGUUCUUCUUUCUCUGUA
UACAGAGAAAGAAGAACAC
3201
2496
No
No
No

3202
UGUUCUUCUUUCUCUGUAA
UUACAGAGAAAGAAGAACA
3203
2497
No
No
No

3204
UUCUUUCUCUGUAUUCCGA
UCGGAAUACAGAGAAAGAA
3205
2502
Yes
No
No

3206
UCUUUCUCUGUAUUCCGAA
UUCGGAAUACAGAGAAAGA
3207
2503
Yes
No
No

3208
UUUCUCUGUAUUCCGAUAA
UUAUCGGAAUACAGAGAAA
3209
2505
Yes
No
No

3210
UUCUCUGUAUUCCGAUACA
UGUAUCGGAAUACAGAGAA
3211
2506
Yes
No
No

3212
UCUCUGUAUUCCGAUACAA
UUGUAUCGGAAUACAGAGA
3213
2507
Yes
No
No

3214
CUCUGUAUUCCGAUACAAA
UUUGUAUCGGAAUACAGAG
3215
2508
Yes
No
No

3216
UGUAUUCCGAUACAAAGUA
UACUUUGUAUCGGAAUACA
3217
2511
Yes
No
No

3218
UAUUCCGAUACAAAGUGUA
UACACUUUGUAUCGGAAUA
3219
2513
Yes
No
No

3220
UUCCGAUACAAAGUGUUGA
UCAACACUUUGUAUCGGAA
3221
2515
Yes
No
No

3222
CCGAUACAAAGUGUUGUAA
UUACAACACUUUGUAUCGG
3223
2517
Yes
No
No

3224
GAUACAAAGUGUUGUAUCA
UGAUACAACACUUUGUAUC
3225
2519
Yes
No
No

3226
AUACAAAGUGUUGUAUCAA
UUGAUACAACACUUUGUAU
3227
2520
Yes
No
No

3228
ACAAAGUGUUGUAUCAAAA
UUUUGAUACAACACUUUGU
3229
2522
Yes
No
No

3230
CAAAGUGUUGUAUCAAAGA
UCUUUGAUACAACACUUUG
3231
2523
Yes
No
No

3232
AAAGUGUUGUAUCAAAGUA
UACUUUGAUACAACACUUU
3233
2524
Yes
No
No

3234
AAGUGUUGUAUCAAAGUGA
UCACUUUGAUACAACACUU
3235
2525
Yes
No
No

3236
AGUGUUGUAUCAAAGUGUA
UACACUUUGAUACAACACU
3237
2526
Yes
No
No

3238
UUGUAUCAAAGUGUGAUAA
UUAUCACACUUUGAUACAA
3239
2530
No
No
No

3240
AUCAAAGUGUGAUAUACAA
UUGUAUAUCACACUUUGAU
3241
2534
No
No
No

3242
AAGUGUGAUAUACAAAGUA
UACUUUGUAUAUCACACUU
3243
2538
No
No
No

3244
GAUAUACAAAGUGUACCAA
UUGGUACACUUUGUAUAUC
3245
2544
No
No
No

3246
AUAUACAAAGUGUACCAAA
UUUGGUACACUUUGUAUAU
3247
2545
No
No
No

3248
UAUACAAAGUGUACCAACA
UGUUGGUACACUUUGUAUA
3249
2546
Yes
No
No

3250
AUACAAAGUGUACCAACAA
UUGUUGGUACACUUUGUAU
3251
2547
No
No
No

3252
AGUGUACCAACAUAAGUGA
UCACUUAUGUUGGUACACU
3253
2553
No
No
No

3254
UGUACCAACAUAAGUGUUA
UAACACUUAUGUUGGUACA
3255
2555
No
No
No

3256
GUACCAACAUAAGUGUUGA
UCAACACUUAUGUUGGUAC
3257
2556
No
No
No

3258
UACCAACAUAAGUGUUGGA
UCCAACACUUAUGUUGGUA
3259
2557
No
No
No

3260
ACCAACAUAAGUGUUGGUA
UACCAACACUUAUGUUGGU
3261
2558
No
No
No

3262
CAACAUAAGUGUUGGUAGA
UCUACCAACACUUAUGUUG
3263
2560
No
No
No

3264
UAAGUGUUGGUAGCACUUA
UAAGUGCUACCAACACUUA
3265
2565
Yes
No
No

3266
GUGUUGGUAGCACUUAAGA
UCUUAAGUGCUACCAACAC
3267
2568
Yes
No
No

3268
UUGGUAGCACUUAAGACUA
UAGUCUUAAGUGCUACCAA
3269
2571
Yes
No
No

3270
UGGUAGCACUUAAGACUUA
UAAGUCUUAAGUGCUACCA
3271
2572
Yes
No
No

3272
GGUAGCACUUAAGACUUAA
UUAAGUCUUAAGUGCUACC
3273
2573
Yes
No
No

3274
GCACUUAAGACUUAUACUA
UAGUAUAAGUCUUAAGUGC
3275
2577
Yes
No
No

3276
CACUUAAGACUUAUACUUA
UAAGUAUAAGUCUUAAGUG
3277
2578
Yes
No
No

3278
CUUAAGACUUAUACUUGCA
UGCAAGUAUAAGUCUUAAG
3279
2580
Yes
No
No

3280
UUAAGACUUAUACUUGCCA
UGGCAAGUAUAAGUCUUAA
3281
2581
Yes
No
No

3282
UAAGACUUAUACUUGCCUA
UAGGCAAGUAUAAGUCUUA
3283
2582
Yes
No
No

3284
AAGACUUAUACUUGCCUUA
UAAGGCAAGUAUAAGUCUU
3285
2583
Yes
No
No

3286
AGACUUAUACUUGCCUUCA
UGAAGGCAAGUAUAAGUCU
3287
2584
Yes
No
No

3288
GACUUAUACUUGCCUUCUA
UAGAAGGCAAGUAUAAGUC
3289
2585
Yes
No
No

3290
CUUAUACUUGCCUUCUGAA
UUCAGAAGGCAAGUAUAAG
3291
2587
No
No
No

3292
UACUUGCCUUCUGAUAGUA
UACUAUCAGAAGGCAAGUA
3293
2591
No
No
No

3294
ACUUGCCUUCUGAUAGUAA
UUACUAUCAGAAGGCAAGU
3295
2592
No
No
No

3296
UGCCUUCUGAUAGUAUUCA
UGAAUACUAUCAGAAGGCA
3297
2595
No
No
No

3298
CUUCUGAUAGUAUUCCUUA
UAAGGAAUACUAUCAGAAG
3299
2598
No
No
No

3300
CUGAUAGUAUUCCUUUAUA
UAUAAAGGAAUACUAUCAG
3301
2601
No
No
No

3302
UGAUAGUAUUCCUUUAUAA
UUAUAAAGGAAUACUAUCA
3303
2602
No
No
No

3304
GAUAGUAUUCCUUUAUACA
UGUAUAAAGGAAUACUAUC
3305
2603
No
No
No

3306
AUAGUAUUCCUUUAUACAA
UUGUAUAAAGGAAUACUAU
3307
2604
No
No
No

3308
UAGUAUUCCUUUAUACACA
UGUGUAUAAAGGAAUACUA
3309
2605
No
No
No

3310
AGUAUUCCUUUAUACACAA
UUGUGUAUAAAGGAAUACU
3311
2606
No
No
No

3312
GUAUUCCUUUAUACACAGA
UCUGUGUAUAAAGGAAUAC
3313
2607
No
No
No

3314
CCUUUAUACACAGUGGAUA
UAUCCACUGUGUAUAAAGG
3315
2612
No
No
No

3316
CUUUAUACACAGUGGAUUA
UAAUCCACUGUGUAUAAAG
3317
2613
No
No
No

3318
UUUAUACACAGUGGAUUGA
UCAAUCCACUGUGUAUAAA
3319
2614
No
No
No

3320
UUAUACACAGUGGAUUGAA
UUCAAUCCACUGUGUAUAA
3321
2615
No
No
No

3322
UAUACACAGUGGAUUGAUA
UAUCAAUCCACUGUGUAUA
3323
2616
No
No
No

3324
AUACACAGUGGAUUGAUUA
UAAUCAAUCCACUGUGUAU
3325
2617
No
No
No

3326
CACAGUGGAUUGAUUAUAA
UUAUAAUCAAUCCACUGUG
3327
2620
No
No
No

3328
ACAGUGGAUUGAUUAUAAA
UUUAUAAUCAAUCCACUGU
3329
2621
No
No
No

3330
CAGUGGAUUGAUUAUAAAA
UUUUAUAAUCAAUCCACUG
3331
2622
No
No
No

3332
AGUGGAUUGAUUAUAAAUA
UAUUUAUAAUCAAUCCACU
3333
2623
No
No
No

3334
UUGAUUAUAAAUAAAUAGA
UCUAUUUAUUUAUAAUCAA
3335
2629
No
No
No

3336
GAUUAUAAAUAAAUAGAUA
UAUCUAUUUAUUUAUAAUC
3337
2631
No
No
No

3338
UUAUAAAUAAAUAGAUGUA
UACAUCUAUUUAUUUAUAA
3339
2633
No
No
No

3340
AAUAAAUAGAUGUGUCUUA
UAAGACACAUCUAUUUAUU
3341
2638
No
No
No

3342
AUAAAUAGAUGUGUCUUAA
UUAAGACACAUCUAUUUAU
3343
2639
No
No
No

3344
UAAAUAGAUGUGUCUUAAA
UUUAAGACACAUCUAUUUA
3345
2640
No
No
No

3346
AAAUAGAUGUGUCUUAACA
UGUUAAGACACAUCUAUUU
3347
2641
No
No
No

3348
AAUAGAUGUGUCUUAACAA
UUGUUAAGACACAUCUAUU
3349
2642
No
No
No

3350
AUAGAUGUGUCUUAACAUA
UAUGUUAAGACACAUCUAU
3351
2643
No
No
No

3352
UAGAUGUGUCUUAACAUAA
UUAUGUUAAGACACAUCUA
3353
2644
No
No
No

TABLE 5

Sense strands with cross-species compatibility with Human and Cyno MLH1

SENSE STRAND SEQ ID NOS

1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424,

1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498, 1510, 1512, 1514, 1516, 1518, 1520, 1522,

1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554,

1556, 1558, 1560, 1562, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586,

1588, 1590, 1592, 1594, 1596, 1598, 1600, 1602, 1604, 1606, 1608, 1610, 1612, 1614, 1616, 1618,

1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1642, 1644, 1646, 1648, 1650, 1652, 1654,

1656, 1658, 1660, 1662, 1664, 1666, 1668, 1670, 1694, 1696, 1698, 1700, 1702, 1704, 1706, 1708,

1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738, 1740,

1742, 1744, 1746, 1748, 1750, 1752, 1754, 1756, 1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772,

1774, 1776, 1778, 1780, 1782, 1806, 1808, 1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826,

1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1862, 1864, 1866, 1868, 1870, 1872,

1874, 1876, 1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896, 1926, 1928, 1930, 1932,

1934, 1936, 1938, 1940, 1950, 1952, 1954, 1956, 1958, 1960, 1962, 1964, 1966, 1968, 1970, 1972,

1974, 1976, 1978, 1980, 1998, 2000, 2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016, 2018, 2020,

2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042, 2044, 2046, 2048, 2050, 2052,

2054, 2056, 2058, 2060, 2062, 2064, 2066, 2068, 2070, 2072, 2074, 2076, 2078, 2080, 2082, 2084,

2086, 2088, 2090, 2092, 2114, 2116, 2118, 2120, 2122, 2124, 2126, 2128, 2150, 2152, 2154, 2156,

2158, 2160, 2162, 2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182, 2184, 2186, 2188,

2190, 2192, 2194, 2196, 2198, 2200, 2202, 2204, 2206, 2208, 2210, 2224, 2226, 2228, 2230, 2232,

2234, 2236, 2238, 2240, 2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256, 2258, 2260, 2262, 2264,

2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282, 2284, 2286, 2288, 2290, 2292, 2294, 2296,

2298, 2300, 2302, 2304, 2308, 2310, 2312, 2314, 2316, 2318, 2320, 2322, 2324, 2326, 2328, 2330,

2332, 2334, 2336, 2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352, 2354, 2356, 2358, 2360, 2362,

2364, 2366, 2368, 2370, 2372, 2394, 2396, 2398, 2420, 2422, 2424, 2426, 2428, 2430, 2432, 2434,

2436, 2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456, 2458, 2460, 2462, 2464, 2466,

2486, 2488, 2490, 2492, 2494, 2496, 2498, 2500, 2502, 2504, 2506, 2508, 2510, 2512, 2536, 2538,

2540, 2542, 2544, 2546, 2548, 2550, 2552, 2554, 2556, 2558, 2560, 2562, 2564, 2566, 2568, 2570,

2572, 2574, 2576, 2578, 2580, 2582, 2584, 2586, 2588, 2590, 2592, 2594, 2596, 2598, 2600, 2602,

2604, 2606, 2608, 2610, 2612, 2614, 2616, 2618, 2620, 2622, 2624, 2626, 2628, 2630, 2632, 2634,

2664, 2666, 2668, 2670, 2672, 2674, 2676, 2678, 2680, 2720, 2722, 2724, 2726, 2728, 2730, 2732,

2734, 2736, 2738, 2740, 2742, 2744, 2746, 2748, 2750, 2752, 2754, 2756, 2758, 2760, 2762, 2764,

2766, 2768, 2770, 2772, 2802, 2804, 2806, 2808, 2810, 2812, 2814, 2816, 2818, 2820, 2822, 2824,

2826, 2828, 2830, 2832, 2834, 2836, 2838, 2840, 2842, 2844, 2846, 2848, 2850, 2852, 2854, 2870,

2872, 2874, 2876, 2878, 2880, 2882, 2884, 2886, 2888, 2890, 2892, 2894, 2896, 2898, 2900, 2902,

2904, 2906, 2908, 2910, 2912, 2914, 2916, 2918, 2920, 2922, 2924, 2926, 2928, 2930, 2932, 2934,

2936, 2938, 2940, 2942, 2944, 2946, 2948, 2966, 2968, 2970, 2972, 2974, 2976, 2978, 2980, 2982,

2984, 2986, 2988, 2990, 3018, 3020, 3022, 3024, 3026, 3028, 3030, 3032, 3034, 3036, 3038, 3040,

3042, 3044, 3046, 3048, 3050, 3052, 3054, 3056, 3058, 3060, 3062, 3064, 3066, 3084, 3086, 3088,

3090, 3092, 3094, 3096, 3098, 3100, 3102, 3104, 3106, 3118, 3120, 3122, 3124, 3126, 3128, 3130,

3132, 3134, 3136, 3138, 3140, 3142, 3144, 3146, 3148, 3150, 3152, 3154, 3156, 3158, 3160, 3162,

3164, 3180, 3204, 3206, 3208, 3210, 3212, 3214, 3216, 3218, 3220, 3222, 3224, 3226, 3228, 3230,

3232, 3234, 3236, 3248, 3264, 3266, 3268, 3270, 3272, 3274, 3276, 3278, 3280, 3282, 3284, 3286,

3288

TABLE 6

Sense strands with cross-species compatibility with Human and Mouse MLH1

SENSE STRAND SEQ ID NOS

1558, 1578, 1636, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738, 1740,

1742, 1744, 1746, 1838, 1840, 1842, 1844, 1846, 1902, 1904, 1906, 1908, 1910, 1940, 2006, 2008,

2010, 2012, 2042, 2044, 2046, 2048, 2078, 2080, 2224, 2226, 2228, 2230, 2232, 2234, 2236, 2238,

2240, 2324, 2326, 2560, 2674, 2676, 2678, 2680, 2746, 2748, 2750, 2752, 2754, 2756, 2758, 2760,

2762, 2764, 2848, 2850, 2870, 2872, 2874, 2876, 2888, 2890, 2892, 2894, 2916, 2918, 2920, 2922,

2924, 2926, 2928, 2930, 2932, 2934, 2936, 2938, 2940, 2942, 2958, 2960, 2962, 3054, 3136, 3138,

3140

TABLE 7

Sense strands with cross-species compatibility with Human and Rat MLH1

SENSE STRAND SEQ ID NOS

1558, 1578, 1636, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1906, 2006, 2008, 2010,

2012, 2014, 2016, 2018, 2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2078,

2080, 2124, 2126, 2128, 2130, 2132, 2236, 2308, 2310, 2634, 2674, 2676, 2678, 2680, 2750, 2872,

2874, 2876, 2886, 2888, 2890, 2892, 2894, 2916, 2918, 2920, 2922, 2924, 2926, 2928, 2930, 2932,

2934, 2936, 2938, 2940, 2942, 3054, 3056, 3058, 3060, 3062

TABLE 8

Sense strands with cross-species compatibility with Human, Cyno, and Mouse MLH1

SENSE STRAND SEQ ID NOS

1558, 1578, 1636, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738, 1740,

1742, 1744, 1746, 1838, 1840, 1842, 1844, 1846, 1940, 2006, 2008, 2010, 2012, 2042, 2044, 2046,

2048, 2078, 2080, 2224, 2226, 2228, 2230, 2232, 2234, 2236, 2238, 2240, 2324, 2326, 2560, 2674,

2676, 2678, 2680, 2746, 2748, 2750, 2752, 2754, 2756, 2758, 2760, 2762, 2764, 2848, 2850, 2870,

2872, 2874, 2876, 2888, 2890, 2892, 2894, 2916, 2918, 2920, 2922, 2924, 2926, 2928, 2930, 2932,

2934, 2936, 2938, 2940, 2942, 3054, 3136, 3138, 3140

TABLE 9

Sense strands with cross-species compatibility with Human, Cyno, and Rat MLH1

SENSE STRAND SEQ ID NOS

1558, 1578, 1636, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 2006, 2008, 2010, 2012, 2014,

2016, 2018, 2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2078, 2080, 2124,

2126, 2128, 2236, 2308, 2310, 2634, 2674, 2676, 2678, 2680, 2750, 2872, 2874, 2876, 2886, 2888,

2890, 2892, 2894, 2916, 2918, 2920, 2922, 2924, 2926, 2928, 2930, 2932, 2934, 2936, 2938, 2940,

2942, 3054, 3056, 3058, 3060, 3062

TABLE 10

Sense strands with cross-species compatibility with Human, Mouse, and Rat MLH1

SENSE STRAND SEQ ID NOS

1558, 1578, 1636, 1838, 1840, 1842, 1844, 1846, 1906, 2006, 2008, 2010, 2012, 2078, 2080, 2236,

2674, 2676, 2678, 2680, 2750, 2872, 2874, 2876, 2888, 2890, 2892, 2894, 2916, 2918, 2920, 2922,

2924, 2926, 2928, 2930, 2932, 2934, 2936, 2938, 2940, 2942, 3054

TABLE 11

Sense strands with cross-species compatibility with Human, Cyno, Mouse, and Rat MLH1

SENSE STRAND SEQ ID NOS

1558, 1578, 1636, 1838, 1840, 1842, 1844, 1846, 2006, 2008, 2010, 2012, 2078, 2080, 2236, 2674,

2676, 2678, 2680, 2750, 2872, 2874, 2876, 2888, 2890, 2892, 2894, 2916, 2918, 2920, 2922, 2924,

2926, 2928, 2930, 2932, 2934, 2936, 2938, 2940, 2942, 3054

Example 2. Antisense Inhibition of MLH1

Inhibition or knockdown of MLH1 can be demonstrated using a cell-based assay. For example, HEK293, NIH3T3, or Hela or another available mammalian cell line with oligonucleotides targeting MLH1 identified above in Example 1 using at least five different dose levels, using transfection reagents such as lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Cells are harvested at multiple time points up to 7 days post transfection for either mRNA or protein analyses. Knockdown of mRNA and protein are determined by RT-qPCR or western blot analyses respectively, using standard molecular biology techniques as previously described (see, for example, as described in Drouet et al., 2014, PLOS One 9(6): e99341). The relative levels of the MLH1 mRNA and protein at the different oligonucleotide levels are compared with a mock oligonucleotide control. The most potent oligonucleotides (for example, those which are capable of at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% or more, reduction in protein levels when compared with controls) are selected for subsequent studies, for example, as described in the examples below.

Human Cell Lines

HeLa cells were obtained from ATCC (ATCC in partnership with LGC Standards, Wesel, Germany, cat. #ATCC-CRM-CCL-2) and cultured in HAM's F12 (#FG0815, Biochrom, Berlin, Germany), supplemented to contain 10% fetal calf serum (1248D, Biochrom GmbH, Berlin, Germany), and 100 U/ml Penicillin/100 μg/ml Streptomycin (A2213, Biochrom GmbH, Berlin, Germany) at 37° C. in an atmosphere with 5% CO₂in a humidified incubator. For transfection of HeLa cells with ASOs, cells were seeded at a density of 15,000 cells/well into 96-well tissue culture plates (#655180, GBO, Germany).

Transfections

In HeLa cells, transfection of ASOs was carried out with Lipofectamine 2000 (Invitrogen/Life Technologies, Karlsruhe, Germany) according to manufacturer's instructions for reverse transfection with 0.25 μL Lipofectamine 2000 per well.

The dual dose screen was performed with ASOs in quadruplicates at 20 nM and 2 nM respectively, with two ASOs targeting AHSA1 (one MOE-ASO and one 2′oMe-ASO) as unspecific controls and a mock transfection. Dose-response experiments were done with ASOs in 5 concentrations transfected in quadruplicates, starting at 20 nM in 5-6-fold dilutions steps down to ˜15-32 pM. Mock transfected cells served as control in dose-response curve (DRC) experiments.

Analysis and Quantitation

After 24 h of incubation with ASOs, medium was removed and cells were lysed in 150 μl Medium-Lysis Mixture (1 volume lysis mixture, 2 volumes cell culture medium) and then incubated at 53° C. for 30 minutes. bDNA assay was performed according to manufacturer's instructions. Luminescence was read using 1420 Luminescence Counter (WALLAC VICTOR Light, Perkin Elmer, Rodgau-Jugesheim, Germany) following 30 minutes incubation at RT in the dark.

The two Ahsa1-ASOs (one 2′-OMe and one MOE-modified) served at the same time as unspecific controls for respective target mRNA expression and as a positive control to analyze transfection efficiency with regards to Ahsa1 mRNA level. By hybridization with an Ahsa1 probeset, the mock transfected wells served as controls for Ahsa1 mRNA level. Transfection efficiency for each 96-well plate and both doses in the dual dose screen were calculated by relating Ahsa1-level with Ahsa1-ASO (normalized to GAPDH) to Ahsa1-level obtained with mock controls.

For each well, the target mRNA level was normalized to the respective GAPDH mRNA level. The activity of a given ASO was expressed as percent mRNA concentration of the respective target (normalized to GAPDH mRNA) in treated cells, relative to the target mRNA concentration (normalized to GAPDH mRNA) averaged across control wells.

The results of the dual-dose screen of ˜480 ASOs targeting MLH1, as well as IC₂₀, IC₅₀and IC₅₀values of approximately 48 positive ASOs from the dual dose screen, are shown in Table 12 below.

TABLE 12

mean % mRNA
SD % mRNA

SEQ ID

Off-target Score
remaining
remaining
IC20
IC50
IC80

NO
Position
Sequence
Human
Cyno
Mouse
Rat
2 nM
20 nM
2 nM
20 nM
(nM)
(nM)
(nM)

7
7
AACTCTGTGGGTTGCTGGGT
2
2
NC
NC
104.35
71.14
13.91
14.16
NA
NA
NA

9
15
AATTTCTCAACTCTGTGGGT
2
1
NC
NC
99.83
100.39
6.85
7.93
NA
NA
NA

10
16
AAATTTCTCAACTCTGTGGG
2
2
NC
NC
100.93
100.50
10.87
7.83
NA
NA
NA

12
18
TCAAATTTCTCAACTCTGTG
2
2
NC
NC
97.45
85.69
8.92
20.36
NA
NA
NA

13
19
GTCAAATTTCTCAACTCTGT
2
2
NC
NC
101.96
80.20
7.23
26.13
NA
NA
NA

19
25
ATGCCAGTCAAATTTCTCAA
2
2
NC
NC
85.74
92.33
8.97
4.37
NA
NA
NA

20
26
AATGCCAGTCAAATTTCTCA
2
2
NC
NC
92.97
92.03
10.93
6.17
NA
NA
NA

21
27
GAATGCCAGTCAAATTTCTC
3
1
NC
NC
98.85
93.56
9.40
3.31
NA
NA
NA

22
28
TGAATGCCAGTCAAATTTCT
2
2
NC
NC
104.98
111.94
12.87
30.18
NA
NA
NA

23
29
TTGAATGCCAGTCAAATTTC
2
2
NC
NC
108.49
93.37
12.54
5.14
NA
NA
NA

24
30
CTTGAATGCCAGTCAAATTT
2
2
NC
NC
102.13
89.79
8.01
4.52
NA
NA
NA

25
31
GCTTGAATGCCAGTCAAATT
2
2
NC
NC
107.69
89.66
9.25
2.29
NA
NA
NA

26
32
AGCTTGAATGCCAGTCAAAT
2
2
NC
NC
115.30
85.50
5.37
3.08
NA
NA
NA

27
33
CAGCTTGAATGCCAGTCAAA
2
2
NC
NC
127.17
80.79
14.29
2.47
NA
NA
NA

54
105
GTGCTCACGTTCTTCCTTCA
2
2
NC
NC
110.43
84.17
10.06
28.87
NA
NA
NA

58
153
GTCTAGATGCTCAACGGAAG
3
1
NC
NC
77.65
70.07
4.17
6.43
NA
NA
NA

59
154
CGTCTAGATGCTCAACGGAA
2
1
NC
NC
77.01
69.92
3.64
1.43
NA
NA
NA

60
155
ACGTCTAGATGCTCAACGGA
2
2
NC
NC
74.53
79.00
8.98
6.49
NA
NA
NA

61
156
AACGTCTAGATGCTCAACGG
3
2
NC
NC
74.97
78.69
6.16
8.53
NA
NA
NA

62
158
GAAACGTCTAGATGCTCAAC
3
2
NC
NC
83.73
77.36
3.52
5.83
NA
NA
NA

63
159
GGAAACGTCTAGATGCTCAA
2
2
NC
NC
77.27
67.40
8.64
12.29
NA
NA
NA

81
196
CCTGCCACGAACGACATTTT
2
2
NC
NC
52.53
23.65
6.81
6.28
NA
NA
NA

82
197
CCCTGCCACGAACGACATTT
3
1
NC
NC
33.75
19.40
5.46
1.79
NA
NA
NA

83
202
ATAACCCCTGCCACGAACGA
3
2
NC
NC
63.35
24.88
6.81
3.22
NA
NA
NA

84
203
AATAACCCCTGCCACGAACG
2
2
NC
NC
53.65
29.25
14.61
4.28
NA
NA
NA

86
205
CGAATAACCCCTGCCACGAA
2
2
NC
NC
48.59
21.32
4.16
1.00
NA
NA
NA

87
229
TTCACCACTGTCTCGTCCAG
2
2
NC
NC
55.00
26.06
7.08
2.51
NA
NA
NA

90
236
GATGCGGTTCACCACTGTCT
3
2
NC
NC
45.96
34.50
1.21
3.76
NA
NA
NA

99
292
TTCTCAATCATCTCTTTGAT
2
1
NC
NC
60.15
23.70
3.42
6.43
NA
NA
NA

100
293
GTTCTCAATCATCTCTTTGA
2
2
NC
NC
35.82
25.52
1.30
3.37
NA
NA
NA

101
294
AGTTCTCAATCATCTCTTTG
2
2
NC
NC
34.56
20.99
2.80
8.30
NA
NA
NA

106
299
TAAACAGTTCTCAATCATCT
2
2
NC
NC
65.12
23.29
1.36
6.86
NA
NA
NA

107
300
CTAAACAGTTCTCAATCATC
2
2
NC
NC
81.44
37.09
5.98
4.60
NA
NA
NA

108
301
TCTAAACAGTTCTCAATCAT
2
1
NC
NC
94.04
41.93
3.64
7.23
NA
NA
NA

109
302
ATCTAAACAGTTCTCAATCA
2
2
NC
NC
83.80
43.08
3.87
6.44
NA
NA
NA

111
304
GCATCTAAACAGTTCTCAAT
2
2
NC
NC
59.23
38.77
3.24
6.32
NA
NA
NA

113
306
TTGCATCTAAACAGTTCTCA
2
3
NC
NC
47.29
27.54
3.93
4.15
NA
NA
NA

114
307
TTTGCATCTAAACAGTTCTC
2
2
NC
NC
60.27
27.21
7.74
4.51
NA
NA
NA

117
310
GATTTTGCATCTAAACAGTT
1
1
2
2
85.40
39.72
9.90
3.90
NA
NA
NA

122
315
TTGTGGATTTTGCATCTAAA
2
2
NC
NC
36.71
6.97
7.26
0.94
NA
NA
NA

123
316
CTTGTGGATTTTGCATCTAA
2
2
NC
NC
32.56
7.26
3.43
1.51
NA
NA
NA

124
317
ACTTGTGGATTTTGCATCTA
2
2
NC
NC
27.77
23.34
1.76
3.38
NA
NA
NA

125
318
TACTTGTGGATTTTGCATCT
2
2
NC
NC
28.15
9.60
2.12
1.27
NA
NA
NA

126
319
ATACTTGTGGATTTTGCATC
2
2
NC
NC
30.43
11.37
7.86
2.72
NA
NA
NA

129
322
TGAATACTTGTGGATTTTGC
2
2
NC
NC
28.88
7.61
2.36
0.45
NA
NA
NA

130
323
TTGAATACTTGTGGATTTTG
2
2
NC
NC
64.65
12.11
3.61
3.43
NA
NA
NA

131
324
CTTGAATACTTGTGGATTTT
2
2
NC
NC
43.96
10.11
3.33
3.46
NA
NA
NA

137
330
CAATCACTTGAATACTTGTG
2
2
NC
NC
31.45
7.57
2.96
0.20
NA
NA
NA

138
331
ACAATCACTTGAATACTTGT
2
2
NC
NC
61.59
17.94
3.03
1.20
NA
NA
NA

140
333
TAACAATCACTTGAATACTT
2
1
NC
NC
90.85
24.99
8.92
1.23
NA
NA
NA

142
335
TTTAACAATCACTTGAATAC
2
2
NC
NC
100.31
41.90
15.66
1.99
NA
NA
NA

143
336
CTTTAACAATCACTTGAATA
2
2
NC
NC
108.97
41.71
4.46
5.93
NA
NA
NA

144
337
TCTTTAACAATCACTTGAAT
2
2
NC
NC
73.97
15.40
7.19
4.30
NA
NA
NA

146
339
CCTCTTTAACAATCACTTGA
2
2
NC
NC
24.05
7.50
2.76
0.61
NA
NA
NA

147
341
TCCCTCTTTAACAATCACTT
2
2
NC
NC
33.48
14.58
3.59
2.42
NA
NA
NA

148
342
CTCCCTCTTTAACAATCACT
2
2
NC
NC
42.95
16.27
4.12
2.50
NA
NA
NA

149
343
CCTCCCTCTTTAACAATCAC
2
2
NC
NC
50.06
17.79
12.35
5.07
NA
NA
NA

150
344
GCCTCCCTCTTTAACAATCA
2
2
NC
NC
45.93
23.38
6.61
9.77
NA
NA
NA

151
345
GGCCTCCCTCTTTAACAATC
2
2
NC
NC
45.03
15.58
7.23
1.62
NA
NA
NA

152
346
AGGCCTCCCTCTTTAACAAT
2
2
NC
NC
34.11
32.22
4.01
3.43
NA
NA
NA

153
357
GAATCAACTTCAGGCCTCCC
2
2
NC
NC
41.96
11.54
6.08
1.44
NA
NA
NA

154
358
TGAATCAACTTCAGGCCTCC
2
3
NC
NC
50.20
6.62
7.94
1.20
NA
NA
NA

155
366
CTTGGATCTGAATCAACTTC
2
2
NC
NC
65.36
9.43
5.30
1.36
NA
NA
NA

156
367
TCTTGGATCTGAATCAACTT
2
2
NC
NC
80.83
11.67
6.59
0.83
NA
NA
NA

157
368
GTCTTGGATCTGAATCAACT
2
1
NC
NC
61.03
15.65
3.50
0.72
NA
NA
NA

158
369
TGTCTTGGATCTGAATCAAC
2
3
NC
NC
75.35
12.67
8.90
1.50
NA
NA
NA

159
370
TTGTCTTGGATCTGAATCAA
2
2
NC
NC
91.57
21.18
8.97
1.65
NA
NA
NA

160
371
ATTGTCTTGGATCTGAATCA
2
2
NC
NC
100.24
31.45
13.98
5.42
NA
NA
NA

172
397
AGATCTTCTTTCCTGATCCC
2
2
NC
NC
33.96
22.36
3.22
2.03
NA
NA
NA

188
413
TTCACATACAATATCCAGAT
2
1
NC
NC
38.68
6.11
3.67
0.64
NA
NA
NA

189
414
TTTCACATACAATATCCAGA
2
2
NC
NC
53.02
9.11
3.58
1.85
NA
NA
NA

190
415
CTTTCACATACAATATCCAG
2
1
NC
NC
70.51
13.27
2.71
1.69
NA
NA
NA

191
416
CCTTTCACATACAATATCCA
2
2
NC
NC
56.82
12.07
1.68
1.04
NA
NA
NA

211
448
TCCTCAAAGGACTGCAGTTT
2
2
NC
NC
21.91
11.33
1.20
0.58
NA
NA
NA

215
461
AATACTGGCTAAATCCTCAA
2
2
2
NC
62.56
22.15
4.11
0.99
NA
NA
NA

216
462
AAATACTGGCTAAATCCTCA
2
1
2
NC
65.69
13.57
5.67
0.91
NA
NA
NA

217
463
GAAATACTGGCTAAATCCTC
3
2
2
NC
76.74
24.80
12.28
5.94
NA
NA
NA

218
464
AGAAATACTGGCTAAATCCT
2
2
2
NC
98.15
30.43
12.12
2.04
NA
NA
NA

219
465
TAGAAATACTGGCTAAATCC
2
2
2
NC
101.50
23.30
10.53
2.98
NA
NA
NA

220
466
GTAGAAATACTGGCTAAATC
2
2
2
NC
88.38
31.18
16.60
1.58
NA
NA
NA

221
467
GGTAGAAATACTGGCTAAAT
2
2
2
NC
70.46
50.62
7.85
2.86
NA
NA
NA

222
468
AGGTAGAAATACTGGCTAAA
1
2
1
NC
74.73
34.51
4.64
7.11
NA
NA
NA

223
469
TAGGTAGAAATACTGGCTAA
1
2
2
NC
56.63
13.53
23.00
3.09
NA
NA
NA

224
470
ATAGGTAGAAATACTGGCTA
2
3
2
NC
53.77
17.28
5.53
0.86
NA
NA
NA

225
471
CATAGGTAGAAATACTGGCT
2
2
2
NC
29.85
16.04
8.85
1.61
NA
NA
NA

226
472
CCATAGGTAGAAATACTGGC
2
3
2
NC
44.14
11.24
7.00
3.21
NA
NA
NA

229
475
AAGCCATAGGTAGAAATACT
2
2
2
NC
88.23
21.03
4.95
1.76
NA
NA
NA

230
476
AAAGCCATAGGTAGAAATAC
2
3
2
NC
126.10
46.70
10.66
4.68
NA
NA
NA

231
477
GAAAGCCATAGGTAGAAATA
2
1
1
NC
106.65
39.76
7.30
0.42
NA
NA
NA

232
478
CGAAAGCCATAGGTAGAAAT
2
2
1
NC
84.03
25.60
22.61
2.02
NA
NA
NA

233
479
TCGAAAGCCATAGGTAGAAA
2
2
NC
NC
75.55
27.07
3.57
1.55
NA
NA
NA

234
480
CTCGAAAGCCATAGGTAGAA
2
2
NC
NC
88.46
16.94
6.53
1.96
NA
NA
NA

235
481
CCTCGAAAGCCATAGGTAGA
2
2
NC
NC
47.28
14.08
11.65
0.84
NA
NA
NA

236
485
CTCACCTCGAAAGCCATAGG
2
2
NC
NC
27.57
21.90
8.01
5.26
NA
NA
NA

237
486
CCTCACCTCGAAAGCCATAG
2
2
NC
NC
25.13
10.37
4.41
3.07
NA
NA
NA

238
487
GCCTCACCTCGAAAGCCATA
2
1
NC
NC
21.60
17.21
1.89
2.24
NA
NA
NA

239
488
AGCCTCACCTCGAAAGCCAT
2
2
NC
NC
39.33
16.62
3.53
1.29
NA
NA
NA

242
491
CAAAGCCTCACCTCGAAAGC
2
1
NC
NC
68.06
20.54
14.63
5.51
NA
NA
NA

243
492
CCAAAGCCTCACCTCGAAAG
2
2
NC
NC
65.39
19.97
8.02
3.06
NA
NA
NA

244
493
GCCAAAGCCTCACCTCGAAA
2
2
NC
NC
31.19
21.00
1.08
1.73
NA
NA
NA

245
525
TAATAGTAACATGAGCCACA
2
2
NC
NC
53.67
10.20
13.67
2.33
NA
NA
NA

246
526
GTAATAGTAACATGAGCCAC
2
1
NC
NC
36.92
49.08
2.19
7.25
NA
NA
NA

247
527
TGTAATAGTAACATGAGCCA
2
3
NC
NC
32.79
30.80
8.50
4.22
NA
NA
NA

248
528
TTGTAATAGTAACATGAGCC
2
2
NC
NC
36.71
14.75
4.93
1.85
NA
NA
NA

249
529
GTTGTAATAGTAACATGAGC
2
2
NC
NC
25.96
54.83
2.94
2.76
NA
NA
NA

270
550
CACTTTCCATCAGCTGTTTT
2
2
NC
NC
85.01
14.38
11.59
3.78
NA
NA
NA

271
551
ACACTTTCCATCAGCTGTTT
2
2
NC
NC
52.03
14.17
1.59
3.52
NA
NA
NA

274
554
TGCACACTTTCCATCAGCTG
2
2
NC
NC
17.31
20.13
2.02
6.11
NA
NA
NA

275
555
ATGCACACTTTCCATCAGCT
2
2
NC
NC
21.62
17.12
3.23
3.11
NA
NA
NA

276
556
TATGCACACTTTCCATCAGC
2
1
NC
NC
40.73
13.28
2.90
1.04
NA
NA
NA

277
557
GTATGCACACTTTCCATCAG
2
2
NC
NC
25.94
38.62
2.55
4.69
NA
NA
NA

278
558
TGTATGCACACTTTCCATCA
2
1
NC
NC
39.11
10.83
0.78
0.92
NA
NA
NA

279
559
CTGTATGCACACTTTCCATC
2
2
NC
NC
28.69
6.52
3.10
0.98
NA
NA
NA

286
573
CTGAGTAACTTGCTCTGTAT
2
2
NC
NC
18.63
7.51
2.27
2.11
NA
NA
NA

287
574
TCTGAGTAACTTGCTCTGTA
1
2
2
2
20.22
20.80
2.72
2.62
NA
NA
NA

288
575
ATCTGAGTAACTTGCTCTGT
2
2
2
2
21.77
22.63
1.03
2.46
NA
NA
NA

289
576
CATCTGAGTAACTTGCTCTG
2
2
2
2
22.93
10.42
2.98
1.07
NA
NA
NA

290
577
CCATCTGAGTAACTTGCTCT
2
2
2
2
23.21
8.99
6.91
2.27
NA
NA
NA

291
578
TCCATCTGAGTAACTTGCTC
2
2
2
3
12.91
8.39
0.94
1.68
0.25
0.82
3.18

292
579
TTCCATCTGAGTAACTTGCT
2
2
2
3
14.52
6.71
1.20
1.41
0.42
1.05
2.86

293
580
TTTCCATCTGAGTAACTTGC
2
2
2
3
15.77
6.93
3.52
0.62
0.45
0.93
2.33

295
582
GTTTTCCATCTGAGTAACTT
2
2
NC
NC
27.05
21.26
4.08
1.98
NA
NA
NA

296
583
AGTTTTCCATCTGAGTAACT
2
2
NC
NC
27.26
22.83
3.20
1.24
NA
NA
NA

297
584
CAGTTTTCCATCTGAGTAAC
2
2
NC
NC
43.93
9.42
6.80
1.05
NA
NA
NA

298
585
TCAGTTTTCCATCTGAGTAA
2
2
NC
NC
25.68
6.86
4.41
1.32
NA
NA
NA

310
632
CGTGATCTGGGTCCCTTGAT
2
2
NC
NC
22.66
9.44
2.16
2.57
NA
NA
NA

311
663
TCGTGGCTATGTTGTAAAAA
3
2
NC
NC
30.05
21.14
2.90
1.76
NA
NA
NA

312
664
CTCGTGGCTATGTTGTAAAA
2
1
NC
NC
31.88
10.71
2.33
1.39
NA
NA
NA

313
665
CCTCGTGGCTATGTTGTAAA
2
2
NC
NC
14.82
8.41
1.74
0.89
2.08
5.22
14.59

314
666
TCCTCGTGGCTATGTTGTAA
2
2
NC
NC
17.40
9.66
1.82
1.58
NA
NA
NA

315
667
CTCCTCGTGGCTATGTTGTA
3
2
NC
NC
17.11
10.01
3.02
0.91
NA
NA
NA

316
668
TCTCCTCGTGGCTATGTTGT
2
3
NC
NC
16.78
8.86
1.33
0.75
0.36
0.95
3.34

317
669
TTCTCCTCGTGGCTATGTTG
2
2
NC
NC
13.93
7.60
1.53
0.72
0.33
0.94
3.17

318
670
TTTCTCCTCGTGGCTATGTT
2
2
NC
NC
36.74
9.22
11.20
1.63
NA
NA
NA

319
671
TTTTCTCCTCGTGGCTATGT
2
2
NC
NC
30.22
7.09
6.50
0.94
NA
NA
NA

320
672
CTTTTCTCCTCGTGGCTATG
2
2
NC
NC
30.01
8.21
6.17
1.37
NA
NA
NA

322
674
AGCTTTTCTCCTCGTGGCTA
2
2
NC
NC
15.61
24.15
1.15
5.01
NA
NA
NA

323
675
AAGCTTTTCTCCTCGTGGCT
2
2
NC
NC
13.29
21.60
0.58
3.30
NA
NA
NA

324
676
AAAGCTTTTCTCCTCGTGGC
2
1
NC
NC
7.91
22.01
0.66
5.22
NA
NA
NA

325
677
TAAAGCTTTTCTCCTCGTGG
2
2
NC
NC
7.62
5.41
0.57
0.70
0.13
0.38
1.25

326
678
TTAAAGCTTTTCTCCTCGTG
3
2
NC
NC
16.56
4.90
1.53
0.44
0.26
0.93
3.32

327
679
TTTAAAGCTTTTCTCCTCGT
2
2
NC
NC
29.15
4.68
0.91
0.45
NA
NA
NA

328
680
TTTTAAAGCTTTTCTCCTCG
2
2
NC
NC
43.20
7.15
4.04
1.27
NA
NA
NA

332
699
CATATTCTTCACTTGGATTT
2
2
NC
NC
57.71
12.58
9.76
1.59
NA
NA
NA

333
700
CCATATTCTTCACTTGGATT
2
2
NC
NC
22.46
15.07
1.49
13.75
NA
NA
NA

334
701
CCCATATTCTTCACTTGGAT
2
2
NC
NC
12.58
8.36
1.90
1.14
0.08
0.40
2.04

335
702
TCCCATATTCTTCACTTGGA
2
3
NC
NC
11.94
7.01
1.30
0.65
0.08
0.50
2.76

336
703
TTCCCATATTCTTCACTTGG
2
2
NC
NC
105.05
106.90
4.22
6.04
NA
NA
NA

337
704
TTTCCCATATTCTTCACTTG
2
2
NC
NC
69.00
13.33
7.64
4.40
NA
NA
NA

342
709
AAAATTTTCCCATATTCTTC
2
2
NC
NC
90.57
56.39
5.40
8.44
NA
NA
NA

345
712
TCCAAAATTTTCCCATATTC
2
2
NC
NC
73.51
23.08
9.29
5.26
NA
NA
NA

383
760
ACTGAGAAACTAATGCCTGC
2
2
NC
NC
30.19
36.06
13.92
6.43
NA
NA
NA

384
761
AACTGAGAAACTAATGCCTG
2
2
2
NC
32.91
18.09
4.29
0.65
NA
NA
NA

385
762
TAACTGAGAAACTAATGCCT
2
2
2
NC
54.54
7.98
4.80
1.50
NA
NA
NA

386
763
TTAACTGAGAAACTAATGCC
1
NC
2
NC
69.97
7.71
4.83
0.91
NA
NA
NA

387
764
TTTAACTGAGAAACTAATGC
1
1
2
NC
91.14
15.15
4.95
1.92
NA
NA
NA

388
765
TTTTAACTGAGAAACTAATG
1
2
2
NC
102.79
24.74
6.79
3.85
NA
NA
NA

397
790
ACATCAGCTACTGTCTCTCC
2
3
NC
NC
58.09
9.95
6.24
1.25
NA
NA
NA

398
791
AACATCAGCTACTGTCTCTC
2
2
NC
NC
64.72
12.13
3.53
2.74
NA
NA
NA

399
792
TAACATCAGCTACTGTCTCT
2
2
NC
NC
61.25
10.52
5.57
1.14
NA
NA
NA

400
793
CTAACATCAGCTACTGTCTC
2
2
NC
NC
58.00
9.48
5.24
1.33
NA
NA
NA

401
794
CCTAACATCAGCTACTGTCT
2
2
NC
NC
31.28
7.21
6.03
1.48
NA
NA
NA

402
795
TCCTAACATCAGCTACTGTC
2
2
NC
NC
27.71
6.73
2.81
0.51
NA
NA
NA

405
798
GTGTCCTAACATCAGCTACT
2
2
NC
NC
22.45
17.51
2.26
1.39
NA
NA
NA

406
799
AGTGTCCTAACATCAGCTAC
2
2
NC
NC
28.59
28.60
1.72
4.24
NA
NA
NA

407
800
TAGTGTCCTAACATCAGCTA
3
3
NC
NC
33.38
8.27
5.20
0.29
NA
NA
NA

408
801
GTAGTGTCCTAACATCAGCT
3
1
NC
NC
15.92
26.00
3.79
1.63
NA
NA
NA

409
802
GGTAGTGTCCTAACATCAGC
2
2
NC
NC
16.90
31.80
1.99
4.13
NA
NA
NA

410
803
GGGTAGTGTCCTAACATCAG
2
2
NC
NC
14.65
29.55
1.45
2.27
NA
NA
NA

411
804
TGGGTAGTGTCCTAACATCA
2
2
NC
NC
14.54
11.44
5.46
2.55
NA
NA
NA

412
805
TTGGGTAGTGTCCTAACATC
2
1
NC
NC
27.50
6.35
0.34
1.21
NA
NA
NA

413
806
ATTGGGTAGTGTCCTAACAT
2
1
NC
NC
29.79
23.52
7.21
3.09
NA
NA
NA

415
808
GCATTGGGTAGTGTCCTAAC
3
1
NC
NC
11.84
11.36
2.10
2.39
0.14
0.36
1.21

416
809
GGCATTGGGTAGTGTCCTAA
2
2
NC
NC
9.48
17.09
0.76
1.03
NA
NA
NA

417
810
AGGCATTGGGTAGTGTCCTA
2
3
NC
NC
12.81
15.60
0.64
1.47
NA
NA
NA

418
811
GAGGCATTGGGTAGTGTCCT
2
3
NC
NC
17.87
26.51
0.54
3.00
NA
NA
NA

419
812
TGAGGCATTGGGTAGTGTCC
2
3
NC
NC
21.17
11.63
3.02
0.77
NA
NA
NA

420
813
TTGAGGCATTGGGTAGTGTC
2
2
NC
NC
26.62
12.47
2.28
1.53
NA
NA
NA

421
814
GTTGAGGCATTGGGTAGTGT
2
2
NC
NC
29.79
17.15
3.24
1.50
NA
NA
NA

458
868
TCTATCAGTTCTCGACTAAC
2
3
1
NC
71.94
17.44
7.71
2.19
NA
NA
NA

473
884
ATCCTCACATCCAATTTCTA
2
2
NC
NC
67.22
23.05
2.68
6.53
NA
NA
NA

474
885
TATCCTCACATCCAATTTCT
2
2
NC
NC
67.80
17.81
4.08
3.67
NA
NA
NA

476
887
TTTATCCTCACATCCAATTT
2
1
NC
NC
84.61
14.33
14.95
4.23
NA
NA
NA

477
888
TTTTATCCTCACATCCAATT
2
2
NC
NC
75.61
16.36
8.29
3.35
NA
NA
NA

478
889
GTTTTATCCTCACATCCAAT
2
2
NC
NC
46.47
12.22
1.58
1.02
NA
NA
NA

482
893
TAGGGTTTTATCCTCACATC
2
2
NC
NC
20.38
6.34
1.89
1.11
NA
NA
NA

483
894
CTAGGGTTTTATCCTCACAT
2
2
NC
NC
11.09
6.25
5.03
1.17
0.09
0.30
1.09

484
895
GCTAGGGTTTTATCCTCACA
3
2
2
2
8.67
19.52
2.89
4.04
NA
NA
NA

485
896
GGCTAGGGTTTTATCCTCAC
2
2
NC
NC
6.96
15.56
4.28
1.74
0.04
0.14
0.64

486
897
AGGCTAGGGTTTTATCCTCA
2
2
NC
NC
11.93
8.49
9.21
1.22
0.05
0.21
1.09

487
898
AAGGCTAGGGTTTTATCCTC
2
3
NC
NC
25.71
13.09
4.84
1.26
NA
NA
NA

490
902
TTTGAAGGCTAGGGTTTTAT
2
1
NC
NC
36.90
9.31
15.37
1.09
NA
NA
NA

491
903
TTTTGAAGGCTAGGGTTTTA
2
3
NC
NC
40.51
8.68
17.94
3.19
NA
NA
NA

493
905
CATTTTGAAGGCTAGGGTTT
2
2
NC
NC
31.27
10.30
10.35
1.51
NA
NA
NA

494
906
TCATTTTGAAGGCTAGGGTT
2
2
NC
NC
19.42
9.09
1.85
0.67
NA
NA
NA

497
909
CATTCATTTTGAAGGCTAGG
2
3
NC
NC
22.09
6.82
5.83
0.49
NA
NA
NA

498
910
CCATTCATTTTGAAGGCTAG
2
2
NC
NC
21.00
7.91
10.54
1.08
NA
NA
NA

499
911
ACCATTCATTTTGAAGGCTA
2
2
NC
NC
11.75
8.10
0.75
1.05
0.12
0.43
1.95

500
912
AACCATTCATTTTGAAGGCT
2
2
NC
NC
14.55
8.40
4.11
1.33
0.12
0.44
1.09

501
913
TAACCATTCATTTTGAAGGC
2
1
NC
NC
21.12
6.92
1.54
0.78
NA
NA
NA

522
935
TGAGTAGTTTGCATTGGATA
3
2
NC
NC
25.31
8.60
2.41
0.53
NA
NA
NA

523
936
CTGAGTAGTTTGCATTGGAT
2
2
NC
NC
14.73
5.87
7.85
0.48
0.18
0.57
2.30

524
937
ACTGAGTAGTTTGCATTGGA
3
2
NC
NC
17.32
22.63
10.91
6.63
NA
NA
NA

525
938
CACTGAGTAGTTTGCATTGG
3
2
NC
NC
18.09
6.98
9.60
0.74
0.21
0.59
1.92

526
939
TCACTGAGTAGTTTGCATTG
2
2
NC
NC
23.66
6.34
5.99
0.88
NA
NA
NA

528
941
CTTCACTGAGTAGTTTGCAT
2
2
NC
NC
20.60
5.79
5.55
0.97
NA
NA
NA

529
942
TCTTCACTGAGTAGTTTGCA
2
2
NC
NC
35.84
5.65
6.31
0.14
NA
NA
NA

530
943
TTCTTCACTGAGTAGTTTGC
2
2
NC
NC
23.87
6.06
8.09
0.54
NA
NA
NA

542
965
GATGAAGAGTAAGAAGATGC
2
2
NC
NC
59.03
27.28
7.69
0.66
NA
NA
NA

545
968
GTTGATGAAGAGTAAGAAGA
2
2
NC
NC
57.72
23.10
6.90
6.32
NA
NA
NA

546
969
GGTTGATGAAGAGTAAGAAG
2
2
NC
NC
28.64
14.90
3.72
2.23
NA
NA
NA

547
970
TGGTTGATGAAGAGTAAGAA
2
2
NC
NC
43.23
11.01
4.88
0.60
NA
NA
NA

548
971
ATGGTTGATGAAGAGTAAGA
2
2
NC
NC
46.69
10.82
3.46
3.97
NA
NA
NA

549
972
GATGGTTGATGAAGAGTAAG
2
3
NC
NC
47.75
24.18
3.27
1.12
NA
NA
NA

551
974
ACGATGGTTGATGAAGAGTA
2
2
NC
NC
42.40
24.00
4.91
0.51
NA
NA
NA

552
975
GACGATGGTTGATGAAGAGT
3
1
NC
NC
51.66
19.10
4.23
3.08
NA
NA
NA

553
976
AGACGATGGTTGATGAAGAG
2
1
NC
NC
54.48
23.21
3.59
3.18
NA
NA
NA

554
977
CAGACGATGGTTGATGAAGA
2
2
NC
NC
50.61
16.47
1.40
0.72
NA
NA
NA

555
988
GTTGATTCTACCAGACGATG
3
2
NC
NC
26.32
16.83
2.41
0.90
NA
NA
NA

556
989
AGTTGATTCTACCAGACGAT
3
3
NC
NC
33.47
20.92
3.68
3.43
NA
NA
NA

557
990
AAGTTGATTCTACCAGACGA
3
2
NC
NC
37.29
13.99
5.68
1.26
NA
NA
NA

558
991
GAAGTTGATTCTACCAGACG
3
2
NC
NC
36.83
15.81
3.45
1.18
NA
NA
NA

559
992
GGAAGTTGATTCTACCAGAC
2
3
NC
NC
25.57
25.66
2.82
2.36
NA
NA
NA

560
993
AGGAAGTTGATTCTACCAGA
2
2
NC
NC
28.87
35.49
2.10
3.49
NA
NA
NA

563
1004
GGCTTTTCTCAAGGAAGTTG
2
1
NC
NC
8.42
23.13
1.20
3.51
NA
NA
NA

564
1005
TGGCTTTTCTCAAGGAAGTT
2
1
NC
NC
14.63
6.07
0.56
0.65
0.16
0.50
1.77

565
1006
ATGGCTTTTCTCAAGGAAGT
2
2
NC
NC
18.29
12.40
2.18
0.59
NA
NA
NA

585
1026
AGGCTGCATACACTGTTTCT
2
2
NC
NC
16.83
19.17
0.67
1.67
NA
NA
NA

586
1027
TAGGCTGCATACACTGTTTC
2
2
NC
NC
24.27
6.40
2.68
0.47
NA
NA
NA

596
1048
GGGTGTGTGTTTTTGGGCAA
2
2
2
NC
30.61
15.73
3.25
1.08
NA
NA
NA

597
1049
TGGGTGTGTGTTTTTGGGCA
2
3
2
NC
30.19
17.13
5.05
3.45
NA
NA
NA

598
1050
ATGGGTGTGTGTTTTTGGGC
2
2
2
NC
36.90
16.65
4.61
0.98
NA
NA
NA

599
1051
AATGGGTGTGTGTTTTTGGG
1
2
2
NC
75.12
40.31
8.71
4.14
NA
NA
NA

600
1052
GAATGGGTGTGTGTTTTTGG
1
2
2
NC
51.82
21.13
2.75
1.33
NA
NA
NA

601
1053
GGAATGGGTGTGTGTTTTTG
1
2
2
NC
71.06
21.58
4.82
1.14
NA
NA
NA

602
1054
AGGAATGGGTGTGTGTTTTT
2
2
2
NC
80.06
23.02
12.04
1.64
NA
NA
NA

603
1055
CAGGAATGGGTGTGTGTTTT
2
2
2
NC
53.62
10.12
5.42
2.81
NA
NA
NA

604
1056
ACAGGAATGGGTGTGTGTTT
2
2
1
NC
37.48
17.43
5.74
1.40
NA
NA
NA

605
1057
TACAGGAATGGGTGTGTGTT
2
2
1
NC
42.23
11.94
5.61
0.34
NA
NA
NA

606
1058
GTACAGGAATGGGTGTGTGT
1
2
1
NC
28.51
10.08
3.84
1.77
NA
NA
NA

607
1059
GGTACAGGAATGGGTGTGTG
2
2
1
NC
15.96
10.82
3.37
0.71
0.12
0.43
2.70

608
1060
AGGTACAGGAATGGGTGTGT
2
2
2
NC
14.46
14.35
2.43
1.39
NA
NA
NA

609
1061
GAGGTACAGGAATGGGTGTG
1
1
1
NC
20.90
11.65
1.55
1.31
NA
NA
NA

610
1062
TGAGGTACAGGAATGGGTGT
2
1
2
NC
30.84
14.99
3.61
1.31
NA
NA
NA

613
1075
CTGATTTCTAAACTGAGGTA
2
2
NC
NC
27.75
14.81
2.61
2.70
NA
NA
NA

619
1100
CACATTAACATCCACATTCT
2
2
NC
NC
68.79
13.92
7.77
0.84
NA
NA
NA

620
1101
GCACATTAACATCCACATTC
2
2
NC
NC
26.29
5.22
3.88
0.61
NA
NA
NA

621
1102
TGCACATTAACATCCACATT
2
2
NC
NC
38.95
9.02
5.64
0.72
NA
NA
NA

622
1103
GTGCACATTAACATCCACAT
2
2
NC
NC
20.30
3.66
3.65
0.50
0.11
0.57
3.11

631
1180
AGCTTGCTCTCGATGTGCTG
2
2
NC
NC
20.00
11.70
0.92
1.03
NA
NA
NA

632
1181
GAGCTTGCTCTCGATGTGCT
2
1
NC
NC
18.61
13.08
1.41
0.35
NA
NA
NA

633
1183
AGGAGCTTGCTCTCGATGTG
2
2
NC
NC
32.71
27.99
2.64
2.85
NA
NA
NA

634
1184
CAGGAGCTTGCTCTCGATGT
2
2
NC
NC
26.82
7.09
5.46
0.23
NA
NA
NA

636
1221
AAGTCTGGGTGAAGTACATC
2
2
NC
NC
46.64
16.14
3.71
0.79
NA
NA
NA

637
1222
AAAGTCTGGGTGAAGTACAT
2
2
NC
NC
52.38
18.35
4.92
1.70
NA
NA
NA

639
1224
GCAAAGTCTGGGTGAAGTAC
2
2
NC
NC
44.44
14.69
4.54
0.85
NA
NA
NA

640
1225
AGCAAAGTCTGGGTGAAGTA
2
2
NC
NC
30.68
13.69
6.16
1.26
NA
NA
NA

641
1226
TAGCAAAGTCTGGGTGAAGT
2
2
NC
NC
62.09
14.36
4.74
3.48
NA
NA
NA

642
1227
GTAGCAAAGTCTGGGTGAAG
2
2
NC
NC
42.69
15.79
7.89
0.84
NA
NA
NA

643
1228
GGTAGCAAAGTCTGGGTGAA
3
1
NC
NC
48.33
17.35
2.47
2.58
NA
NA
NA

644
1229
TGGTAGCAAAGTCTGGGTGA
2
2
NC
NC
87.84
51.60
14.42
2.89
NA
NA
NA

645
1230
CTGGTAGCAAAGTCTGGGTG
2
2
NC
NC
64.82
14.18
2.07
1.58
NA
NA
NA

646
1231
CCTGGTAGCAAAGTCTGGGT
2
2
NC
NC
50.97
9.68
2.00
0.56
NA
NA
NA

648
1233
GTCCTGGTAGCAAAGTCTGG
2
1
NC
NC
103.16
104.11
21.01
5.51
NA
NA
NA

649
1234
AGTCCTGGTAGCAAAGTCTG
2
2
NC
NC
29.71
22.82
2.31
17.24
NA
NA
NA

650
1235
AAGTCCTGGTAGCAAAGTCT
2
1
NC
NC
36.20
12.72
2.05
2.73
NA
NA
NA

651
1236
CAAGTCCTGGTAGCAAAGTC
2
2
NC
NC
66.63
8.37
12.78
3.25
NA
NA
NA

652
1237
GCAAGTCCTGGTAGCAAAGT
2
2
NC
NC
103.63
96.70
3.66
11.00
NA
NA
NA

655
1264
GATTTAACCATCTCCCCAGA
2
2
NC
NC
70.20
10.46
16.79
3.94
NA
NA
NA

699
1327
ATCTGGTGGGCATAGACCTT
2
2
NC
NC
30.23
7.23
13.94
2.66
NA
NA
NA

701
1342
GAATCTGTACGAACCATCTG
3
2
NC
NC
45.66
8.44
5.65
1.13
NA
NA
NA

702
1343
GGAATCTGTACGAACCATCT
3
2
NC
NC
27.72
21.88
6.29
2.90
NA
NA
NA

703
1344
GGGAATCTGTACGAACCATC
3
2
NC
NC
24.62
19.88
7.13
3.82
NA
NA
NA

704
1345
CGGGAATCTGTACGAACCAT
2
2
NC
NC
17.58
5.61
12.65
0.43
0.43
0.89
2.07

705
1346
CCGGGAATCTGTACGAACCA
2
1
NC
NC
16.59
6.79
10.96
0.64
0.18
0.58
2.05

706
1348
TCCCGGGAATCTGTACGAAC
3
2
NC
NC
24.79
5.60
6.98
0.72
NA
NA
NA

707
1360
TCAAGCTTCTGTTCCCGGGA
2
2
NC
NC
12.85
3.94
9.71
1.28
0.12
0.37
1.21

708
1361
ATCAAGCTTCTGTTCCCGGG
3
2
NC
NC
18.31
4.97
12.17
1.68
0.05
0.31
2.21

709
1362
CATCAAGCTTCTGTTCCCGG
3
2
NC
NC
21.42
3.53
15.57
1.24
0.16
0.56
2.02

714
1385
TTTGCTCAGAGGCTGCAGAA
2
2
NC
NC
35.98
7.53
20.55
1.36
NA
NA
NA

715
1386
GTTTGCTCAGAGGCTGCAGA
2
2
NC
NC
23.47
16.64
10.07
2.36
NA
NA
NA

729
1436
AATATCTGTCTTATCCTCTG
2
2
NC
NC
32.04
9.29
15.39
1.91
NA
NA
NA

730
1437
AAATATCTGTCTTATCCTCT
2
2
NC
NC
56.32
14.47
15.52
6.36
NA
NA
NA

731
1438
GAAATATCTGTCTTATCCTC
2
1
NC
NC
59.58
10.23
12.09
2.51
NA
NA
NA

733
1440
TAGAAATATCTGTCTTATCC
2
2
NC
NC
58.24
6.97
14.67
1.93
NA
NA
NA

734
1441
CTAGAAATATCTGTCTTATC
2
2
NC
NC
46.68
4.87
22.95
1.53
NA
NA
NA

735
1442
ACTAGAAATATCTGTCTTAT
2
1
NC
NC
64.72
20.25
12.11
2.66
NA
NA
NA

736
1443
CACTAGAAATATCTGTCTTA
2
2
NC
NC
59.89
8.56
11.79
1.46
NA
NA
NA

737
1444
CCACTAGAAATATCTGTCTT
2
3
NC
NC
42.87
7.05
13.20
0.77
NA
NA
NA

738
1445
GCCACTAGAAATATCTGTCT
2
2
NC
NC
19.98
7.94
12.63
0.87
NA
NA
NA

739
1446
TGCCACTAGAAATATCTGTC
2
2
NC
NC
19.72
5.09
12.98
1.18
0.30
0.88
2.79

740
1447
CTGCCACTAGAAATATCTGT
2
2
NC
NC
37.06
9.49
17.50
0.79
NA
NA
NA

741
1448
CCTGCCACTAGAAATATCTG
2
2
NC
NC
83.03
28.38
11.66
10.64
NA
NA
NA

744
1452
TAGCCCTGCCACTAGAAATA
2
2
NC
NC
60.02
13.91
18.36
5.76
NA
NA
NA

749
1471
ATCTCCTCATCTTGCTGCCT
2
2
NC
NC
33.49
8.67
16.34
4.13
NA
NA
NA

752
1476
CAAGCATCTCCTCATCTTGC
2
2
NC
NC
13.59
4.15
10.09
1.06
0.25
0.75
2.33

753
1477
TCAAGCATCTCCTCATCTTG
2
2
NC
NC
27.55
4.49
15.73
1.24
NA
NA
NA

754
1478
TTCAAGCATCTCCTCATCTT
2
2
NC
NC
67.59
13.44
15.97
4.55
NA
NA
NA

757
1481
GAGTTCAAGCATCTCCTCAT
2
2
NC
NC
62.60
11.36
19.21
2.88
NA
NA
NA

762
1510
TTTTTGGCAGCCACTTCAGC
2
1
NC
NC
114.79
103.00
20.71
7.21
NA
NA
NA

763
1511
ATTTTTGGCAGCCACTTCAG
2
2
NC
NC
98.71
68.38
5.39
9.84
NA
NA
NA

765
1513
TGATTTTTGGCAGCCACTTC
2
2
NC
NC
36.01
5.20
10.64
0.75
NA
NA
NA

766
1514
CTGATTTTTGGCAGCCACTT
2
2
NC
NC
30.25
4.45
8.05
1.22
NA
NA
NA

768
1516
CTCTGATTTTTGGCAGCCAC
2
2
NC
NC
13.07
4.62
1.60
0.22
0.22
0.68
3.04

769
1517
GCTCTGATTTTTGGCAGCCA
2
2
NC
NC
8.44
8.18
1.89
0.27
0.14
0.53
2.32

788
1539
CCTTTGTTGTATCCCCCTCC
2
2
NC
NC
20.21
4.38
5.39
1.58
0.34
1.01
3.42

790
1541
CCCCTTTGTTGTATCCCCCT
2
2
NC
NC
20.62
5.37
3.13
0.98
NA
NA
NA

791
1542
TCCCCTTTGTTGTATCCCCC
2
2
NC
NC
23.48
7.41
3.97
2.54
NA
NA
NA

792
1543
GTCCCCTTTGTTGTATCCCC
2
1
NC
NC
100.47
88.37
8.84
6.17
NA
NA
NA

819
1570
GGTCCTCTCTTCTCTGACAT
2
2
NC
NC
58.63
11.34
7.04
2.35
NA
NA
NA

827
1603
TCCCGATGTCTCTTTCTGGG
2
2
NC
NC
16.05
3.67
9.04
0.41
0.16
0.52
1.89

828
1604
TTCCCGATGTCTCTTTCTGG
2
3
NC
NC
33.73
4.04
13.16
0.43
NA
NA
NA

829
1605
CTTCCCGATGTCTCTTTCTG
2
2
NC
NC
53.70
8.42
13.79
0.91
NA
NA
NA

830
1606
TCTTCCCGATGTCTCTTTCT
2
2
NC
NC
77.10
8.94
15.70
1.81
NA
NA
NA

831
1607
ATCTTCCCGATGTCTCTTTC
2
2
NC
NC
60.00
8.02
12.80
2.53
NA
NA
NA

832
1608
AATCTTCCCGATGTCTCTTT
2
1
NC
NC
65.71
6.74
13.39
1.53
NA
NA
NA

833
1609
GAATCTTCCCGATGTCTCTT
2
2
NC
NC
52.67
5.55
14.38
1.31
NA
NA
NA

834
1610
AGAATCTTCCCGATGTCTCT
2
2
NC
NC
46.94
8.07
12.68
0.51
NA
NA
NA

835
1611
CAGAATCTTCCCGATGTCTC
3
2
NC
NC
45.90
5.58
11.41
0.83
NA
NA
NA

840
1617
CCACATCAGAATCTTCCCGA
2
2
NC
NC
42.29
6.08
12.23
0.95
NA
NA
NA

841
1618
TCCACATCAGAATCTTCCCG
2
2
NC
NC
23.05
8.39
1.20
4.40
NA
NA
NA

842
1619
TTCCACATCAGAATCTTCCC
2
2
NC
NC
38.94
8.19
2.71
1.41
NA
NA
NA

845
1622
CATTTCCACATCAGAATCTT
2
3
NC
NC
80.41
16.41
4.15
3.07
NA
NA
NA

847
1624
ACCATTTCCACATCAGAATC
2
2
NC
NC
68.04
10.71
5.25
0.84
NA
NA
NA

850
1627
TCCACCATTTCCACATCAGA
2
2
2
NC
35.73
7.84
4.97
0.77
NA
NA
NA

852
1629
CTTCCACCATTTCCACATCA
2
3
NC
NC
50.97
15.33
2.65
2.10
NA
NA
NA

853
1630
TCTTCCACCATTTCCACATC
2
2
NC
NC
69.10
22.59
9.13
3.49
NA
NA
NA

857
1634
ATCATCTTCCACCATTTCCA
2
3
NC
NC
56.50
17.20
3.57
2.80
NA
NA
NA

859
1636
GAATCATCTTCCACCATTTC
2
2
NC
NC
51.18
9.63
4.18
0.25
NA
NA
NA

861
1655
TGCAGTCATTTCCTTTCGGG
2
2
NC
NC
8.10
4.68
1.74
0.31
0.16
0.47
1.74

866
1660
CAAGCTGCAGTCATTTCCTT
2
2
NC
NC
31.03
5.85
5.88
1.02
NA
NA
NA

867
1661
ACAAGCTGCAGTCATTTCCT
2
1
NC
NC
32.41
5.48
10.00
0.49
NA
NA
NA

868
1662
TACAAGCTGCAGTCATTTCC
2
2
NC
NC
41.44
6.10
8.54
1.49
NA
NA
NA

869
1663
GTACAAGCTGCAGTCATTTC
2
2
NC
NC
31.87
5.92
9.46
0.78
NA
NA
NA

870
1664
GGTACAAGCTGCAGTCATTT
2
2
NC
NC
19.62
6.14
4.64
0.99
NA
NA
NA

871
1665
GGGTACAAGCTGCAGTCATT
2
2
NC
NC
11.39
7.58
0.71
1.44
0.13
0.76
4.61

872
1685
GTTAATGATCCTTCTCCGGG
3
2
NC
NC
16.00
7.44
2.40
0.54
0.10
0.60
3.62

877
1690
GTGAGGTTAATGATCCTTCT
2
1
NC
NC
13.70
6.50
1.42
0.79
0.27
0.86
3.52

878
1691
AGTGAGGTTAATGATCCTTC
2
2
NC
NC
13.57
5.41
2.04
0.84
0.36
1.09
4.03

879
1692
TAGTGAGGTTAATGATCCTT
2
1
NC
NC
19.27
5.60
2.90
0.43
0.33
1.28
5.18

880
1693
CTAGTGAGGTTAATGATCCT
2
1
NC
NC
26.95
8.81
6.37
2.31
NA
NA
NA

881
1694
ACTAGTGAGGTTAATGATCC
2
1
NC
NC
19.52
9.33
1.78
2.03
NA
NA
NA

882
1695
CACTAGTGAGGTTAATGATC
2
2
NC
NC
18.49
6.98
3.33
2.10
0.20
0.70
2.77

884
1708
TGGAGACTCAAAACACTAGT
2
2
NC
NC
19.02
7.16
5.41
3.14
NA
NA
NA

885
1709
CTGGAGACTCAAAACACTAG
2
2
NC
NC
12.85
4.67
1.06
1.02
0.18
0.57
2.04

886
1710
CCTGGAGACTCAAAACACTA
2
1
NC
NC
16.65
5.77
2.87
1.31
0.25
0.85
3.32

887
1711
TCCTGGAGACTCAAAACACT
2
2
NC
NC
21.75
5.92
2.38
1.14
NA
NA
NA

889
1713
CTTCCTGGAGACTCAAAACA
2
2
NC
NC
45.46
9.02
3.36
1.57
NA
NA
NA

890
1714
TCTTCCTGGAGACTCAAAAC
2
2
NC
NC
50.79
12.75
4.92
7.80
NA
NA
NA

891
1715
TTCTTCCTGGAGACTCAAAA
2
1
NC
NC
93.36
103.76
4.23
9.89
NA
NA
NA

893
1717
ATTTCTTCCTGGAGACTCAA
2
2
NC
NC
55.08
6.98
5.19
0.87
NA
NA
NA

894
1718
AATTTCTTCCTGGAGACTCA
2
2
NC
NC
61.08
7.27
12.91
0.98
NA
NA
NA

901
1725
GCTCATTAATTTCTTCCTGG
2
2
NC
NC
20.13
5.24
3.78
0.52
0.09
0.53
2.92

923
1762
TGGTTATGCAACATCTCCCG
2
1
NC
NC
17.35
18.34
5.47
1.94
NA
NA
NA

924
1763
GTGGTTATGCAACATCTCCC
2
2
NC
NC
16.20
19.58
4.19
1.63
NA
NA
NA

925
1764
AGTGGTTATGCAACATCTCC
2
2
NC
NC
23.69
18.86
5.61
1.94
NA
NA
NA

926
1765
GAGTGGTTATGCAACATCTC
2
2
NC
NC
20.41
17.59
3.21
2.30
NA
NA
NA

927
1766
GGAGTGGTTATGCAACATCT
2
1
NC
NC
17.13
17.59
2.07
2.18
NA
NA
NA

928
1767
AGGAGTGGTTATGCAACATC
2
1
NC
NC
23.83
14.79
3.38
1.89
NA
NA
NA

929
1768
AAGGAGTGGTTATGCAACAT
2
2
NC
NC
42.95
13.46
5.84
1.42
NA
NA
NA

930
1769
GAAGGAGTGGTTATGCAACA
2
1
NC
NC
41.94
14.80
3.77
4.79
NA
NA
NA

931
1770
CGAAGGAGTGGTTATGCAAC
2
1
NC
NC
23.28
9.91
4.22
1.39
NA
NA
NA

936
1789
TGAGGATTCACACAGCCCAC
2
2
2
2
17.04
13.00
2.05
1.38
NA
NA
NA

937
1790
CTGAGGATTCACACAGCCCA
2
2
1
1
15.87
12.83
1.05
0.64
NA
NA
NA

938
1791
ACTGAGGATTCACACAGCCC
2
2
2
2
18.66
12.90
1.15
2.32
NA
NA
NA

939
1792
CACTGAGGATTCACACAGCC
2
NC
2
2
31.16
9.98
2.79
1.29
NA
NA
NA

975
1855
AGTTCTTCACTAAGCTTGGT
2
2
NC
NC
29.52
19.93
4.25
1.72
NA
NA
NA

977
1857
ACAGTTCTTCACTAAGCTTG
2
2
NC
NC
39.47
11.25
3.26
0.49
NA
NA
NA

981
1874
AATGAGTATCTGGTAGAACA
2
2
2
NC
33.56
13.02
2.09
1.89
NA
NA
NA

982
1875
AAATGAGTATCTGGTAGAAC
2
2
2
NC
49.15
8.98
12.33
2.27
NA
NA
NA

983
1876
TAAATGAGTATCTGGTAGAA
1
1
2
1
55.92
10.30
5.29
2.68
NA
NA
NA

984
1877
ATAAATGAGTATCTGGTAGA
1
2
1
NC
38.93
8.65
9.76
0.97
NA
NA
NA

985
1878
CATAAATGAGTATCTGGTAG
2
2
1
NC
21.43
8.65
2.82
0.30
NA
NA
NA

986
1879
TCATAAATGAGTATCTGGTA
2
2
2
NC
14.28
7.79
1.81
0.61
0.29
0.77
2.32

1019
1962
TCTCTGGACTATCTAAGGCA
2
2
NC
NC
22.18
9.04
3.66
1.64
NA
NA
NA

1023
1979
TTCCTCTGTCCAGCCACTCT
2
1
NC
NC
50.08
15.27
4.30
3.94
NA
NA
NA

1024
1981
TCTTCCTCTGTCCAGCCACT
2
2
NC
NC
44.05
13.91
9.38
2.13
NA
NA
NA

1025
1982
ATCTTCCTCTGTCCAGCCAC
2
2
NC
NC
37.17
11.97
2.26
1.04
NA
NA
NA

1026
1983
CATCTTCCTCTGTCCAGCCA
2
1
NC
NC
32.70
11.63
4.73
0.83
NA
NA
NA

1027
1985
ACCATCTTCCTCTGTCCAGC
2
2
NC
NC
50.53
26.55
3.60
11.81
NA
NA
NA

1028
1986
GACCATCTTCCTCTGTCCAG
2
2
NC
NC
21.33
16.03
3.72
5.90
NA
NA
NA

1029
1989
TGGGACCATCTTCCTCTGTC
2
2
NC
NC
19.62
18.29
3.08
1.29
NA
NA
NA

1030
1990
TTGGGACCATCTTCCTCTGT
2
2
NC
NC
22.13
16.50
4.81
1.37
NA
NA
NA

1031
1991
TTTGGGACCATCTTCCTCTG
2
2
NC
NC
22.09
15.25
4.49
0.96
NA
NA
NA

1032
1992
CTTTGGGACCATCTTCCTCT
2
1
NC
NC
28.68
15.17
4.36
3.55
NA
NA
NA

1034
1994
TTCTTTGGGACCATCTTCCT
2
2
NC
NC
60.24
14.44
3.65
4.90
NA
NA
NA

1036
1996
CCTTCTTTGGGACCATCTTC
2
2
NC
NC
48.98
12.78
3.77
4.72
NA
NA
NA

1037
1997
TCCTTCTTTGGGACCATCTT
2
1
NC
NC
52.70
12.42
9.46
1.56
NA
NA
NA

1038
2004
CAGCAAGTCCTTCTTTGGGA
2
2
NC
NC
10.45
9.16
1.21
2.08
0.08
0.28
1.26

1039
2005
TCAGCAAGTCCTTCTTTGGG
2
2
NC
NC
20.27
8.64
1.87
2.15
NA
NA
NA

1040
2006
TTCAGCAAGTCCTTCTTTGG
2
1
NC
NC
27.79
11.33
4.38
2.46
NA
NA
NA

1041
2007
ATTCAGCAAGTCCTTCTTTG
2
2
NC
NC
33.30
14.07
3.99
1.39
NA
NA
NA

1083
2056
AAATAGTCTGCAAGCATCTC
1
2
2
NC
41.38
11.90
4.33
0.52
NA
NA
NA

1084
2057
GAAATAGTCTGCAAGCATCT
2
2
2
2
38.58
13.29
10.20
0.46
NA
NA
NA

1085
2058
AGAAATAGTCTGCAAGCATC
2
2
2
2
29.84
12.15
4.81
1.58
NA
NA
NA

1086
2059
GAGAAATAGTCTGCAAGCAT
2
2
2
3
27.63
15.67
4.88
0.97
NA
NA
NA

1087
2060
AGAGAAATAGTCTGCAAGCA
1
NC
2
2
33.86
14.04
6.29
1.40
NA
NA
NA

1089
2062
AAAGAGAAATAGTCTGCAAG
2
2
NC
NC
58.41
17.12
7.29
1.75
NA
NA
NA

1101
2081
CCCTTCCTCATCAATTTCCA
2
1
NC
NC
31.25
15.64
3.22
3.31
NA
NA
NA

1104
2084
GTTCCCTTCCTCATCAATTT
2
2
NC
NC
57.38
29.32
9.91
8.48
NA
NA
NA

1105
2085
GGTTCCCTTCCTCATCAATT
2
2
NC
NC
53.96
27.59
5.68
2.75
NA
NA
NA

1107
2087
CAGGTTCCCTTCCTCATCAA
2
2
NC
NC
49.11
27.64
7.95
7.29
NA
NA
NA

1109
2089
ATCAGGTTCCCTTCCTCATC
2
1
2
2
37.95
16.43
3.84
2.36
NA
NA
NA

1110
2090
AATCAGGTTCCCTTCCTCAT
2
2
2
1
66.42
16.39
6.57
1.24
NA
NA
NA

1111
2091
CAATCAGGTTCCCTTCCTCA
1
2
1
1
68.17
14.98
7.69
1.22
NA
NA
NA

1112
2092
CCAATCAGGTTCCCTTCCTC
2
2
1
1
55.69
19.51
3.53
1.71
NA
NA
NA

1118
2122
GGCACATAGTTGTCAATCAG
2
1
NC
NC
21.89
18.52
0.89
1.27
NA
NA
NA

1119
2123
GGGCACATAGTTGTCAATCA
2
3
NC
NC
20.24
17.60
1.01
2.95
NA
NA
NA

1120
2151
GAATGAAGATAGGCAGTCCC
2
NC
2
2
50.96
24.90
7.53
4.47
NA
NA
NA

1121
2152
AGAATGAAGATAGGCAGTCC
1
2
2
2
58.01
24.14
9.59
1.04
NA
NA
NA

1122
2153
AAGAATGAAGATAGGCAGTC
1
2
2
2
53.74
21.10
4.80
1.67
NA
NA
NA

1123
2154
GAAGAATGAAGATAGGCAGT
2
2
2
1
38.75
25.89
4.63
5.64
NA
NA
NA

1131
2186
TTCTTCGTCCCAATTCACCT
2
2
NC
NC
38.44
15.09
1.32
1.88
NA
NA
NA

1132
2187
TTTCTTCGTCCCAATTCACC
2
2
NC
NC
58.53
13.07
4.21
1.69
NA
NA
NA

1133
2188
TTTTCTTCGTCCCAATTCAC
2
2
NC
NC
62.52
11.90
2.87
2.21
NA
NA
NA

1134
2189
CTTTTCTTCGTCCCAATTCA
2
1
NC
NC
50.58
14.22
3.94
1.36
NA
NA
NA

1136
2191
TCCTTTTCTTCGTCCCAATT
2
2
NC
NC
29.80
9.87
5.01
1.91
NA
NA
NA

1138
2193
ATTCCTTTTCTTCGTCCCAA
2
2
NC
NC
24.10
9.76
1.86
1.37
NA
NA
NA

1139
2194
CATTCCTTTTCTTCGTCCCA
2
2
NC
NC
24.26
12.60
2.76
1.60
NA
NA
NA

1140
2195
ACATTCCTTTTCTTCGTCCC
2
2
NC
NC
23.58
13.32
3.03
1.54
NA
NA
NA

1141
2196
AACATTCCTTTTCTTCGTCC
2
2
NC
NC
27.14
15.58
5.19
6.93
NA
NA
NA

1142
2197
AAACATTCCTTTTCTTCGTC
2
2
NC
NC
41.22
12.84
5.46
2.51
NA
NA
NA

1143
2198
AAAACATTCCTTTTCTTCGT
2
2
NC
NC
50.01
16.16
2.75
1.08
NA
NA
NA

1144
2199
CAAAACATTCCTTTTCTTCG
2
1
NC
NC
58.85
17.98
2.27
4.10
NA
NA
NA

1151
2206
AGGCTTTCAAAACATTCCTT
2
2
NC
NC
15.91
23.64
1.46
4.96
NA
NA
NA

1152
2207
GAGGCTTTCAAAACATTCCT
2
2
NC
NC
15.81
17.27
0.99
1.57
NA
NA
NA

1161
2216
TTCTTTACTGAGGCTTTCAA
2
2
NC
NC
53.76
11.00
4.27
0.65
NA
NA
NA

1162
2217
ATTCTTTACTGAGGCTTTCA
2
3
NC
NC
38.54
11.47
4.34
0.84
NA
NA
NA

1163
2218
CATTCTTTACTGAGGCTTTC
2
2
NC
NC
31.98
13.25
2.97
6.33
NA
NA
NA

1186
2252
TATGTACTGCTTCCGGATGG
2
2
NC
NC
95.26
56.34
6.20
3.23
NA
NA
NA

1187
2253
ATATGTACTGCTTCCGGATG
3
2
NC
NC
28.54
16.64
3.08
0.51
NA
NA
NA

1188
2254
GATATGTACTGCTTCCGGAT
3
2
NC
NC
14.73
12.13
1.00
1.15
NA
NA
NA

1189
2255
AGATATGTACTGCTTCCGGA
3
2
NC
NC
13.10
15.70
2.36
1.00
NA
NA
NA

1190
2256
CAGATATGTACTGCTTCCGG
3
3
NC
NC
13.11
17.10
2.53
1.89
NA
NA
NA

1191
2257
TCAGATATGTACTGCTTCCG
2
1
NC
NC
17.38
16.72
2.61
4.11
NA
NA
NA

1192
2258
CTCAGATATGTACTGCTTCC
2
2
NC
NC
69.79
31.46
8.14
3.15
NA
NA
NA

1194
2260
TCCTCAGATATGTACTGCTT
2
2
NC
NC
16.09
11.05
2.51
0.85
NA
NA
NA

1195
2261
CTCCTCAGATATGTACTGCT
2
2
NC
NC
25.07
9.13
4.41
1.25
NA
NA
NA

1197
2264
CGACTCCTCAGATATGTACT
2
1
NC
NC
24.65
17.60
4.57
1.73
NA
NA
NA

1199
2266
GTCGACTCCTCAGATATGTA
3
2
NC
NC
28.41
15.85
10.67
1.47
NA
NA
NA

1200
2267
GGTCGACTCCTCAGATATGT
3
2
NC
NC
21.13
23.68
5.91
8.80
NA
NA
NA

1201
2268
GGGTCGACTCCTCAGATATG
3
2
NC
NC
20.89
20.52
1.26
3.02
NA
NA
NA

1204
2302
ATGGAGCCAGGCACTTCACT
2
1
NC
NC
19.49
21.47
2.53
2.55
NA
NA
NA

1205
2303
AATGGAGCCAGGCACTTCAC
2
2
NC
NC
93.76
101.43
5.12
3.90
NA
NA
NA

1207
2306
TGGAATGGAGCCAGGCACTT
2
3
NC
NC
29.60
12.81
6.39
1.54
NA
NA
NA

1209
2309
GTTTGGAATGGAGCCAGGCA
2
2
NC
NC
16.63
15.24
1.52
1.59
NA
NA
NA

1214
2314
CAGGAGTTTGGAATGGAGCC
2
3
NC
NC
21.19
27.52
1.29
6.83
NA
NA
NA

1215
2324
AGTCCACTTCCAGGAGTTTG
2
2
NC
NC
26.71
20.02
1.48
1.35
NA
NA
NA

1216
2325
CAGTCCACTTCCAGGAGTTT
2
2
NC
NC
31.06
18.06
2.49
1.71
NA
NA
NA

1218
2329
TCCACAGTCCACTTCCAGGA
2
2
NC
NC
11.76
17.94
1.42
2.33
NA
NA
NA

1220
2331
GTTCCACAGTCCACTTCCAG
2
3
NC
NC
19.73
20.38
3.60
2.39
NA
NA
NA

1221
2332
TGTTCCACAGTCCACTTCCA
2
2
NC
NC
24.30
16.77
5.32
1.82
NA
NA
NA

1222
2333
GTGTTCCACAGTCCACTTCC
2
2
NC
NC
19.42
20.48
2.47
2.57
NA
NA
NA

1223
2334
TGTGTTCCACAGTCCACTTC
2
1
NC
NC
18.25
19.92
3.39
1.35
NA
NA
NA

1239
2385
CTGTGAAATGTTTAGGAGGC
2
2
NC
NC
17.75
16.86
2.28
1.34
NA
NA
NA

1240
2386
TCTGTGAAATGTTTAGGAGG
2
2
NC
NC
24.18
18.40
4.91
1.27
NA
NA
NA

1241
2387
TTCTGTGAAATGTTTAGGAG
2
2
NC
NC
22.14
10.77
4.67
1.23
NA
NA
NA

1244
2390
ATCTTCTGTGAAATGTTTAG
2
2
NC
NC
26.27
13.45
4.96
0.97
NA
NA
NA

1247
2393
TCCATCTTCTGTGAAATGTT
2
2
NC
NC
13.38
12.40
2.16
1.25
NA
NA
NA

1257
2427
ATAGATCAGGCAGGTTAGCA
2
1
NC
NC
19.89
12.32
2.46
1.14
NA
NA
NA

1258
2428
TATAGATCAGGCAGGTTAGC
2
3
NC
NC
14.37
13.81
1.03
0.70
NA
NA
NA

1259
2429
GTATAGATCAGGCAGGTTAG
2
2
NC
NC
14.45
11.68
2.07
0.68
NA
NA
NA

1260
2430
TGTATAGATCAGGCAGGTTA
2
2
NC
NC
87.68
94.97
8.52
4.62
NA
NA
NA

1262
2432
TTTGTATAGATCAGGCAGGT
2
2
NC
NC
20.44
14.15
1.28
0.62
NA
NA
NA

1263
2433
CTTTGTATAGATCAGGCAGG
2
2
NC
NC
13.38
11.53
0.70
0.95
0.14
0.55
5.60

1264
2434
ACTTTGTATAGATCAGGCAG
2
2
NC
NC
13.58
13.18
0.30
1.02
NA
NA
NA

1265
2435
GACTTTGTATAGATCAGGCA
2
2
NC
NC
11.27
11.30
0.53
1.50
0.07
0.33
2.30

1266
2436
AGACTTTGTATAGATCAGGC
3
2
NC
NC
10.32
10.49
1.98
2.17
0.05
0.15
0.72

1267
2437
AAGACTTTGTATAGATCAGG
2
2
NC
NC
14.64
10.64
3.42
0.73
0.08
0.42
3.39

1268
2438
AAAGACTTTGTATAGATCAG
2
1
NC
NC
27.08
13.81
5.51
4.33
NA
NA
NA

1269
2439
CAAAGACTTTGTATAGATCA
2
2
NC
NC
17.03
11.10
11.35
2.93
NA
NA
NA

1270
2449
TAACACCTCTCAAAGACTTT
2
1
NC
NC
46.54
21.29
5.71
9.16
NA
NA
NA

1273
2452
ATTTAACACCTCTCAAAGAC
2
3
NC
NC
51.39
22.12
7.97
6.64
NA
NA
NA

1274
2453
TATTTAACACCTCTCAAAGA
2
2
NC
NC
63.35
21.72
10.62
11.87
NA
NA
NA

1275
2454
ATATTTAACACCTCTCAAAG
2
3
NC
NC
56.98
20.59
6.68
4.03
NA
NA
NA

1277
2456
CCATATTTAACACCTCTCAA
2
2
NC
NC
30.16
12.91
6.23
5.01
NA
NA
NA

1278
2457
ACCATATTTAACACCTCTCA
2
1
NC
NC
22.23
13.08
2.74
4.47
NA
NA
NA

1314
2513
CAACACTTTGTATCGGAATA
3
1
NC
NC
27.79
11.30
2.74
2.91
NA
NA
NA

1315
2524
ACACTTTGATACAACACTTT
2
2
NC
NC
27.55
10.61
6.34
2.30
NA
NA
NA

1343
2576
AAGTATAAGTCTTAAGTGCT
2
2
NC
NC
45.01
19.13
5.01
2.14
NA
NA
NA

Example 3. In Vitro Screen for Reduced Expansion

Expansion of DNA triplet repeats can be replicated in vitro using patient-derived cells lines and DNA-damaging agents. Human fibroblasts from Huntington's (GM04281, GM04687 and GM04212) or Friedreich's Ataxia patients (GM03816 and GM02153) or Myotonic dystrophy) (GM04602, GM03987 and GM03989) are purchased from Coriell Cell Repositories and are maintained in medium following the manufacturer's instructions (Kovtum et al., 2007 Nature, 447(7143): 447-452; Li et al., 2016 Biopreservation and Biobanking 14(4):324-29; Zhang et al., 2013 Mol Ther 22(2): 312-320). To induce CAG-repeat expansion in vitro, fibroblast cells are treated with oxidizing agents such as hydrogen peroxide (H₂O₂), potassium chromate (K2CrO₄) or potassium bromate (KBrO₃) for up to 2 hrs (Kovtum et al., ibid). Cells are washed, and medium replace to allow cells to recover for 3 days. The treatment is repeated up to twice more before cells are harvested and DNA isolated. CAG repeat length is determined using methods described below. The effect of dsRNA agents on altering CAG-repeat expansion is measured at different concentrations is compared with controls (mock-transfected and/or control dsRNA at the same concentration as the experimental agent).

Expansion of DNA triplet repeats can be replicated in vitro using patient-derived cell lines. Induced pluripotent stem cells (iPSC) derived from Human fibroblasts from Huntington's Patients (CS09iHD-109n1) are purchased from Cedars-Sinai RMI Induced Pluripotent Stem Cell Core and are maintained following the manufacturer's recommendations (https://www.cedars-sinai.org/content/dam/cedars-sinai/research/documents/biomanufacturing/recommended-guidelines-for-handling-ipscsv1.pdf). The CAG repeat from an iPSC line with 109 CAGs shows an increase in CAG repeat size over time, with an average expansion of 4 CAG repeats over 70 days in dividing iPS cells (Goold et al., 2019 Human Molecular Genetics February 15; 28(4): 650-661).

CS09iHD-109n1 iPSC are treated with either LNP-formulated siRNA or ASO for continuous knockdown of target mRNA and CAG repeat expansion is determined by DNA fragment analysis described below. SiRNAs or ASOs are added to cells in varying concentrations every 3 to 15 days and knockdown of mRNA is determined by RT-qPCR using standard molecular biology techniques. DNA and mRNA are isolated from cells according to standard techniques at t=0.14 days, 28 days, 42 days, 56 days and 80 days. The differences in expansion between treatment and control are compared according to a linear repeated-measures model, and at each time point according to Tukey's post-hoc tests.

Example 4. In Vitro Screening of MLH1 Knockdown

Inhibition or knockdown of MLH1 can be demonstrated using a cell-based assay. For example, HEK293, NIH3T3, or Hela or another available mammalian cell line with dsRNA agents targeting MLH1 identified above in Example 1 using at least five different dose levels, using transfection reagents such as lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Cells are harvested at multiple time points up to 7 days post transfection for either mRNA or protein analyses. Knockdown of mRNA and protein are determined by RT-qPCR or western blot analyses respectively, using standard molecular biology techniques as previously described (see, for example, as described in Drouet et al., 2014, PLOS One 9(6): e99341). The relative levels of the MLH1 mRNA and protein at the different dsRNA levels are compared with a mock oligonucleotide control.

Some siRNA duplexes were evaluated through mRNA knockdown at 10 nM and 0.5 nM, 24 hours after transfection of HeLa cells. The extent of mRNA knockdown by the siRNA duplexes was analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) using TaqMan Gene Expression probes. mRNA expression was calculated via delta-delta Ct(ΔΔCT) method were target expression was doubly normalized to express of the reference gene beta-glucuronidase (GUSB) and cells treated with non-targeting control siRNA.

In Table 13 below, the 3′ U of the antisense oligonucleotide can be any nucleotide (e.g., U, A, G, C, T). In some aspects, the 3′ U of the antisense oligonucleotide in Table 13 is U.

TABLE 13

mean % mRNA
SD % mRNA

SEQ

SEQ

remaining
remaining

ID NO
Sense
Antis
ID NO
Pos
Cyno
Mouse
Rat
0.5 nM
10 nM
0.5 nM
10 nM

1418
GUUGAGAAAUUUGACUGGA
UCCAGUCAAAUUUCUCAAC
1419
24
Yes
No
No
93.57
96.04
16.45
13.53

1424
UUGACUGGCAUUCAAGCUA
UAGCUUGAAUGCCAGUCAA
1425
34
Yes
No
No
84.26
81.83
2.35
4.03

1486
GGCACUUCCGUUGAGCAUA
UAUGCUCAACGGAAGUGCC
1487
149
Yes
No
No
74.89
77.21
10.69
9.46

1492
UUCCGUUGAGCAUCUAGAA
UUCUAGAUGCUCAACGGAA
1493
154
Yes
No
No
70.80
71.54
4.18
4.35

1494
CCGUUGAGCAUCUAGACGA
UCGUCUAGAUGCUCAACGG
1495
156
Yes
No
No
68.36
76.85
34.25
3.81

1496
CGUUGAGCAUCUAGACGUA
UACGUCUAGAUGCUCAACG
1497
157
Yes
No
No
73.04
75.42
6.64
15.70

1514
GGCGCCAAAAUGUCGUUCA
UGAACGACAUUUUGGCGCC
1515
190
Yes
No
No
72.07
53.44
6.64
9.75

1516
CGCCAAAAUGUCGUUCGUA
UACGAACGACAUUUUGGCG
1517
192
Yes
No
No
52.78
57.01
6.55
20.77

1520
GUUCGUGGCAGGGGUUAUA
UAUAACCCCUGCCACGAAC
1521
204
Yes
No
No
101.97
103.73
17.37
30.90

1534
GACGAGACAGUGGUGAACA
UGUUCACCACUGUCUCGUC
1535
232
Yes
No
No
97.77
94.78
17.23
37.47

1536
UAUCCAGCGGCCAGCUAAA
UUUAGCUGGCCGCUGGAUA
1537
270
Yes
No
No
53.67
46.12
6.09
2.32

1544
GCCAGCUAAUGCUAUCAAA
UUUGAUAGCAUUAGCUGGC
1545
279
Yes
No
No
51.95
40.60
1.45
6.83

1546
CCAGCUAAUGCUAUCAAAA
UUUUGAUAGCAUUAGCUGG
1547
280
Yes
No
No
41.77
37.24
6.69
3.42

1560
GCUAUCAAAGAGAUGAUUA
UAAUCAUCUCUUUGAUAGC
1561
289
Yes
No
No
45.40
42.88
1.75
6.91

1562
CUAUCAAAGAGAUGAUUGA
UCAAUCAUCUCUUUGAUAG
1563
290
Yes
No
No
29.69
41.07
4.51
7.30

1564
GAGAUGAUUGAGAACUGUA
UACAGUUCUCAAUCAUCUC
1565
298
Yes
No
No
63.95
51.27
7.12
6.95

1584
UUUAGAUGCAAAAUCCACA
UGUGGAUUUUGCAUCUAAA
1585
315
Yes
No
No
57.43
40.72
2.13
9.46

1598
CCACAAGUAUUCAAGUGAA
UUCACUUGAAUACUUGUGG
1599
329
Yes
No
No
36.95
42.89
1.04
46.13

1604
AAGUAUUCAAGUGAUUGUA
UACAAUCACUUGAAUACUU
1605
333
Yes
No
No
106.88
90.20
8.68
28.46

1608
GUAUUCAAGUGAUUGUUAA
UUAACAAUCACUUGAAUAC
1609
335
Yes
No
No
26.99
34.32
5.14
5.86

1622
GAGGCCUGAAGUUGAUUCA
UGAAUCAACUUCAGGCCUC
1623
359
Yes
No
No
41.86
41.02
11.50
8.02

1624
CUGAAGUUGAUUCAGAUCA
UGAUCUGAAUCAACUUCAG
1625
364
Yes
No
No
62.65
40.21
8.63
6.58

1630
AGUUGAUUCAGAUCCAAGA
UCUUGGAUCUGAAUCAACU
1631
368
Yes
No
No
36.04
31.21
2.32
1.66

1660
UAUUGUAUGUGAAAGGUUA
UAACCUUUCACAUACAAUA
1661
420
Yes
No
No
33.45
26.72
1.93
2.18

1710
GUCCUUUGAGGAUUUAGCA
UGCUAAAUCCUCAAAGGAC
1711
456
Yes
No
No
54.86
47.22
12.21
6.05

1720
UGAGGAUUUAGCCAGUAUA
UAUACUGGCUAAAUCCUCA
1721
462
Yes
Yes
No
37.65
35.63
1.37
4.95

1722
GAGGAUUUAGCCAGUAUUA
UAAUACUGGCUAAAUCCUC
1723
463
Yes
Yes
No
84.59
72.97
20.93
6.80

1726
GGAUUUAGCCAGUAUUUCA
UGAAAUACUGGCUAAAUCC
1727
465
Yes
Yes
No
46.04
37.96
13.78
9.69

1728
GAUUUAGCCAGUAUUUCUA
UAGAAAUACUGGCUAAAUC
1729
466
Yes
Yes
No
40.05
30.46
4.27
2.12

1732
UAGCCAGUAUUUCUACCUA
UAGGUAGAAAUACUGGCUA
1733
470
Yes
Yes
No
34.45
36.15
1.36
19.05

1734
AGCCAGUAUUUCUACCUAA
UUAGGUAGAAAUACUGGCU
1735
471
Yes
Yes
No
63.67
56.73
9.01
5.58

1736
GCCAGUAUUUCUACCUAUA
UAUAGGUAGAAAUACUGGC
1737
472
Yes
Yes
No
41.16
42.84
9.25
5.56

1748
UCUACCUAUGGCUUUCGAA
UUCGAAAGCCAUAGGUAGA
1749
481
Yes
No
No
70.21
64.36
5.76
3.82

1752
UACCUAUGGCUUUCGAGGA
UCCUCGAAAGCCAUAGGUA
1753
483
Yes
No
No
80.86
54.12
10.60
15.67

1756
GCUUUCGAGGUGAGGCUUA
UAAGCCUCACCUCGAAAGC
1757
491
Yes
No
No
43.16
36.30
3.80
11.79

1758
UUUCGAGGUGAGGCUUUGA
UCAAAGCCUCACCUCGAAA
1759
493
Yes
No
No
83.41
66.19
13.00
27.78

1762
GCUUUGGCCAGCAUAAGCA
UGCUUAUGCUGGCCAAAGC
1763
505
Yes
No
No
106.24
93.09
21.64
22.82

1764
AGCAUAAGCCAUGUGGCUA
UAGCCACAUGGCUUAUGCU
1765
514
Yes
No
No
106.58
96.16
45.99
59.73

1772
UGUGGCUCAUGUUACUAUA
UAUAGUAACAUGAGCCACA
1773
525
Yes
No
No
50.08
35.84
4.25
6.11

1836
AUACAGAGCAAGUUACUCA
UGAGUAACUUGCUCUGUAU
1837
573
Yes
No
Yes
30.97
39.43
2.52
16.42

1878
CAAUCAAGGGACCCAGAUA
UAUCUGGGUCCCUUGAUUG
1879
630
Yes
No
No
56.87
41.98
4.06
9.71

1884
UCACGGUGGAGGACCUUUA
UAAAGGUCCUCCACCGUGA
1885
647
Yes
No
No
33.49
27.17
2.89
3.07

1954
GACAGUAGCUGAUGUUAGA
UCUAACAUCAGCUACUGUC
1955
795
Yes
No
No
32.82
32.14
2.51
5.84

1960
AGUAGCUGAUGUUAGGACA
UGUCCUAACAUCAGCUACU
1961
798
Yes
No
No
43.70
36.61
4.16
5.59

1966
AGCUGAUGUUAGGACACUA
UAGUGUCCUAACAUCAGCU
1967
801
Yes
No
No
44.74
39.31
4.65
16.78

1972
GUUAGGACACUACCCAAUA
UAUUGGGUAGUGUCCUAAC
1973
808
Yes
No
No
44.93
42.64
11.77
8.81

1974
UUAGGACACUACCCAAUGA
UCAUUGGGUAGUGUCCUAA
1975
809
Yes
No
No
103.18
78.48
20.86
9.68

1976
GACACUACCCAAUGCCUCA
UGAGGCAUUGGGUAGUGUC
1977
813
Yes
No
No
69.33
52.48
8.17
4.57

1998
ACAAUAUUCGCUCCAUCUA
UAGAUGGAGCGAAUAUUGU
1999
839
Yes
No
No
49.88
39.06
2.66
3.41

2008
UUCGCUCCAUCUUUGGAAA
UUUCCAAAGAUGGAGCGAA
2009
845
Yes
Yes
Yes
43.55
32.91
3.67
3.13

2012
CGCUCCAUCUUUGGAAAUA
UAUUUCCAAAGAUGGAGCG
2013
847
Yes
Yes
Yes
69.62
75.70
14.97
14.19

2024
UGGAAAUGCUGUUAGUCGA
UCGACUAACAGCAUUUCCA
2025
858
Yes
No
Yes
33.29
38.07
1.72
11.60

2034
AUGCUGUUAGUCGAGAACA
UGUUCUCGACUAACAGCAU
2035
863
Yes
No
Yes
77.58
61.87
15.13
12.09

2038
GCUGUUAGUCGAGAACUGA
UCAGUUCUCGACUAACAGC
2039
865
Yes
No
Yes
62.22
42.47
6.34
3.74

2052
UCGAGAACUGAUAGAAAUA
UAUUUCUAUCAGUUCUCGA
2053
873
Yes
No
No
62.28
58.83
12.05
43.16

2054
CGAGAACUGAUAGAAAUUA
UAAUUUCUAUCAGUUCUCG
2055
874
Yes
No
No
91.42
71.70
7.53
26.83

2080
GUGAGGAUAAAACCCUAGA
UCUAGGGUUUUAUCCUCAC
2081
896
Yes
Yes
Yes
47.27
45.01
5.14
4.49

2086
AACCCUAGCCUUCAAAAUA
UAUUUUGAAGGCUAGGGUU
2087
906
Yes
No
No
43.18
47.49
4.25
28.46

2114
UAUCCAAUGCAAACUACUA
UAGUAGUUUGCAUUGGAUA
2115
935
Yes
No
No
96.44
79.53
9.28
27.00

2116
AUCCAAUGCAAACUACUCA
UGAGUAGUUUGCAUUGGAU
2117
936
Yes
No
No
60.35
46.08
4.80
18.11

2120
CCAAUGCAAACUACUCAGA
UCUGAGUAGUUUGCAUUGG
2121
938
Yes
No
No
94.31
73.07
30.27
33.72

2122
CAAUGCAAACUACUCAGUA
UACUGAGUAGUUUGCAUUG
2123
939
Yes
No
No
32.31
33.92
3.24
8.37

2158
ACUCUUCAUCAACCAUCGA
UCGAUGGUUGAUGAAGAGU
2159
975
Yes
No
No
59.69
42.41
14.57
8.92

2162
CUUCAUCAACCAUCGUCUA
UAGACGAUGGUUGAUGAAG
2163
978
Yes
No
No
41.71
34.15
2.74
1.68

2176
CAUCGUCUGGUAGAAUCAA
UUGAUUCUACCAGACGAUG
2177
988
Yes
No
No
56.14
43.45
8.79
10.68

2178
AUCGUCUGGUAGAAUCAAA
UUUGAUUCUACCAGACGAU
2179
989
Yes
No
No
40.77
32.37
8.28
3.44

2248
GUACCUCAGUUUAGAAAUA
UAUUUCUAAACUGAGGUAC
2249
1074
Yes
No
No
37.30
33.90
5.96
2.15

2310
GCACAUCGAGAGCAAGCUA
UAGCUUGCUCUCGAUGUGC
2311
1182
Yes
No
Yes
56.04
38.24
6.45
4.05

2340
UGUACUUCACCCAGACUUA
UAAGUCUGGGUGAAGUACA
2341
1223
Yes
No
No
77.09
56.42
13.29
12.82

2356
CUUUGCUACCAGGACUUGA
UCAAGUCCUGGUAGCAAAG
2357
1238
Yes
No
No
98.04
80.27
14.90
3.59

2358
UUGCUACCAGGACUUGCUA
UAGCAAGUCCUGGUAGCAA
2359
1240
Yes
No
No
94.29
55.78
15.61
20.46

2370
GGGAGAUGGUUAAAUCCAA
UUGGAUUUAACCAUCUCCC
2371
1268
Yes
No
No
38.12
26.38
1.01
3.25

2432
CCCACCAGAUGGUUCGUAA
UUACGAACCAUCUGGUGGG
2433
1337
Yes
No
No
40.19
31.25
5.99
1.58

2436
CCAGAUGGUUCGUACAGAA
UUCUGUACGAACCAUCUGG
2437
1341
Yes
No
No
35.85
31.67
2.51
4.02

2466
AGCAAACCCCUGUCCAGUA
UACUGGACAGGGGUUUGCU
2467
1399
Yes
No
No
62.03
44.71
31.33
10.33

2496
AGGGCUAGGCAGCAAGAUA
UAUCUUGCUGCCUAGCCCU
2497
1465
Yes
No
No
36.05
30.39
2.88
3.45

2540
CCCCAGAAAGAGACAUCGA
UCGAUGUCUCUUUCUGGGG
2541
1602
Yes
No
No
43.11
33.88
4.72
2.55

2570
GUGGAAGAUGAUUCCCGAA
UUCGGGAAUCAUCUUCCAC
2571
1642
Yes
No
No
40.79
35.26
5.71
3.57

2588
AUGACUGCAGCUUGUACCA
UGGUACAAGCUGCAGUCAU
2589
1666
Yes
No
No
40.79
41.66
6.77
13.47

2598
GAGAAGGAUCAUUAACCUA
UAGGUUAAUGAUCCUUCUC
2599
1689
Yes
No
No
41.92
32.99
3.77
3.17

2604
AAGGAUCAUUAACCUCACA
UGUGAGGUUAAUGAUCCUU
2605
1692
Yes
No
No
82.45
60.49
10.10
14.81

2608
AUCAUUAACCUCACUAGUA
UACUAGUGAGGUUAAUGAU
2609
1696
Yes
No
No
35.09
23.03
2.87
4.80

2612
CAUUAACCUCACUAGUGUA
UACACUAGUGAGGUUAAUG
2613
1698
Yes
No
No
54.06
38.14
10.29
2.12

2616
UUAACCUCACUAGUGUUUA
UAAACACUAGUGAGGUUAA
2617
1700
Yes
No
No
39.24
29.90
2.18
4.45

2618
AACCUCACUAGUGUUUUGA
UCAAAACACUAGUGAGGUU
2619
1702
Yes
No
No
72.65
47.22
11.56
4.86

2622
CCUCACUAGUGUUUUGAGA
UCUCAAAACACUAGUGAGG
2623
1704
Yes
No
No
52.37
36.54
3.88
6.67

2628
CACUAGUGUUUUGAGUCUA
UAGACUCAAAACACUAGUG
2629
1707
Yes
No
No
52.90
52.38
10.72
21.32

2664
GGAGAUGUUGCAUAACCAA
UUGGUUAUGCAACAUCUCC
2665
1764
Yes
No
No
63.04
42.75
7.90
5.27

2674
GUGGGCUGUGUGAAUCCUA
UAGGAUUCACACAGCCCAC
2675
1789
Yes
Yes
Yes
92.81
65.64
13.29
11.58

2678
GGGCUGUGUGAAUCCUCAA
UUGAGGAUUCACACAGCCC
2679
1791
Yes
Yes
Yes
98.93
53.41
25.78
15.13

2720
ACCACCAAGCUUAGUGAAA
UUUCACUAAGCUUGGUGGU
2721
1852
Yes
No
No
50.64
36.04
5.62
6.29

2744
ACUGUUCUACCAGAUACUA
UAGUAUCUGGUAGAACAGU
2745
1872
Yes
No
No
75.65
47.68
19.99
4.48

2746
UGUUCUACCAGAUACUCAA
UUGAGUAUCUGGUAGAACA
2747
1874
Yes
Yes
No
37.39
30.49
2.26
4.63

2748
GUUCUACCAGAUACUCAUA
UAUGAGUAUCUGGUAGAAC
2749
1875
Yes
Yes
No
62.87
44.70
5.44
1.70

2752
ACCAGAUACUCAUUUAUGA
UCAUAAAUGAGUAUCUGGU
2753
1880
Yes
Yes
No
51.16
37.87
2.32
4.35

2802
CCAUGCUUGCCUUAGAUAA
UUAUCUAAGGCAAGCAUGG
2803
1955
Yes
No
No
51.62
35.17
3.88
2.12

2804
CAUGCUUGCCUUAGAUAGA
UCUAUCUAAGGCAAGCAUG
2805
1956
Yes
No
No
73.57
32.96
20.89
5.76

2806
GCUUGCCUUAGAUAGUCCA
UGGACUAUCUAAGGCAAGC
2807
1959
Yes
No
No
63.25
44.04
1.71
6.37

2808
UUGCCUUAGAUAGUCCAGA
UCUGGACUAUCUAAGGCAA
2809
1961
Yes
No
No
54.15
40.82
6.07
5.83

2810
UGCCUUAGAUAGUCCAGAA
UUCUGGACUAUCUAAGGCA
2811
1962
Yes
No
No
48.62
37.54
3.54
10.64

2836
ACUUGCUGAAUACAUUGUA
UACAAUGUAUUCAGCAAGU
2837
2016
Yes
No
No
44.99
38.51
6.91
5.96

2840
GCUGAAUACAUUGUUGAGA
UCUCAACAAUGUAUUCAGC
2841
2020
Yes
No
No
97.12
72.48
23.44
20.00

2870
GAGAUGCUUGCAGACUAUA
UAUAGUCUGCAAGCAUCUC
2871
2056
Yes
Yes
No
61.83
61.40
6.02
14.69

2882
CAGACUAUUUCUCUUUGGA
UCCAAAGAGAAAUAGUCUG
2883
2066
Yes
No
No
38.09
32.29
5.14
5.55

2894
GGGAACCUGAUUGGAUUAA
UUAAUCCAAUCAGGUUCCC
2895
2098
Yes
Yes
Yes
88.30
69.86
4.74
13.87

2898
UUGGAUUACCCCUUCUGAA
UUCAGAAGGGGUAAUCCAA
2899
2108
Yes
No
No
69.26
46.23
20.07
9.56

2902
GGAUUACCCCUUCUGAUUA
UAAUCAGAAGGGGUAAUCC
2903
2110
Yes
No
No
73.70
46.35
2.58
10.95

2906
UACCCCUUCUGAUUGACAA
UUGUCAAUCAGAAGGGGUA
2907
2114
Yes
No
No
65.00
44.45
12.11
6.66

2916
CUUUGGAGGGACUGCCUAA
UUAGGCAGUCCCUCCAAAG
2917
2144
Yes
Yes
Yes
42.51
35.02
4.80
4.51

2922
UGGAGGGACUGCCUAUCUA
UAGAUAGGCAGUCCCUCCA
2923
2147
Yes
Yes
Yes
39.91
42.49
12.67
12.87

2924
GGAGGGACUGCCUAUCUUA
UAAGAUAGGCAGUCCCUCC
2925
2148
Yes
Yes
Yes
48.79
42.07
5.76
3.21

2928
GGGACUGCCUAUCUUCAUA
UAUGAAGAUAGGCAGUCCC
2929
2151
Yes
Yes
Yes
59.78
48.89
14.17
11.75

2936
GCCUAUCUUCAUUCUUCGA
UCGAAGAAUGAAGAUAGGC
2937
2157
Yes
Yes
Yes
56.69
41.63
15.43
8.74

2938
CCUAUCUUCAUUCUUCGAA
UUCGAAGAAUGAAGAUAGG
2939
2158
Yes
Yes
Yes
61.36
58.55
19.37
11.66

2940
CUAUCUUCAUUCUUCGACA
UGUCGAAGAAUGAAGAUAG
2941
2159
Yes
Yes
Yes
36.21
34.45
4.66
6.48

2944
AUCUUCAUUCUUCGACUAA
UUAGUCGAAGAAUGAAGAU
2945
2161
Yes
No
No
50.60
44.76
10.87
3.93

2948
UCGACUAGCCACUGAGGUA
UACCUCAGUGGCUAGUCGA
2949
2172
Yes
No
No
48.27
35.28
3.15
3.15

2966
GGUGAAUUGGGACGAAGAA
UUCUUCGUCCCAAUUCACC
2967
2187
Yes
No
No
54.54
42.28
6.74
0.98

3020
CAUCCGGAAGCAGUACAUA
UAUGUACUGCUUCCGGAUG
3021
2253
Yes
No
No
43.64
31.75
2.52
3.07

3022
UCCGGAAGCAGUACAUAUA
UAUAUGUACUGCUUCCGGA
3023
2255
Yes
No
No
47.86
33.85
6.19
3.23

3024
CCGGAAGCAGUACAUAUCA
UGAUAUGUACUGCUUCCGG
3025
2256
Yes
No
No
42.93
31.75
4.03
4.48

3026
CGGAAGCAGUACAUAUCUA
UAGAUAUGUACUGCUUCCG
3027
2257
Yes
No
No
41.58
34.48
1.21
8.42

3028
GGAAGCAGUACAUAUCUGA
UCAGAUAUGUACUGCUUCC
3029
2258
Yes
No
No
78.30
43.97
20.65
12.26

3064
GGACUGUGGAACACAUUGA
UCAAUGUGUUCCACAGUCC
3065
2339
Yes
No
No
113.80
62.77
26.58
15.98

3066
GACUGUGGAACACAUUGUA
UACAAUGUGUUCCACAGUC
3067
2340
Yes
No
No
33.65
31.90
3.61
13.34

3084
GCCUUGCGCUCACACAUUA
UAAUGUGUGAGCGCAAGGC
3085
2365
Yes
No
No
56.61
43.91
10.56
2.89

3088
CUUGCGCUCACACAUUCUA
UAGAAUGUGUGAGCGCAAG
3089
2367
Yes
No
No
50.33
46.74
1.45
9.10

3120
CUAACCUGCCUGAUCUAUA
UAUAGAUCAGGCAGGUUAG
3121
2429
Yes
No
No
41.88
39.74
15.98
8.53

3122
UAACCUGCCUGAUCUAUAA
UUAUAGAUCAGGCAGGUUA
3123
2430
Yes
No
No
44.20
43.04
4.66
4.40

3124
CCUGCCUGAUCUAUACAAA
UUUGUAUAGAUCAGGCAGG
3125
2433
Yes
No
No
37.49
109.90
7.18
26.42

3130
GCCUGAUCUAUACAAAGUA
UACUUUGUAUAGAUCAGGC
3131
2436
Yes
No
No
39.21
110.46
8.85
30.44

3134
UGAUCUAUACAAAGUCUUA
UAAGACUUUGUAUAGAUCA
3135
2439
Yes
No
No
35.25
33.81
3.13
3.06

3136
AUCUAUACAAAGUCUUUGA
UCAAAGACUUUGUAUAGAU
3137
2441
Yes
Yes
No
61.56
44.19
12.10
7.84

3148
AAAGUCUUUGAGAGGUGUA
UACACCUCUCAAAGACUUU
3149
2449
Yes
No
No
57.89
39.65
4.14
2.41

3164
AGAGGUGUUAAAUAUGGUA
UACCAUAUUUAACACCUCU
3165
2459
Yes
No
No
54.13
46.33
4.74
3.48

3210
UUCUCUGUAUUCCGAUACA
UGUAUCGGAAUACAGAGAA
3211
2506
Yes
No
No
46.41
36.80
4.59
4.07

3212
UCUCUGUAUUCCGAUACAA
UUGUAUCGGAAUACAGAGA
3213
2507
Yes
No
No
67.04
55.75
11.62
9.15

3216
UGUAUUCCGAUACAAAGUA
UACUUUGUAUCGGAAUACA
3217
2511
Yes
No
No
46.22
37.21
3.81
7.69

3248
UAUACAAAGUGUACCAACA
UGUUGGUACACUUUGUAUA
3249
2546
Yes
No
No
71.38
48.20
9.41
3.61

3272
GGUAGCACUUAAGACUUAA
UUAAGUCUUAAGUGCUACC
3273
2573
Yes
No
No
122.92
100.71
19.58
19.84

3274
GCACUUAAGACUUAUACUA
UAGUAUAAGUCUUAAGUGC
3275
2577
Yes
No
No
34.74
31.11
4.11
4.59

3276
CACUUAAGACUUAUACUUA
UAAGUAUAAGUCUUAAGUG
3277
2578
Yes
No
No
41.93
95.87
9.34
19.12

3278
CUUAAGACUUAUACUUGCA
UGCAAGUAUAAGUCUUAAG
3279
2580
Yes
No
No
52.78
47.77
7.09
23.95

3288
GACUUAUACUUGCCUUCUA
UAGAAGGCAAGUAUAAGUC
3289
2585
Yes
No
No
41.83
38.98
3.14
19.53

Example 5

Genomic DNA Extraction and Quantitation of CAG Repeat Length by Small Pool-PCR (sp-PCR) Analyses

Genomic DNA is purified using standard Proteinase K digestions and extracted using DNAzol (Invitrogen) following the manufacturer's instructions. CAG repeat length is determined by small pool-PCR analyses as previously described (Mario Gomes-Pereira and Darren Monckton, 2017, Front Cell Neuro 11:153). In brief, DNA is digested with HindIII, diluted to a final concentration between 1-6 pg/μl and approximately 10 μg was used in subsequent PCR reactions. Primer flanking Exon 1 of the human HTT are used to amplify the CAG alleles and the PCR product is resolved by electrophoresis. Subsequently, Southern blot hybridization is performed, and the CAG alleles are observed by autoradiography OR visualized with ethidium bromide staining. CAG length can be measured directly by sequencing on a MiSeQ or appropriate machine. The change in CAG repeat number in various treatment groups in comparison with controls is calculated using simple descriptive statistics (e.g. mean±standard deviation).

Genomic DNA Extraction and Quantitation of CAG Repeat Length by DNA Fragment Analyses

Genomic DNA is purified using DNAeasy Blood and Tissue Kit (Qiagen) following the manufacturer's instructions. DNA is quantified by Qubit dsDNA assay (ThemoScientific) and CAG repeat length is determined by fragment analysis by Laragen (Culver City, Calif.)

Example 6. Mouse Studies
Natural History Studies in HD Mouse Models:

The HD mouse R6/2 line is transgenic for the 5′ end of the human HD gene (HTT) carrying approximately 120 CAG repeat expansions. HTT is ubiquitously expressed. Transgenic mice exhibit a progressive neurological phenotype that mimics many of the pathological features of HD, including choreiform-like movements, involuntary stereotypic movements, tremor, and epileptic seizures, as well as nonmovement disorder components, including unusual vocalization. They urinate frequently and exhibit loss of body weight and muscle bulk through the course of the disease. Neurologically these mice develop Neuronal Intranuclear Inclusions (NII) which contain both the huntingtin and ubiquitin proteins. Previously unknown, these NII have subsequently been identified in HD patients. The age of onset for development of HD symptoms in R6/2 mice has been reported to occur between 9 and 11 weeks (Mangiarini et al., 1996 Cell 87: 493-506).

Somatic expansions were reported in R6/2 mice striatum, cortex and liver. Somatic instability increased with higher constitutive length (Larson et al, Neurobiology of Disease 76 (2015) 98-111). A natural history study in R6/2 mice with 120 CAG repeats was performed. Their genotype and length of CAG expansion was determined. R6/2 mice at 4, 8, 12 and 16 weeks of age (4 male and 4 female mice per age group) were sacrificed. Striatum, cerebellum, cortex, liver, kidney, heart, spleen, lung, duodenum, colon, quadricep, CSF and plasma were collected and snap frozen in liquid nitrogen. Genomic DNA was extracted, the length of CAG repeats measured, and the instability index was calculated from striatum, cerebellum, cortex, liver and kidney according to Lee et al. BMC Systems Biology 2010, 4:29). At 12 and 16 weeks of age, the striatum showed a significant increase of somatic expansion as measured by the instability index (****p<0.0001, One-way ANOVA) (FIG. 1). No changes in somatic expansion were observed across all ages in the R6/2 mouse cerebellum (FIG. 2)

Mouse models recapitulating many of the features of trinucleotide repeat expansion diseases including, HD, FA and DM1, are readily available from commercial venders and academic institutions (Polyglutamine Disorders, Advances in Experimental Medicine and Biology, Vol 1049, 2018: Editors Clevio Nobrega and Lois Pereira de Almeida, Springer). All mouse experiments are conducted in accordance with local IACUC guidelines. Three examples of different diseased mouse models and how they could be used to investigate the usefulness of pharmacological intervention against MLH1 for somatic expansion are included below.

In Huntington's research, several transgenic and knock-in mouse models were generated to investigate the underlying pathological mechanisms involved in the disease. For example, the R6/2 transgenic mouse contains a transgene of ˜1.9 kb of human HTT containing 144 copies of the CAG repeat (Mangiarini et al., 1996 Cell 87: 493-506) while the HdhQ111 model was generated by replacing the mouse HTT exon 1 with a human exon1 containing 111 copies of the CAG repeat (Wheeler et al., 2000 Hum Mol Genet 9:503-513). Both the R6/2 and HdhQ111 models replicate many of the features of human HD including motor and behavioral dysfunctions, neuronal loss, as well as the expansion of CAG repeats in the striatum (Pouladi et al., 2013, Nature Reviews Neuroscience 14: 708-721; Mangiarini et al., 1997 Nature Genet 15: 197-200; Wheeler et al., Hum Mol Genet 8: 115-122).

R6/2 mice are genotyped using DNA derived from tail snips at weaning and the CAG repeat size is determined. Mice are randomized into groups (n=12/group) at weaning at 4 wks old and dosed with monthly (week 4 and 8) ICV injection of either PBS (control) or up to a 500 μg dose of oligos targeting MLH1. A series of oligos targeting different regions of MLH1 can be tested to identify the most efficacious oligo sequence in vivo. At 12 weeks of age, mice are euthanized, and tissues extracted for analyses. The list of tissues includes, but not restricted to, striatum, cortex, cerebellum, and liver. Genomic DNA is extracted and the length of CAG repeats measured as described below. CSF and plasma are collected for biomarker analysis. Additional pertinent mouse models of HD can be considered.

In Friedreich Ataxia, the YG8 FRDA transgenic mouse model is commonly used to understand the pathology (Al-Mandawi et al., 2006 Genomics 88(5)580-590; Bourn et al., 2012 PLOS One 7(10); e47085). This model was generated through the insertion of a human YAC transgenic containing in the background of a null FRDA mouse. The YG8 model demonstrates somatic expansion of the GAA triplet repeat expansion in neuronal tissues with only mild motor defects. YG8 FRDA mice are genotyped using DNA derived from tail snips at weaning and the CAG repeat size is determined using methods. To determine if MLH1 plays a role in somatic expansion of the disease allele, hemizygous YG8 FRDA animals are administered ICV with oligos targeting knockdown of MLH1 identified above.

Approximately 2 months later, animals are euthanized and tissues collected for molecular analyses. Suitable tissues are heart, quadriceps, dorsal root ganglia (DRG's), cerebellum, kidney, and liver. Genomic DNA is extracted, and the length of CAG repeats measured as described above in Example 5.

In Myotonic Dystrophy, the DM300-328 transgenic mouse model is suitable for investigating the pathology behind DM1. This mouse model has a large human genomic sequence (˜45 kb) containing over 300 CTG repeats and displays both the somatic expansion and degenerative muscle changes observed in human DM1 (Seznec et al., 2000; Tome et al., 2009 PLOS Genetics 5(5): e1000482; Pandey et al., 2015 J Pharmacol Exp Ther 355:329-340). DM300-328 mice are genotyped using DNA derived from tail snips at weaning and the CAG repeat size is determined. To determine if MLH1 plays a role in somatic expansion of the disease allele in myotonic dystrophy, DM300-328 transgenic animals are administered ASOs targeting knockdown of MLH1 by either subcutaneous injections (sc), intraperitoneal (ip) or intravenous tail injections (iv). Mice are administered ASOs up to 2×/week for maximum 8 weeks of treatment. Animals are euthanized at multiple time points and tissues collected for molecular analyses. Suitable tissues are quadriceps, heart, diaphragm, cortex, cerebellum, sperm, kidney, and liver. Genomic DNA is extracted and the length of CAG repeats measured and compared with parallel controls.

The HdhQ111 mouse model for Huntington Disease is a heterozygous knock-in line, in which the majority of exon 1 and part of intron 1 on one allele of the huntingtin gene (i.e., HTT or Huntington Disease gene) are replaced with human DNA containing ˜111 CAG repeats. In this example, ASOs to knock down MLH1 activity or levels is administered. After a treatment period, brain tissue from treated or untreated mice is isolated (e.g., striatum tissue) and analyzed using qRT-PCR as previously described to determine RNA levels of MLH1. Huntingtin gene repeat analysis is performed using mouse tissues (e.g., striatum tissue) after a treatment period using a human-specific PCR assay that amplifies the HTT CAG repeat from the knock-in allele but does not amplify the mouse sequence (i.e., the wild type allele). In this protocol, the forward primer is fluorescently labeled (e.g., with 6-FAM as described previously, for example Pinto R M, Dragileva E, Kirby A, et al. Mismatch repair genes MLH1 and MSH3 modify CAG instability in Huntington's disease mice: genome-wide and candidate approaches. PLoS Genet. 2013; 9(10):e1003930.), and products can be resolved using an analyzer with comparison against an internal size standard to generate CAG repeat size distribution traces. Repeat size is determined from the peak with the greatest intensity from a control tissue (e.g., tail tissue in a mouse) and from an affected tissue (e.g., brain striatum tissue or brain cortex tissue). Immunohistochemistry is carried out with polyclonal anti-huntingtin antibody (e.g., EM48) on paraffin-embedded or otherwise prepared sections of brain tissue and can be quantified using a standardized staining index to capture both nuclear staining intensity and number of stained nuclei. A decrease in repeat size in affected tissue indicates that the agent that reduces the level and/or activity of MLH1 is capable of decreasing the repeat which are responsible for the toxic and/or defective gene products in Huntington's disease.

Other Aspects

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.

While the invention has been described in connection with specific aspects thereof, it will be understood that invention is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and can be applied to the essential features hereinbefore set forth, and follows in the scope of the claimed.

E1. A single-stranded antisense oligonucleotide of 10-30 linked nucleosides in length, wherein the antisense oligonucleotide comprises a region of at least 10 contiguous nucleobases having at least 80% complementarity to an MLH1 gene.

E2. The antisense oligonucleotide of E2, wherein the antisense oligonucleotide comprises:

(a) a DNA core sequence comprising linked deoxyribonucleosides;

(b) a 5′ flanking sequence comprising linked nucleosides; and

E3. A single-stranded antisense oligonucleotide of 10-30 linked nucleosides in length for inhibiting expression of a human MLH1 gene in a cell, wherein the antisense oligonucleotide comprises a region of at least 10 contiguous nucleobases having at least 80% complementarity to an MLH1 gene.

E4. The antisense oligonucleotide of E4, wherein the antisense oligonucleotide comprises:

(a) a DNA core comprising linked deoxyribonucleosides;

(b) a 5′ flanking sequence comprising linked nucleosides; and

E5. The antisense oligonucleotide of any one of E1-E4, wherein the region of at least 10 nucleobases has at least 90% complementary to an MLH1 gene.

E6. The antisense oligonucleotide of any one of E1-E5, wherein the region of at least 10 nucleobases has at least 95% complementary to an MLH1 gene.

E7. The antisense oligonucleotide of any one of E1-E6, wherein the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 193-258, 289-607, 629-734, 757-836, 865-1125, 1177-1206, 1218-1286, 1324-1408, 1433-1747, 1759-1814, 1852-1901, 1959-2029, 2053-2240, 2250-2356, 2382-2479, 2510-2546, or 2573-2598 of the MLH1 gene.

E8. The antisense oligonucleotide of any one of E1-E6, wherein the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 193-251, 289-607, 629-734, 757-836, 865-1125, 1177-1206, 1218-286, 1324-1408, 1433-1747, 1759-1814, 1852-1901, 1959-2029, 2053-2240, 2250-2356, 2382-2479, 2510-2546, or 2573-2598 of the MLH1 gene

E9. The antisense oligonucleotide of any one of E1-E6, wherein the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 193-251, 289-607, 629-734, 758-836, 865-1125, 1177-1206, 1218-1286, 1324-1408, 1433-1747, 1759-1814, 1852-1901, 1959-2029, 2053-2240, 2250-2356, 2382-2479, 2510-2546, or 2573-2598 of the MLH1 gene.

E10. The antisense oligonucleotide of any one of E1-E6, wherein the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 312-391, 410-508, 522-607, 629-726, 759-1125, 1177-1206, 1221-1286, 1324-1407, 1433-1747, 1764-1814, 1854-1901, 1959-2029, 2053-2113, 2184-2240, 2251-2283, 2303-2351, 2384-2479, or 2510-2546 of the MLH1 gene.

E11. The antisense oligonucleotide of any one of E1-E6, wherein the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 575-602, 662-724, 805-830, 891-960, 1002-1027, 1056-1081, 1100-1125, 1342-1384, 1443-1498, 1513-1561, 1600-1625, 1652-1747, 1876-1901, 2001-2026, or 2430-2459 of the MLH1 gene.

E12. The antisense oligonucleotide of any one of E1-E6, wherein the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 307-332, 458-500, 571-602, 758-787, 865-890, 892-917, 1045-1084, 1624-1649, 1786-1813, 1871-1901, 2053-2081, 2086-2114, or 2149-2176 of the MLH1 gene.

E13. The antisense oligonucleotide of any one of E1-E6, wherein the region of at least 10 nucleobases is complementary to an MLH1 gene corresponding to a sequence of reference mRNA NM_000249.3 at one or more of positions 575-602, 1056-1081, or 1876-1901 of the MLH1 gene.

E14. The antisense oligonucleotide of any one of E1-E6, wherein the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 6-1393.

E15. The antisense oligonucleotide of any one of E1-E6, wherein the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 81-84, 86-87, 90, 99-101, 106-107, 111, 113-114, 117, 122-126, 129-131, 137-138, 140, 144, 146-160, 172, 188-191, 211, 215-220, 222-226, 229, 231-239, 242-249, 270-271, 274-279, 286-293, 295-298, 310-320, 322-328, 332-335, 337, 345, 383-388, 397-402, 405-413, 415-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-598, 600-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699, 701-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 1118-1123, 1131-1134, 1136, 1138-1139, 1140-1144, 1151-1152, 1161-1163, 1187-1192, 1194-1195, 1197, 1199-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, or 1343.

E16. The antisense oligonucleotide of any one of E1-E6, wherein the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 81-84, 86-87, 99-101, 106, 113-114, 122-126, 129-131, 137-138, 140, 144, 146-159, 172, 188-191, 211, 215-217, 219, 223-226, 229, 232-239, 242-249, 270-271, 274-279, 286-293, 295-298, 310-320, 322-328, 332-335, 337, 345, 384-388, 397-402, 405-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 1118-1123, 1131-1134, 1136, 1138-1144, 1151-1152, 1161-1163, 1187-1191, 1194-1195, 1197, 1199-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, or 1343.

E17. The antisense oligonucleotide of any one of E1-E6, wherein the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 81-87, 99-101, 106, 113-114, 122-126, 129-131, 137-138, 140, 144, 146, 147, 148-151, 153-159, 172, 188-191, 211215-217, 219, 223-226, 229, 232-239, 242-245, 248-249, 270-271274-276, 278-279, 286-293, 295-298, 310-320, 322-338, 332-335, 337, 345, 384-386, 387-388, 397-402, 405-413, 415-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-598, 600-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699, 701-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 1118-1123, 1131-1134, 1136, 1138-1144, 1151-1152, 1161-1163, 1187-1191, 1194-1195, 1197, 1199, 1200-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, or 1343.

E18. The antisense oligonucleotide of any one of E1-E6, wherein the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 122, 123, 125-126, 129-130, 131, 137, 146-147, 153-156, 158, 188-191, 211, 216, 223, 226, 235, 237, 245, 248, 270-271, 276, 278-279, 286, 289-293, 297-298, 310, 312-320, 323-328, 332, 334-335, 337, 385-386, 397-402, 407, 410-412, 415-417, 419-420, 476, 478, 482-487, 490-491, 493-494, 497-501, 522-523, 525-526, 528-530, 546-548, 557, 563-565, 586, 603, 605-610, 613, 619-622, 631-632, 634, 639-641, 645-646, 650-651, 655, 699, 701, 704-709, 714, 729-731, 733-734, 736-740, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 847, 850, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 928-931, 936-939, 977, 981-986, 1019, 1024-1026, 1034, 1036-1041, 1083-1085, 1087, 1111, 1132-1134, 1136, 1138-1140, 1142, 1161-1163, 1188-1190, 1194-1195, 1207, 1218, 1241, 1244, 1247, 1257-1259, 1262-1269, 1277-1278, or 1314-1315.

E19. The antisense oligonucleotide of any one of E1-E6, wherein the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 291-293, 313, 316-317, 325-326, 334-335, 415, 483, 485-486, 499-500, 523, 525, 564, 607, 622, 704-705, 707-709, 739, 752, 768-769, 788, 827, 861, 871-872, 877-879, 882, 885, 886, 901, 986, 1038, 1263, or 1265-1267.

E20. The antisense oligonucleotide of any one of E1-E6, wherein the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 117, 215-226, 229-232, 287-293, 384-385, 387-388, 458, 484, 596-610, 850, 936-938, 981-986, 1083-1086, 1109-1112, or 1121-1123.

E21. The antisense oligonucleotide of any one of E1-E6, wherein the nucleobase sequence of the antisense oligonucleotide consists of any one of SEQ ID NOs: 6-1393.

E22. The antisense oligonucleotide of any one of E1-E6, wherein the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 81-87, 90, 99-101, 106-107, 111, 113-114, 117, 122-126, 129-131, 137-138, 140, 144, 146-160, 172, 188-191, 211, 215-220, 22-226, 229, 231-239, 242-249, 270-271, 274-279, 286-293, 295-297, 298, 310-320, 322-328, 332-335, 337, 345, 383-388, 397-402, 405-413, 415-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-598, 600-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699, 701-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 118-1123, 1131-1134, 1136, 1138-1144, 1151-1152, 1161-1163, 1187-1192, 1194-1195, 1197, 1199-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1222, 1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, or 1343.

E23. The antisense oligonucleotide of any one of E1-E6, wherein the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 81-84, 86-87, 99-101, 106, 113-114, 122-126, 129-131, 137-138, 140, 144, 146-159, 172, 188-191, 211, 215-217, 219, 223-226, 229, 232-239, 242-249, 270-271, 274-279, 286-293, 295-298, 310-320, 322-328, 332-335, 337, 345, 384-388, 397-402, 405-413, 415-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-598, 600-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699, 701-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 1118-1123, 1131-1134, 1136, 1138-1144, 1151-1152, 1161-1163, 1187-1191, 1194-1195, 1197, 1199-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, or 1343.

E24. The antisense oligonucleotide of any one of E1-E6, wherein the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 81-84, 86-87, 99-101, 106, 113-114, 122-126, 129-131, 137-138, 140, 144, 146-151, 153-159, 172, 188-191, 211, 215-217, 219223-226, 229, 232-239, 242-245, 248-249, 270-271, 274-276, 278-279, 286-293, 295-298, 310-320, 322-328, 332-335, 337, 345, 384-388, 397-402, 405-413, 415-421, 458, 473-474, 476-478, 482-487, 490-491, 493-494, 497-501, 522-525, 526, 528-530, 542, 545-549, 551-560, 563-565, 585-586, 596-598, 600-610, 613, 619-622, 631-634, 636-637, 639-643, 645-646, 649-651, 655, 699, 701-709, 714-715, 729-731, 733-741, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 845, 847, 850, 852-853, 857, 859, 861, 866-872, 877-882, 884-887, 889-890, 893-894, 901, 923-931, 936-939, 975, 977, 981-986, 1019, 1023-1032, 1034, 1036-1041, 1083-1087, 1089, 1101, 1104-1105, 1107, 1109-1112, 1118-1123, 1131-1134, 1136, 1138-1144, 1151-1152, 1161-1163, 1187-1191, 1194-1195, 1197, 1199-1201, 1204, 1207, 1209, 1214-1216, 1218, 1220-1223, 1239-1241, 1244, 1247, 1257-1259, 1262-1270, 1273-1275, 1277-1278, 1314-1315, or 1343.

E25. The antisense oligonucleotide of any one of E1-E6, wherein the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 122-123, 125-126, 129-131, 137, 146-147, 153-156, 158, 188-191, 211, 216, 223, 226, 235, 237, 245, 248, 270-271, 276, 278-279, 286, 289-293, 297-298, 310, 312-320, 323-328, 332, 334-335, 337, 385-386, 397-402, 407, 410-412, 415-417, 419-420, 476, 478, 482-487, 490-491, 493-494, 497-501, 522-523, 525-526, 528-530, 546-548, 557, 563-565, 586, 603, 605-610, 613, 619-622, 631-632, 634, 639-641, 645-646, 650-651, 655, 699, 701, 704-709, 714, 729-731, 733-734, 736-740, 744, 749, 752-754, 757, 765-766, 768-769, 788, 790-791, 819, 827-835, 840-842, 847, 850, 859, 861, 866-867, 868-872, 877-882, 884-887, 889-890, 893-894, 901, 928-931, 936-939, 977, 981-986, 1019, 1024-1026, 1034, 1036-1041, 1083-1085, 1087, 1111, 1132-1134, 1136, 1138-1140, 1142, 1161-1163, 1188-1190, 1194-1195, 1207, 1218, 1241, 1244, 1247, 1257-1259, 1262-1269, 1277-1278, or 1314-1315.

E26. The antisense oligonucleotide of any one of E1-E6, wherein the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 291-293, 313, 316-317, 325-326, 334-335, 415, 483, 485-486, 499-500, 523, 525, 564, 607, 622, 704-705, 707-709, 739, 752, 768-769, 788, 827, 861, 871-872, 877-879, 882, 885, 886, 901, 986, 1038, 1263, or 1265-1267.

E27. The antisense oligonucleotide of any one of E1-E6, wherein the antisense oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 117, 215-226, 229-232, 287-293, 384-385, 387-388, 458-484, 596-610, 850, 936-938, 981-986, 1083-1086, 1109-1112 or 1121-1123.

E28. The antisense oligonucleotide of any one of E1-E27, wherein the antisense oligonucleotide exhibits at least 50% mRNA inhibition at a 20 nM oligonucleotide concentration when determined using a cell assay when compared with a control cell.

E29. The antisense oligonucleotide of any one of E1-E27, wherein the antisense oligonucleotide exhibits at least 60% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell.

E30. The antisense oligonucleotide of any one of E1-E27, wherein the antisense oligonucleotide exhibits at least 70% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell.

E31. The antisense oligonucleotide of any one of E1-E27, wherein the antisense oligonucleotide exhibits at least 85% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell.

E32. The antisense oligonucleotide of any one of E1-E27, wherein the antisense oligonucleotide exhibits at least 50% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell.

E33. The antisense oligonucleotide of any one of E1-E27, wherein the antisense oligonucleotide exhibits at least 60% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell.

E34. The antisense oligonucleotide of any one of E1-E27, wherein the antisense oligonucleotide exhibits at least 70% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell.

E35. The antisense oligonucleotide of any one of E1-E27, wherein the antisense oligonucleotide exhibits at least 85% mRNA inhibition at a 20 nM antisense oligonucleotide concentration when determined using a cell assay when compared with a control cell.

E36. The antisense oligonucleotide of any one of E1-E35, wherein the antisense oligonucleotide comprises at least one alternative internucleoside linkage.

E37. The antisense oligonucleotide of E36, wherein the at least one alternative internucleoside linkage is a phosphorothioate internucleoside linkage.

E38. The antisense oligonucleotide of E36, wherein the at least one alternative internucleoside linkage is a 2′-alkoxy internucleoside linkage.

E39. The antisense oligonucleotide of E36, wherein the at least one alternative internucleoside linkage is an alkyl phosphate internucleoside linkage.

E40. The antisense oligonucleotide of any one of E1-E39, wherein the antisense oligonucleotide comprises at least one alternative nucleobase.

E41. The antisense oligonucleotide of E40, wherein the alternative nucleobase is 5′-methylcytosine, pseudouridine, or 5-methoxyuridine.

E42. The antisense oligonucleotide of any one of E1-E41, wherein the antisense oligonucleotide comprises at least one alternative sugar moiety.

E43. The antisense oligonucleotide of E42, wherein the alternative sugar moiety is 2′-OMe or a bicyclic nucleic acid.

E44. The antisense oligonucleotide of any one of E1-E43, wherein the antisense oligonucleotide further comprises a ligand conjugated to the 5′ end or the 3′ end of the antisense oligonucleotide through a monovalent or branched bivalent or trivalent linker.

E45. The antisense oligonucleotide of any one of E1-E44, wherein the antisense oligonucleotide comprises a region complementary to at least 17 contiguous nucleotides of a MLH1 gene.

E46. The antisense oligonucleotide of E45, wherein the antisense oligonucleotide comprises a region complementary to at least 19 contiguous nucleotides of a MLH1 gene.

E47. The antisense oligonucleotide of any one of E1-E44, wherein the antisense oligonucleotide comprises a region complementary to 19 to 23 contiguous nucleotides of a MLH1 gene.

E48. The antisense oligonucleotide of any one of E1-E44, wherein the antisense oligonucleotide comprises a region complementary to 19 contiguous nucleotides of a MLH1 gene.

E49. The antisense oligonucleotide of any one of E1-E44, wherein the antisense oligonucleotide comprises a region complementary to 20 contiguous nucleotides of a MLH1 gene.

E50. The antisense oligonucleotide of any one of E1-E44, wherein the antisense oligonucleotide is from about 15 to 25 nucleosides in length.

E51. The antisense oligonucleotide of any one of E1-E44, wherein the antisense oligonucleotide is 20 nucleosides in length.

E52. A pharmaceutical composition comprising one or more of the antisense oligonucleotides of any one of E1-E51 and a pharmaceutically acceptable carrier or excipient.

E53. A composition comprising one or more of the antisense oligonucleotides of any one of E1-E51 and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, ora liposome.

E54. A method of inhibiting transcription of MLH1 in a cell, the method comprising contacting the cell with one or more of the antisense oligonucleotides of any one of E1-E51, the pharmaceutical composition of E52, or the composition of E53 for a time sufficient to obtain degradation of an mRNA transcript of a MLH1 gene, inhibits expression of the MLH1 gene in the cell.

E55. A method of treating, preventing, or delaying the progression a trinucleotide repeat expansion disorder in a subject in need thereof, the method comprising administering to the subject one or more of the antisense oligonucleotides of any one of E1-E51, the pharmaceutical composition of E52, or the composition of E53.

E56. A method of reducing the level and/or activity of MLH1 in a cell of a subject identified as having a trinucleotide repeat expansion disorder, the method comprising contacting the cell with one or more of the antisense oligonucleotides of any one of E1-E51, the pharmaceutical composition of E52, or the composition of E53.

E57. A method for inhibiting expression of an MLH1 gene in a cell comprising contacting the cell with one or more of the antisense oligonucleotides of any one of E1-E51, the pharmaceutical composition of E52, or the composition of E53 and maintaining the cell for a time sufficient to obtain degradation of a mRNA transcript of an MLH1 gene, thereby inhibiting expression of the MLH1 gene in the cell.

E58. A method of decreasing trinucleotide repeat expansion in a cell, the method comprising contacting the cell with one or more of the antisense oligonucleotides of any one of E1-E51, the pharmaceutical composition of E52, or the composition of E53.

E59. The method of E57 or E58, wherein the cell is in a subject.

E60. The method of any one of E55, E56, and E59, wherein the subject is a human.

E61. The method of any one of E55-E59, wherein the cell is a cell of the central nervous system or a muscle cell.

E62. The method of any one of E54, E55, and E59-E61, wherein the subject is identified as having a trinucleotide repeat expansion disorder.

E63. The method of any one of E55, E56, and E58-E62, wherein the trinucleotide repeat expansion disorder is a polyglutamine disease.

E64. The method of E63, wherein the polyglutamine disease is selected from the group consisting of dentatorubropallidoluysian atrophy, Huntington's disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 17, and Huntington's disease-like 2.

E65. The method of any one of E55-E62, wherein the trinucleotide repeat expansion disorder is a non-polyglutamine disease.

E66. The method of E65, wherein the non-polyglutamine disease is selected from the group consisting of fragile X syndrome, fragile X-associated tremor/ataxia syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy type 1, spinocerebellar ataxia type 8, spinocerebellar ataxia type 12, oculopharyngeal muscular dystrophy, Fragile X-associated premature ovarian failure, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy.

E67. One or more antisense oligonucleotides of any one of E1-E51, the pharmaceutical composition of E52, or the composition of E53 for use in the prevention or treatment of a trinucleotide repeat expansion disorder.

E68. The antisense oligonucleotide, pharmaceutical composition, or composition for the use of E67, wherein the trinucleotide repeat expansion disorder is selected from the group consisting of dentatorubropallidoluysian atrophy, Huntington's disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 17, Huntington's disease-like 2, fragile X syndrome, fragile X-associated tremor/ataxia syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy type 1, spinocerebellar ataxia type 8, spinocerebellar ataxia type 12, oculopharyngeal muscular dystrophy, Fragile X-associated premature ovarian failure, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy.

E69. The antisense oligonucleotide, pharmaceutical composition, or composition for the use of E67 or E68, wherein the trinucleotide repeat expansion disorder is Huntington's disease.

E70. The antisense oligonucleotide, pharmaceutical composition, or composition for the use of E67 or E68, wherein the trinucleotide repeat expansion disorder is Friedreich's ataxia.

E71. The antisense oligonucleotide, pharmaceutical composition, or composition for the use of E67 or E68, wherein the trinucleotide repeat expansion disorder is myotonic dystrophy type 1.

E72. The antisense oligonucleotide, pharmaceutical composition, or composition for the use of any of E67-E71, wherein the antisense oligonucleotide, pharmaceutical composition, or composition is administered intrathecally.

E73. The antisense oligonucleotide, pharmaceutical composition, or composition for the use of any of E67-E71, wherein the antisense oligonucleotide, pharmaceutical composition, or composition is administered intraventricularly.

E74. The antisense oligonucleotide, pharmaceutical composition, or composition for use of any of E67-E71, wherein the antisense oligonucleotide, pharmaceutical composition, or composition is administered intramuscularly.

E75. A method of treating, preventing, or delaying the progression a disorder in a subject in need thereof wherein the subject is suffering from trinucleotide repeat expansion disorder, comprising administering to said subject one or more of the antisense oligonucleotides of any one of E1-E51, the pharmaceutical composition of E52, or the composition of E53.

E76. The method of E75, further comprising administering an additional therapeutic agent.

E77. The method of E76, wherein the additional therapeutic agent is an oligonucleotide that hybridizes to an mRNA encoding the Huntingtin gene.

E78. A method of preventing or delaying the progression of a trinucleotide repeat expansion disorder in a subject, the method comprising administering to the subject one or more of the antisense oligonucleotides of any one of E1-E51, the pharmaceutical composition of E52, or the composition of E53 in an amount effective to delay progression of a trinucleotide repeat expansion disorder of the subject.

E79. The method of E78, wherein the trinucleotide repeat expansion disorder is selected from the group consisting of dentatorubropallidoluysian atrophy, Huntington's disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 17, Huntington's disease-like 2, fragile X syndrome, fragile X-associated tremor/ataxia syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy type 1, spinocerebellar ataxia type 8, spinocerebellar ataxia type 12, oculopharyngeal muscular dystrophy, Fragile X-associated premature ovarian failure, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy.

E80. The method of E78 or E79, wherein the trinucleotide repeat expansion disorder is Huntington's disease.

E81. The method of E78 or E79, wherein the trinucleotide repeat expansion disorder is Friedrich's ataxia.

E82. The method of E78 or E79, wherein the trinucleotide repeat expansion disorder is myotonic Dystrophy type 1.

E83. The method of E78 or E79, further comprising administering an additional therapeutic agent.

E84. The method of E83, wherein the additional therapeutic agent is an oligonucleotide that hybridizes to an mRNA encoding the Huntington gene.

E85. The method of any of E78-E84, wherein progression of the trinucleotide repeat expansion disorder is delayed by at least 120 days, for example, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years or more, when compared with a predicted progression.

E86. One or more antisense oligonucleotides of any one of E1-E51, the pharmaceutical composition of E52, or the composition of E53, for use in preventing or delaying progression of a trinucleotide repeat expansion disorder in a subject.

E87. The antisense oligonucleotide, pharmaceutical composition, or composition for the use of E86, wherein the trinucleotide repeat expansion disorder is selected from the group consisting of dentatorubropallidoluysian atrophy, Huntington's disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 17, Huntington's disease-like 2, fragile X syndrome, fragile X-associated tremor/ataxia syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy type 1, spinocerebellar ataxia type 8, spinocerebellar ataxia type 12, oculopharyngeal muscular dystrophy, Fragile X-associated premature ovarian failure, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy.

E88. The antisense oligonucleotide, pharmaceutical composition, or composition for the use of E86 or E87, wherein the trinucleotide repeat expansion disorder is Huntington's disease.

E89. The antisense oligonucleotide, pharmaceutical composition, or composition for the use of E86 or E87, wherein the trinucleotide repeat expansion disorder is Friedrich's ataxia.

E90. The antisense oligonucleotide, pharmaceutical composition, or composition for the use of E86 or E87, wherein the trinucleotide repeat expansion disorder is Myotonic dystrophy type 1.

E91. The antisense oligonucleotide, pharmaceutical composition, or composition for the use of any one of E86-E90, wherein progression of the trinucleotide repeat expansion disorder is delayed by at least 120 days, for example, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years or more, when compared with a predicted progression.

E92. A double-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand is complementary to at least 15 contiguous nucleobases of an MLH1 gene, and wherein the dsRNA comprises a duplex structure of between 15 and 30 linked nucleosides in length.

E93. A dsRNA for reducing expression of MLH1 in a cell, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand is complementary to at least 15 contiguous nucleobases of an MLH1 gene, and wherein the dsRNA comprises a duplex structure of between 15 and 30 linked nucleosides in length.

E94. The dsRNA of E92 or E93 comprising a duplex structure of between 19 and 23 linked nucleosides in length.

E95. The dsRNA of any one of E92-E94, further comprising a loop region joining the sense strand and antisense strand, wherein the loop region is characterized by a lack of base pairing between nucleobases within the loop region.

E96. The dsRNA of any one of E92-E95, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MLH1 gene corresponding to reference mRNA NM_000249.3 at one or more of positions 153-176, 267-388, 417-545, 792-995, 1639-1727, 1849-1900, 2105-2207, 2337-2387, 2426-2479 and 2508-2600 of the MLH1 gene.

E97. The dsRNA of any one of E92-E95, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MLH1 gene corresponding to reference mRNA NM_000249.3 at one or more of positions 326-388, 459-511, 805-878, 903-926, 1639-1720, and 2141-2192 of the MLH1 gene.

E98. The dsRNA of any one of E92-E95, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MLH1 gene corresponding to reference mRNA NM_000249.3 at one or more of positions 267-388, 417-545, 805-878, 903-995, 1639-1727, 1849-1900, 2141-2207, 2337-2387, and 2426-2479 of the MLH1 gene.

E99. The dsRNA of any one of E92-E95, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MLH1 gene corresponding to reference mRNA NM_000249.3 at one or more of positions 153-176, 267-388, 417-545, 792-995, 1639-1727, 1849-1900, 2105-2207, 2337-2387, and 2426-2479 of the MLH1 gene.

E100. The dsRNA of any one of E92-E95, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MLH1 gene corresponding to reference mRNA NM_000249.3 at one or more of positions 332-355, 459-545, 836-859, 1849-1900, 2141-2164, and 2426-2449 of the MLH1 gene.

E101. The dsRNA of any one of E92-E95, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MLH1 gene corresponding to reference mRNA NM_000249.3 at one or more of positions 267-388, 417-545, 805-995, 1639-1722, 1849-1900, 2105-2207, 2337-2387, 2426-2479, and 2508-2600 of the MLH1 gene.

E102. The dsRNA of any one of E92-E95, wherein the antisense strand comprises an antisense nucleobase sequence selected from a list in Table 4, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T), and the sense strand comprises a sense nucleobase sequence complementary to the antisense nucleobase sequence, wherein the.

E103. The dsRNA of any one of E92-E95, wherein the antisense nucleobase sequence consists of an antisense strand in Table 4, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T), and the sense nucleobase sequence consists of a sequence complementary to the antisense nucleobase sequence.

E104. The dsRNA of any one of any one of E92-E95, wherein the sense strand comprises a sense nucleobase sequence selected from a list in Table 4, and the antisense strand comprises an antisense nucleobase sequence complementary to the sense nucleobase sequence.

E105. The dsRNA of any one of any one of E92-E95, wherein the sense nucleobase sequence consists of a sense sequence in Table 4, and the antisense nucleobase sequence consists of a sequence complementary to the sense nucleobase sequence.

E106. The dsRNA of any one of E92-E95, wherein the sense strand comprises a sense nucleobase sequence selected from any one of the lists in Tables 5-11, and the antisense strand comprises an antisense nucleobase sequence complementary to the sense nucleobase sequence.

E107. The dsRNA of any one of E92-E95, wherein the sense nucleobase sequence consists of a sense sequence in any one of Tables 5-11, and the antisense nucleobase sequence consists of a sequence complementary to the sense nucleobase sequence.

E108. The dsRNA of any one of E92-E95, wherein the antisense strand comprises an antisense nucleobase sequence selected from a list in Table 13, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T), and the sense strand comprises a sense nucleobase sequence complementary to the antisense nucleobase sequence.

E109. The dsRNA of any one of E92-E95, wherein the antisense nucleobase sequence consists of an antisense sequence in Table 13, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T), and the sense nucleobase sequence consists of a sequence complementary to the antisense nucleobase sequence.

E110. The dsRNA of any one of E92-E95, wherein the sense strand comprises a sense nucleobase sequence selected from a list in Table 13, and the antisense strand comprises an antisense nucleobase sequence complementary to the sense nucleobase sequence.

E111. The dsRNA of any one of E92-E95, wherein the sense nucleobase sequence consists of a sense sequence in Table 13, and the antisense nucleobase sequence consists of a sequence complementary to the sense nucleobase sequence.

E112. The dsRNA of any one of E92-E111, wherein the dsRNA comprises at least one alternative nucleobase, at least one alternative internucleoside linkage, and/or at least one alternative sugar moiety.

E113. The dsRNA of E112, wherein the at least one alternative internucleoside linkage is a phosphorothioate internucleoside linkage.

E114. The dsRNA of E112, wherein the at least one alternative internucleoside linkage is a 2′-alkoxy internucleoside linkage.

E115. The dsRNA of E112, wherein the at least one alternative internucleoside linkage is an alkyl phosphate internucleoside linkage.

E116. The dsRNA of E112, wherein the at least one alternative nucleobase is 5′-methylcytosine, pseudouridine, or 5-methoxyuridine.

E117. The dsRNA of E112, wherein the alternative sugar moiety is 2′-OMe or a bicyclic nucleic acid.

E118. The dsRNA of any one of E92-E117, wherein the dsRNA comprises at least one 2′-OMe sugar moiety and at least one phosphorothioate internucleoside linkage.

E119. The dsRNA of any one of E92-E118, wherein the dsRNA further comprises a ligand conjugated to the 3′ end of the sense strand through a monovalent or branched bivalent or trivalent linker.

E120. The dsRNA of any one of E92-E119, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1486, 1492, 1494, 1496, 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, 3164, 3210, 3212, 3216, 3248, 3272, 3274, 3276, 3278, and 3288.

E121. The dsRNA of any one of E92-E119, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, and 2966.

E122. The dsRNA of any one of E92-E119, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, and 3164.

E123. The dsRNA of any one of E92-E119, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1486, 1492, 1494, 1496, 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, and 3164.

E124. The dsRNA of any one of E92-E119, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1584, 1598, 1604, 1608, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1998, 2008, 2012, 2024, 2720, 2744, 2746, 2748, 2752, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 3120, 3122, 3124, 3130, 3134, 3136, and 3148.

E125. The dsRNA of any one of E92-E119, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, 3164, 3210, 3212, 3216, 3248, 3272, 3274, 3276, 3278, and 3288.

E126. The dsRNA of any one of E92-E119, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1487, 1493, 1495, 1497, 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, 3165, 3211, 3213, 3217, 3249, 3273, 3275, 3277, 3279, and 3289, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

E127. The dsRNA of any one of E92-E119, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, and 2967, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

E128. The dsRNA of any one of E92-E119, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, and 3165, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

E129. The dsRNA of any one of E92-E119, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1487, 1493, 1495, 1497, 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, and 3165, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

E130. The dsRNA of any one of E92-E119, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1585, 1599, 1605, 1609, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1999, 2009, 2013, 2025, 2721, 2745, 2747, 2749, 2753, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 3121, 3123, 3125, 3131, 3135, 3137, and 3149, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

E131. The dsRNA of any one of E92-E119, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, 3165, 3211, 3213, 3217, 3249, 3273, 3275, 3277, 3279, and 3289, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

E132. The dsRNA of any one of E92-E119, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1486, 1492, 1494, 1496, 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, 3164, 3210, 3212, 3216, 3248, 3272, 3274, 3276, 3278, and 3288.

E133. The dsRNA of any one of E92-E119, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, and 2966.

E134. The dsRNA of any one of E92-E119, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, and 3164.

E135. The dsRNA of any one of E92-E119, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1486, 1492, 1494, 1496, 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, and 3164.

E136. The dsRNA of any one of E92-E119, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1584, 1598, 1604, 1608, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1998, 2008, 2012, 2024, 2720, 2744, 2746, 2748, 2752, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 3120, 3122, 3124, 3130, 3134, 3136, and 3148.

E137. The dsRNA of any one of E92-E119, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1536, 1544, 1546, 1560, 1562, 1564, 1584, 1598, 1604, 1608, 1622, 1624, 1630, 1660, 1710, 1720, 1722, 1726, 1728, 1732, 1734, 1736, 1748, 1752, 1756, 1758, 1762, 1764, 1772, 1954, 1960, 1966, 1972, 1974, 1976, 1998, 2008, 2012, 2024, 2034, 2038, 2052, 2054, 2080, 2086, 2114, 2116, 2120, 2122, 2158, 2162, 2176, 2178, 2570, 2588, 2598, 2604, 2608, 2612, 2616, 2618, 2622, 2628, 2720, 2744, 2746, 2748, 2752, 2894, 2898, 2902, 2906, 2916, 2922, 2924, 2928, 2936, 2938, 2940, 2944, 2948, 2966, 3064, 3066, 3084, 3088, 3120, 3122, 3124, 3130, 3134, 3136, 3148, 3164, 3210, 3212, 3216, 3248, 3272, 3274, 3276, 3278, and 3288.

E138. The dsRNA of any one of E92-E119, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1487, 1493, 1495, 1497, 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, 3165, 3211, 3213, 3217, 3249, 3273, 3275, 3277, 3279, and 3289, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

E139. The dsRNA of any one of E92-E119, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, and 2967, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

E140. The dsRNA of any one of E92-E119, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, and 3165, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

E141. The dsRNA of any one of E92-E119, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1487, 1493, 1495, 1497, 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, and 3165, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

E142. The dsRNA of any one of E92-E119, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1585, 1599, 1605, 1609, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1999, 2009, 2013, 2025, 2721, 2745, 2747, 2749, 2753, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 3121, 3123, 3125, 3131, 3135, 3137, and 3149, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

E143. The dsRNA of any one of E92-E119, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 1537, 1545, 1547, 1561, 1563, 1565, 1585, 1599, 1605, 1609, 1623, 1625, 1631, 1661, 1711, 1721, 1723, 1727, 1729, 1733, 1735, 1737, 1749, 1753, 1757, 1759, 1763, 1765, 1773, 1955, 1961, 1967, 1973, 1975, 1977, 1999, 2009, 2013, 2025, 2035, 2039, 2053, 2055, 2081, 2087, 2115, 2117, 2121, 2123, 2159, 2163, 2177, 2179, 2571, 2589, 2599, 2605, 2609, 2613, 2617, 2619, 2623, 2629, 2721, 2745, 2747, 2749, 2753, 2895, 2899, 2903, 2907, 2917, 2923, 2925, 2929, 2937, 2939, 2941, 2945, 2949, 2967, 3065, 3067, 3085, 3089, 3121, 3123, 3125, 3131, 3135, 3137, 3149, 3165, 3211, 3213, 3217, 3249, 3273, 3275, 3277, 3279, and 3289, wherein the 5′ nucleotide represented by U of the antisense oligonucleotide is any nucleotide (e.g., U, A, G, C, T).

E144. The dsRNA of any one of E92-E143, wherein the dsRNA exhibits at least 50% mRNA inhibition at a 0.5 nM dsRNA concentration when determined using a cell assay when compared with a control cell.

E145. The dsRNA of any one of E92-E143, wherein the dsRNA exhibits at least 40% mRNA inhibition at a 0.5 nM dsRNA concentration when determined using a cell assay when compared with a control cell.

E146. The dsRNA of any one of E92-E143, wherein the dsRNA exhibits at least 30% mRNA inhibition at a 0.5 nM dsRNA concentration when determined using a cell assay when compared with a control cell.

E147. The dsRNA of any one of E92-E143, wherein the dsRNA exhibits at least 60% mRNA inhibition at a 10 nM dsRNA concentration when determined using a cell assay when compared with a control cell.

E148. The dsRNA of any one of E92-E143, wherein the dsRNA exhibits at least 50% mRNA inhibition at a 10 nM dsRNA concentration when determined using a cell assay when compared with a control cell.

E149. The dsRNA of any one of E92-E148, wherein the antisense strand is complementary to at least 17 contiguous nucleotides of an MLH1 gene.

E150. The dsRNA of any one of E92-E148, wherein the antisense strand is complementary to at least 19 contiguous nucleotides of an MLH1 gene.

E151. The dsRNA of any one of E92-E148, wherein the antisense strand is complementary to 19 contiguous nucleotides of an MLH1 gene.

E152. The dsRNA of any one of E92-E151, wherein the antisense strand and/or the sense strand comprises a 3′ overhang of at least 1 linked nucleoside; or a 3′ overhang of at least 2 linked nucleosides.

E153. A pharmaceutical composition comprising the dsRNA of any one of E92-E152 and a pharmaceutically acceptable carrier.

E154. A composition comprising the dsRNA of any one of E92-E152 and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome.

E155. A vector encoding at least one strand of the dsRNA of any one of E92-E152.

E156. A cell comprising the vector of E155.

E157. A method of reducing transcription of MLH1 in a cell, the method comprising contacting the cell with the dsRNA of any one of E92-E152, the pharmaceutical composition of E153, the composition of E154, the vector of E155, or the cell of E156 for a time sufficient to obtain degradation of an mRNA transcript of MLH1, thereby reducing expression of MLH1 in the cell.

E158. A method of treating, preventing, or delaying progression of a trinucleotide repeat expansion disorder in a subject in need thereof, the method comprising administering to the subject the dsRNA of any one of E92-E152, the pharmaceutical composition of E153, the composition of E154, the vector of E155, or the cell of E156.

E159. A method of reducing the level and/or activity of MLH1 in a cell of a subject identified as having a trinucleotide repeat expansion disorder, the method comprising contacting the cell with the dsRNA of any one of E92-E152, the pharmaceutical composition of E153, the composition of E154, the vector of E155, or the cell of E156.

E160. A method for reducing expression of MLH1 in a cell comprising contacting the cell with the dsRNA of any one of E92-E152, the pharmaceutical composition of E153, the composition of E154, the vector of E155, or the cell of E156 and maintaining the cell for a time sufficient to obtain degradation of an mRNA transcript of MLH1, thereby reducing expression of MLH1 in the cell.

E161. A method of decreasing trinucleotide repeat expansion in a cell, the method comprising contacting the cell with the dsRNA of any one of E92-E152, the pharmaceutical composition of E153, the composition of E154, the vector of E155, or the cell of E156.

E162. The method of E160 or E161, wherein the cell is in a subject.

E163. The method of any one of E158, E159, and E162, wherein the subject is a human.

E164. The method of any one of E158-E162, wherein the cell is a cell of the central nervous system or a muscle cell.

E165. The method of any one of E158, E166, and E162-E164, wherein the subject is identified as having a trinucleotide repeat expansion disorder.

E166. The method of any one of E158, E159, and E161-E163, wherein the trinucleotide repeat expansion disorder is a polyglutamine disease.

E167. The method of E166, wherein the polyglutamine disease is selected from the group consisting of dentatorubropallidoluysian atrophy, Huntington's disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 17, and Huntington's disease-like 2.

E168. The method of any one of E158, E159, and E161-E163, wherein the trinucleotide repeat expansion disorder is a non-polyglutamine disease.

E169. The method of E168, wherein the non-polyglutamine disease is selected from the group consisting of fragile X syndrome, fragile X-associated tremor/ataxia syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy type 1, spinocerebellar ataxia type 8, spinocerebellar ataxia type 12, oculopharyngeal muscular dystrophy, Fragile X-associated premature ovarian failure, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy.

E170. A dsRNA of any one of E92-E152, the pharmaceutical composition of E153, the composition of E154, the vector of E155, or the cell of E156 for use in prevention or treatment of a trinucleotide repeat expansion disorder.

E171. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E170, wherein the trinucleotide repeat expansion disorder is selected from the group consisting of dentatorubropallidoluysian atrophy, Huntington's disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 17, Huntington's disease-like 2, fragile X syndrome, fragile X-associated tremor/ataxia syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy type 1, spinocerebellar ataxia type 8, spinocerebellar ataxia type 12, oculopharyngeal muscular dystrophy, Fragile X-associated premature ovarian failure, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy.

E172. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E170 or E171, wherein the trinucleotide repeat expansion disorder is Huntington's disease.

E173. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E170 or E171, wherein the trinucleotide repeat expansion disorder is Friedreich's ataxia.

E174. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E170 or E171, wherein the trinucleotide repeat expansion disorder is myotonic dystrophy type 1.

E175. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of any of E170-E174, wherein the dsRNA, pharmaceutical composition, composition, vector, or cell is administered intrathecally.

E176. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of any of E170-E174, wherein the dsRNA, pharmaceutical composition, composition, vector, or cell is administered intraventricularly.

E177. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell for use of any of E170-E174, wherein the dsRNA, pharmaceutical composition, composition, vector, or cell is administered intramuscularly.

E178. A method of treating, preventing, or delaying progression of a disorder in a subject in need thereof wherein the subject is suffering from trinucleotide repeat expansion disorder, comprising administering to said subject the dsRNA of any one of E92-E152, the pharmaceutical composition of E153, the composition of E154, the vector of E155, or the cell of E158.

E179. The method of E178, further comprising administering a second therapeutic agent.

E180. The method of E179, wherein the second therapeutic agent is an oligonucleotide that hybridizes to an mRNA encoding the Huntingtin gene.

E181. A method of preventing or delaying progression of a trinucleotide repeat expansion disorder in a subject, the method comprising administering to the subject the dsRNA of any one of E92-E152, the pharmaceutical composition of E153, the composition of E154, the vector of E155, or the cell of E156 in an amount effective to delay progression of a trinucleotide repeat expansion disorder of the subject.

E182. The method of E181, wherein the trinucleotide repeat expansion disorder is selected from the group consisting of dentatorubropallidoluysian atrophy, Huntington's disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 17, Huntington's disease-like 2, fragile X syndrome, fragile X-associated tremor/ataxia syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy type 1, spinocerebellar ataxia type 8, spinocerebellar ataxia type 12, oculopharyngeal muscular dystrophy, Fragile X-associated premature ovarian failure, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy.

E183. The method of E181 or E182, wherein the trinucleotide repeat expansion disorder is Huntington's disease.

E184. The method of E181 or E182, wherein the trinucleotide repeat expansion disorder is Friedrich's ataxia.

E185. The method of E181 or E182, wherein the trinucleotide repeat expansion disorder is myotonic dystrophy type 1.

E186. The method of E181 or E182, further comprising administering a second therapeutic agent.

E187. The method of E186, wherein the second therapeutic agent is an oligonucleotide that hybridizes to an mRNA encoding the Huntingtin gene.

E188. The method of any of E181-E187, wherein progression of the trinucleotide repeat expansion disorder is delayed by at least 120 days, for example, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years or more, when compared with a predicted progression.

E189. A dsRNA of any one of E92-E152, the pharmaceutical composition of E153, the composition of E154, the vector of E155, or the cell of E156 for use in preventing or delaying progression of a trinucleotide repeat expansion disorder in a subject.

E190. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E189, wherein the trinucleotide repeat expansion disorder is selected from the group consisting of dentatorubropallidoluysian atrophy, Huntington's disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 17, Huntington's disease-like 2, fragile X syndrome, fragile X-associated tremor/ataxia syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy type 1, spinocerebellar ataxia type 8, spinocerebellar ataxia type 12, oculopharyngeal muscular dystrophy, Fragile X-associated premature ovarian failure, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy.

E191. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E189 or E190, wherein the trinucleotide repeat expansion disorder is Huntington's disease.

E192. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E189 or E190, wherein the trinucleotide repeat expansion disorder is Friedrich's ataxia.

E193. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E189 or E190, wherein the trinucleotide repeat expansion disorder is myotonic dystrophy type 1.

E194. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of any one of E189-E193, wherein progression of the trinucleotide repeat expansion disorder is delayed by at least 120 days, for example, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years or more, when compared with a predicted progression.

Number	Date	Country
62774771	Dec 2018	US
62778686	Dec 2018	US
62876987	Jul 2019	US

METHODS FOR THE TREATMENT OF TRINUCLEOTIDE REPEAT EXPANSION DISORDERS ASSOCIATED WITH MLH1 ACTIVITY

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