METHODS FOR THE TREATMENT OF NUCLEOTIDE REPEAT EXPANSION DISORDERS ASSOCIATED WITH MSH3 ACTIVITY

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The content of the electronically submitted sequence listing in ASCII text file (Name 4398_026 PC02_Seqlisting_ST25; Size: 611,414 Bytes; and Date of Creation: May 7, 2021) filed with the application is incorporated herein by reference in its entirety.

BACKGROUND

Nucleotide repeat expansion disorders (e.g., trinucleotide repeat expansion disorders) are genetic disorders caused by nucleotide repeat expansions (e.g., trinucleotide repeats). Nucleotide repeat expansions (e.g., 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 nucleotide repeats (e.g., trinucleotide repeats) can result in defective or toxic gene products, impair RNA transcription, and/or cause toxic effects by forming toxic mRNA transcripts.

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

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the dose response curve for dsRNA of SENSE OLIGO NO: 156/ANTISENSE OLIGO NO: 157 tested at 10 nM and 0.5 nM.

FIG. 2 shows the dose response curve for of SENSE OLIGO NO: 906/ANTISENSE OLIGO NO: 907 tested at 10 nM and 0.5 nM.

FIG. 3 shows the dose response curve for dsRNA of SENSE OLIGO NO: 968/ANTISENSE OLIGO NO: 969 tested at 10 nM and 0.5 nM.

FIG. 4 shows the dose response curve for dsRNA of SENSE OLIGO NO: 1392/ANTISENSE OLIGO NO: 1393 tested at 10 nM and 0.5 nM.

FIG. 5 shows the dose response curve for dsRNA of SENSE OLIGO NO: 1874/SEQ ID NO: 1875, tested at 10 nM and 0.5 nM.

FIG. 6 shows the dose response curve for dsRNA of SENSE OLIGO NO: 1366/ANTISENSE OLIGO NO: 1367, tested at 10 nM and 0.5 nM.

FIG. 7 shows the non linear regression curves depicting mean, standard deviation, and RQ values for each the dsRNA shown, at ten concentrations. (See Example 7.)

FIG. 8A shows the dose response curve for dsRNA of SENSE OLIGO NO: 420/ANTISENSE OLIGO NO: 421 at ten concentrations. (See Example 7.)

FIG. 8B shows the dose response curve for dose response curve for dsRNA of SENSE OLIGO NO: 1302/ANTISENSE OLIGO NO: 1303 at ten concentrations. (See Example 7.)

FIG. 8C shows the dose response curve for dsRNA of SENSE OLIGO NO: 550/ANTISENSE OLIGO NO: 551 at ten concentrations. (See Example 7.)

FIG. 8D shows the dose response curve for dsRNA of SENSE OLIGO NO: 672/ANTISENSE OLIGO NO: 673 at ten concentrations. (See Example 7.)

FIGS. 9A-9I show the IC₅₀analysis for the target knock down measured by qPCR for siRNAs with highest activity in the dual-dose screen. The X-axis represents the concentration of siRNA transfected and the Y-axis represents the relative MSH3 target remaining. (See Example 8.)

FIG. 9A shows the IC₅₀analysis for the target knock down measured by qPCR for siRNA of SENSE OLIGO NO: 1366/ANTISENSE OLIGO NO: 1367.

FIG. 9B shows the IC₅₀analysis for the target knock down measured by qPCR for siRNA of SENSE OLIGO NO: 1874/ANTISENSE OLIGO NO: 1875.

FIG. 9C shows the IC₅₀analysis for the target knock down measured by qPCR for siRNA of SENSE OLIGO NO: 388/ANTISENSE OLIGO NO: 389.

FIG. 9D shows the IC₅₀analysis for the target knock down measured by qPCR for siRNA of SENSE OLIGO NO: 392/ANTISENSE OLIGO NO: 393.

FIG. 9E shows the IC₅₀analysis for the target knock down measured by qPCR for siRNA of SENSE OLIGO NO: 402/ANTISENSE OLIGO NO: 403.

FIG. 9F shows the IC₅₀analysis for the target knock down measured by qPCR for siRNA of SENSE OLIGO NO: 1302/ANTISENSE OLIGO NO: 1303.

FIG. 9G shows the IC₅₀analysis for the target knock down measured by qPCR for siRNA of SENSE OLIGO NO: 648/ANTISENSE OLIGO NO: 649.

FIG. 9H shows the IC₅₀analysis for the target knock down measured by qPCR for siRNA of SENSE OLIGO NO: 656/ANTISENSE OLIGO NO: 657.

FIG. 9I shows the IC₅₀analysis for the target knock down measured by qPCR for siRNA of SENSE OLIGO NO: 832/ANTISENSE OLIGO NO: 833.

FIG. 10 shows the fold change in MSH3 expression relative to a luciferase control from four plates. The X-axis represents the concentration of siRNA transfection on different plates and the Y-axis represents the percentage of target remaining.

SUMMARY OF THE DISCLOSURE

The present disclosure features useful compositions and methods to treat nucleotide repeat expansion disorders (e.g., 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 MSH3 activity.

Some aspects of this disclosure are directed to 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 MSH3 gene, and wherein the dsRNA comprises a duplex structure of between 15 and 30 linked nucleosides in length.

In some aspects, this disclosure is directed to a dsRNA for reducing expression of MSH3 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 MSH3 gene, and wherein the dsRNA comprises a duplex structure of between 15 and 30 linked nucleosides in length.

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 MSH3 gene corresponding to a sequence of reference mRNA NM 002439.4 at one or more of positions 566-589, 678-701, 875-989, 1019-1088, 1370-1393, 1466-1569, 1721-1849, 1879-2038, 2086-2171, 2783-2806, 2847-2922, 3043-3119, 3241-3314, 3330-3353, or 3701-3792 of the MSH3 gene.

In some aspects, the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM 002439.4 at one or more of positions 876-989, 1019-1088, 1370-1393, 1889-1938, or 3241-3314 of the MSH3 gene.

In some aspects, the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM 002439.4 at one or more of positions 876-989, 1019-1088, 1370-1393, 1466-1569, 1756-1849, 1879-2038, 2086-2171, 2783-2806, 2847-2922, 3043-3119, 3241-3314, 3330-3353, or 3701-3792 of the MSH3 gene.

In some aspects, the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM 002439.4 at one or more of positions 678-701, 875-989, 1019-1088, 1370-1393, 1466-1569, 1721-1849, 1879-2038, 2086-2171, 2783-2806, 2847-2922, 3043-3119, 3241-3314, 3330-3353, or 3701-3792 of the MSH3 gene.

In some aspects, the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM 002439.4 at one or more of positions 875-989, 1019-1088, 1370-1393, 1466-1569, 1721-1849, 1879-1970, 2783-2806, 2847-2922, 3043-3119, 3241-3314, 3330-3353, or 3703-3792 of the MSH3 gene.

In some aspects, the antisense strand comprises an antisense nucleobase sequence selected from Table 3, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), 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 3, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), 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 Table 3, 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 strand in Table 3, 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 Tables 4-10, 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 strand in any one of Tables 4-10, 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 Table 11, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), 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 11, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), 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 Table 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 strand in Table 11, 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, at least one alternative internucleoside linkage is a phosphorothioate internucleoside linkage. In some aspects, at least one alternative internucleoside linkage is a 2′-alkoxy internucleoside linkage. In some aspects, at least one alternative internucleoside linkage is an alkyl phosphate internucleoside linkage. In some aspects, at least one alternative nucleobase is 5′-methylcytosine, pseudouridine, or 5-methoxyuridine. In some aspects, at least one 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: 140, 156, 234, 240, 246, 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.

In some aspects, the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 2264, 2290, 2308, or 2318.

In some aspects, the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.

In some aspects, the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 234, 240, 246, 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.

In some aspects, the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.

In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 2265, 2291, 2309, or 2319, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).

In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).

In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).

In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).

In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 2264, 2290, 2308, or 2318.

In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.

In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 234, 240, 246, 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.

In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.

In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 141, 157, 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691.

In some aspects, the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).

In some aspects, the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).

In some aspects, the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).

In some aspects, the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 141, 157, 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).

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 70% 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 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 MSH3 gene. In some aspects, the antisense strand is complementary to at least 19 contiguous nucleotides of an MSH3 gene. In some aspects, the antisense strand is complementary to 19 contiguous nucleotides of an MSH3 gene. In some aspects, the sense strand is complementary to at least 17 contiguous nucleotides of an MSH3 gene. In some aspects, the sense strand is complementary to at least 19 contiguous nucleotides of an MSH3 gene. In some aspects, the sense strand is complementary to 19 contiguous nucleotides of an MSH3 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.

In some aspects, this disclosure is directed to a pharmaceutical composition comprising one or more of the dsRNAs described herein and a pharmaceutically acceptable carrier.

In some aspects, this disclosure is directed to a composition comprising one or more of the dsRNAs described herein and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome.

In some aspects, this disclosure is directed to a vector encoding at least one strand of any one of the dsRNAs described herein.

In some aspects, this disclosure is directed to a cell comprising the vector encoding at least one strand of any one of the dsRNAs described herein.

In some aspects, this disclosure is directed to a method of reducing transcription of MSH3 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 the dsRNAs described herein, a composition of one or more of the dsRNAs described herein and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome, the vector encoding at least one strand of any one of the dsRNAs described herein, or the cell comprising the vector encoding at least one strand of any one of the dsRNAs described herein, for a time sufficient to obtain degradation of an mRNA transcript of MSH3, thereby reducing expression of MSH3 in the cell.

In some aspects, this disclosure 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 contacting the cell with one or more of the dsRNAs described herein, a pharmaceutical composition of one or more the dsRNAs described herein, a composition of one or more of the dsRNAs described herein and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome, the vector encoding at least one strand of any one of the dsRNAs described herein, or the cell comprising the vector encoding at least one strand of any one of the dsRNAs described herein.

In some aspects, this disclosure is directed to a method of reducing the level and/or activity of MSH3 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 the dsRNAs described herein, a composition of one or more of the dsRNAs described herein and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome, the vector encoding at least one strand of any one of the dsRNAs described herein, or the cell comprising the vector encoding at least one strand of any one of the dsRNAs described herein.

In some aspects, this disclosure is directed to a method for reducing expression of MSH3 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 the dsRNAs described herein, a composition of one or more of the dsRNAs described herein and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome, the vector encoding at least one strand of any one of the dsRNAs described herein, or the cell comprising the vector encoding at least one strand of any one of the dsRNAs described herein, thereby reducing expression of MSH3 in the cell.

In some aspects, this disclosure is directed to a method of decreasing nucleotide repeat expansion (e.g., 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 the dsRNAs described herein, a composition of one or more of the dsRNAs described herein and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome, the vector encoding at least one strand of any one of the dsRNAs described herein, or the cell comprising the vector encoding at least one strand of any one of the dsRNAs described herein.

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 nucleotide repeat expansion disorder (e.g., a trinucleotide repeat expansion). In some aspects, the nucleotide repeat expansion disorder (e.g., a trinucleotide repeat expansion) 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, and Huntington's disease-like 2. In some aspects, the nucleotide repeat expansion disorder (e.g., a trinucleotide repeat expansion disorder) is Huntington's disease.

In some aspects, the inucleotide repeat expansion disorder (e.g., a 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, and early infantile epileptic encephalopathy. In some aspects, the nucleotide repeat expansion disorder (e.g., a trinucleotide repeat expansion disorder) is Friedreich's ataxia. In some aspects, the nucleotide repeat expansion disorder (e.g., trinucleotide repeat expansion disorder) is myotonic dystrophy type 1.

In some aspects, this disclosure is directed to one or more of the dsRNAs described herein, a pharmaceutical composition of one or more the dsRNAs described herein, a composition of one or more of the dsRNAs described herein and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome, the vector encoding at least one strand of any one of the dsRNAs described herein, or the cell comprising the vector encoding at least one strand of any one of the dsRNAs described herein for use in prevention or treatment of a nucleotide repeat expansion disorder (e.g., a trinucleotide repeat expansion disorder).

In some aspects, the one or more of the dsRNAs described herein, the pharmaceutical composition of one or more the dsRNAs described herein, the composition of one or more of the dsRNAs described herein and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome, the vector encoding at least one strand of any one of the dsRNAs described herein, or the cell comprising the vector encoding at least one strand of any one of the dsRNAs described herein is administered intrathecally.

In some aspects, this disclosure is directed to a method of treating, preventing, or delaying progression of a disorder in a subject in need thereof wherein the subject is suffering from a nucleotide repeat expansion disorder (e.g., a trinucleotide repeat expansion disorder), comprising administering to said subject one or more of the dsRNAs described herein, the pharmaceutical composition of one or more the dsRNAs described herein, the composition of one or more of the dsRNAs described herein and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome, the vector encoding at least one strand of any one of the dsRNAs described herein, or the cell comprising the vector encoding at least one strand of any one of the dsRNAs described herein.

In some aspects, the method of treating, preventing, or delaying progression of a disorder in a subject further comprises administering at least one additional therapeutic agent. In some aspects, the at least one additional therapeutic agent is another oligonucleotide, or pharmaceutically acceptable salt thereof, that hybridizes to an mRNA encoding the Huntingtin gene.

In some aspects, the method of treating, preventing, or delaying progression of a disorder in a subject delays progression of the nucleotide repeat expansion disorder (e.g., a trinucleotide repeat expansion disorder) 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.

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%, and 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, 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, intraocular routes, subcutaneous routes, intra cisterna magna routes, intravenous routes, intramuscular routes, intracerebroventricular routes, intrathecal 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 an additional therapeutic agent of the combination can be administered orally.

As used herein, the term “MSH3” refers to MutS Homolog 3, 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 MSH3 that maintain at least one in vivo or in vitro activity of a native MSH3. The term encompasses full-length unprocessed precursor forms of MSH3 as well as mature forms resulting from post-translational cleavage of the signal peptide. MSH3 is encoded by the MSH3 gene. The nucleic acid sequence of an exemplary Homo sapiens (human) MSH3 gene is set forth in NCBI Reference NM_002439.4 or in SEQ ID NO: 1. The term “MSH3” also refers to natural variants of the wild-type MSH3 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 MSH3, which is set forth in NCBI Reference No. NP_002430.3 or in SEQ ID NO: 2. The nucleic acid sequence of an exemplary Mus musculus (mouse) MSH3 gene is set forth in NCBI Reference No. NM_010829.2 or in SEQ ID NO: 3. The nucleic acid sequence of an exemplary Rattus norvegicus (rat) MSH3 gene is set forth in NCBI Reference No. NM_001191957.1 or in SEQ ID NO: 4. The nucleic acid sequence of an exemplary Macaca fascicularis (cyno) MSH3 gene is set forth in NCBI Reference No. XM_005557283.2 or in SEQ ID NO: 5.

The term “MSH3” as used herein also refers to a particular polypeptide expressed in a cell by naturally occurring DNA sequence variations of the MSH3 gene, such as a single nucleotide polymorphism in the MSH3 gene. Numerous SNPs within the MSH3 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 MSH3 gene can be found at, NCBI dbSNP Accession Nos.: rs1650697, rs70991108, rs10168, rs26279, rs26282, rs26779, rs26784, rs32989, rs33003, rs33008, rs33013, rs40139, rs181747, rs184967, rs245346, rs245397, rs249633, rs380691, rs408626, rs442767, rs836802, rs836808, rs863221, rs1105525, rs1428030, rs1478834, rs1650694, rs1650737, rs1677626, rs1677658, rs1805355, rs2897298, rs3045983, rs3797897, rs4703819, rs6151627, rs6151640, rs6151662, rs6151670, rs6151735, rs6151838, rs7709909, rs7712332, rs10079641, rs12513549, and rs12522132.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an MSH3 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 dsRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a MSH3 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 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or from about 15-30 nucleotides, 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 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 an oligonucleotide 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 dsRNA featured herein by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide 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 featured 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.

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 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 (e.g., A-LNA, 5mC L-NA, G-LNA, T-LNA)) 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 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., MSH3). “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 MSH3 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 MSH3 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” dsRNA or “chimera” is a dsRNA which contains two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleoside or nucleotide.

The duplex region 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 MSH3 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-III-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” 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 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 a dsRNA (e.g. the termini of region A or C). In some aspects, the conjugate or dsRNA conjugate can comprise a linker region which is positioned between the dsRNA and the conjugate moiety. In some aspects, the linker between the conjugate and 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” 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 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 a RISC pathway. “Substantially complementary” can refer to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding MSH3). For example, a polynucleotide is complementary to at least a part of an MSH3 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding MSH3. However, where two oligonucleotides 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 oligonucleotide that is substantially complementary to all or a portion of a gene, primary transcript, a sequence (e.g., a target sequence, e.g., an MSH3 nucleotide sequence), or processed mRNA, so as to interfere with expression of the endogenous gene (e.g., MSH3). 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 oligonucleotide.

As used herein, an “agent that reduces the level and/or activity of MSH3” refers to any polynucleotide agent (e.g., a dsRNA) that reduces the level of or inhibits expression of MSH3 in a cell or subject. By “reducing the level of MSH3,” “reducing expression of MSH3,” and “reducing transcription of MSH3” is meant decreasing the level, decreasing the expression, or decreasing the transcription of MSH3 in a cell or subject, e.g., by administering a dsRNA to the cell or subject. The level of MSH3 can be measured using any method known in the art (e.g., by measuring the levels of MSH3 mRNA or levels of MSH3 protein in a cell or a subject). The reduction can be a decrease in the level, expression, or transcription of MSH3 of about 5% or more (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) in a cell or subject compared to prior to treatment. The MSH3 can be any MSH3 gene (such as, e.g., a mouse MSH3 gene, a rat MSH3 gene, a monkey MSH3 gene, or a human MSH3 gene) as well as variants or mutants of a MSH3 gene that encode a MSH3 protein. Thus, the MSH3 gene can be a wild-type MSH3 gene, a mutant MSH3 gene, or a transgenic MSH3 gene in the context of a genetically manipulated cell, group of cells, or organism.

By “reducing the activity of MSH3” is meant decreasing the level of an activity related to MSH3 (e.g., by reducing the amount of nucleotide repeats in a gene associated with a nucleotide repeat expansion disorder, e.g., a trinucleotide repeat expansion disorder, that is related to MSH3 activity). The activity level of MSH3 can be measured using any method known in the art (e.g., by directly sequencing a gene associated with a nucleotide repeat expansion disorder to measure the levels of nucleotide repeats).

By “reducing the level of MSH3” is meant decreasing the level of MSH3 in a cell or subject, e.g., by administering an oligonucleotide, or pharmaceutically acceptable salt thereof, to the cell or subject. The level of MSH3 can be measured using any method known in the art (e.g., by measuring the levels of MSH3 mRNA or levels of MSH3 protein in a cell or a subject).

By “modulating the activity of a MutSβ heterodimer comprising MSH3” is meant altering the level of an activity related to a MutSβ heterodimer, or a related downstream effect. The activity level of a MutSβ 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., MSH3). Non-limiting examples of inhibitors include polynucleotides (e.g., 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.

The phrase “contacting a cell with a dsRNA,” as used herein, includes contacting a cell by any possible means. Contacting a cell with a dsRNA includes contacting a cell in vitro with the dsRNA or contacting a cell in vivo with the dsRNA. The contacting can be done directly or indirectly. Thus, for example, the dsRNA can be put into physical contact with the cell by the individual performing the method, or alternatively, the 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 dsRNA. Contacting a cell in vivo can be done, for example, by injecting the dsRNA into or near the tissue where the cell is located, or by injecting the 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 dsRNA can contain and/or be coupled to a ligand that directs the dsRNA to a site of interest. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell can be contacted in vitro with a dsRNA and subsequently transplanted into a subject.

In one aspect, contacting a cell with a dsRNA includes “introducing” or “delivering the dsRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of a dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing a dsRNA into a cell can be in vitro and/or in vivo. For example, for in vivo introduction, dsRNA 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., a dsRNA or a plasmid 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 dsRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the 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 MSH3 (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 nucleotide repeat expansion disorder (e.g., a trinucleotide repeat expansion disorder), it is an amount of the agent that reduces the level and/or activity of MSH3 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 MSH3. The amount of a given agent that reduces the level and/or activity of MSH3 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 MSH3 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 MSH3 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 a dsRNA that, when administered to a subject having or predisposed to have a nucleotide repeat expansion disorder (e.g., 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 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 MSH3 (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 nucleotide repeat expansion disorders (e.g., trinucleotide repeat expansion 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 a dsRNA that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. dsRNAs employed in the methods 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 strand that is substantially complementary to all or a portion of a gene, primary transcript, a sequence (e.g., a target sequence, e.g., an MSH3 nucleotide sequence), or processed mRNA, so as to interfere with expression of the endogenous gene (e.g., MSH3). 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 dsRNA.

An “amount effective to reduce nucleotide repeat expansion” of a particular gene refers to an amount of the agent that reduces the level and/or activity of MSH3 (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 nucleotide repeat expansion of a particular gene (e.g., a gene associated with a nucleotide repeat expansion disorder, e.g., a trinucleotide repeat expansion disorder described herein).

As used herein, the term “a subject identified as having a nucleotide repeat expansion disorder” refers to a subject identified as having a molecular or pathological state, disease or condition of or associated with a nucleotide repeat expansion disorder, such as the identification of a nucleotide repeat expansion disorder or symptoms thereof, or to identification of a subject having or suspected of having a nucleotide 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. Nucleotide repeats are common in the human genome and are not normally associated with disease. In some cases, however, the number of repeats expands beyond a stable threshold and can lead to disease, with the severity of symptoms generally correlated with the number of repeats. Nucleotide 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 (DNA core sequences), 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., MSH3), 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 10%, 15%, 20%, 50%, 75%, 100%, or 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; for intraocular administration (e.g., for intravitreal or subretinal administration); or in any other pharmaceutically acceptable formulation.

In some aspects, provided herein are pharmaceutical compositions that are formulated for intracerebroventricular injection.

A “pharmaceutically acceptable excipient,” as used herein, refers to 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 nucleotide or 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 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.

DETAILED DESCRIPTION

The present inventors have found that inhibition or depletion of MSH3 level and/or activity in a cell is effective in the treatment of a nucleotide repeat expansion disorder (e.g., a trinucleotide repeat expansion disorder). Accordingly, useful compositions and methods to treat nucleotide repeat expansion disorders (e.g., a trinucleotide repeat expansion disorders), e.g., in a subject in need thereof are provided herein.

I. Nucleotide Repeat Expansion Disorders

Nucleotide repeat expansion disorders (e.g., 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.

Nucleotide repeat expansion disorders (e.g., 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 nucleotide repeat expansions in non-coding regions, as in Myotonic dystrophy, or by the expansion of nucleotide repeats other than CAG that can be in coding or non-coding regions such as the CGG repeat expansion responsible for Fragile X Syndrome.

Nucleotide repeat expansion disorders (e.g., 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, nucleotide repeat expansion disorders (e.g., 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.

Nucleotide repeat expansion disorders (e.g., trinucleotide repeat expansion disorders) are well known in the art. For example, frontotemporal dementia (FTD) is a hexanucleotide repeat string of nucleotides GGGGCC that is repeated many more times in an individual than an individual without FTD. Additionally, an individual having spinocerebellar ataxia type 36 (SCA36) has many more GGCCTG repeats than an individual without SCA36.

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

TABLE 1

Exemplary Trinucleotide Repeat Expansion Disorders

Nucleotide

Disorder
Gene
Repeat

ARX-nonsyndromic X-linked
ARX
GCG

mental retardation (XLMR)

Baratela-Scott Syndrome
XYLT1
GGC

Blepharophimosis/Ptosis (BPES)
FOXL2
GCG

Epicanthus inversus syndrome

(BPES) types I and 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
ATN1
CAG

atrophy (DRPLA)/Haw River

syndrome

Early infantile epileptic
ARX
GCG

encephalopathy (Ohtahara

syndrome)

FRA2A syndrome
AFF3
CGC

FRA7A syndrome
ZNF713
CGG

Fragile X mental retardation
AFF2/FMR2
GCC

(FRAX-E)

Fragile X Syndrome (FXS)
FMR1
CGG

Fragile X-associated Primary
FMR1
CGG

Ovarian Insufficiency (FXPOI)

Fragile X-associated Tremor Ataxia
FMR1
CGG

Syndrome (FXTAS)

Friedreich ataxia (FRDA)
FXN
GAA

Fuchs' Corneal Endothelial
TCF4
CTG

Dystrophy (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)

KCNN3-associated (e.g.,
KCNN3
CAG

schizophrenia)

Multiple Skeletal dysplasias
COMP
GAC

Myotonic Dystrophy type 1 (DM1)
DMPK
CTG

Myotonic Dystrophy type 2 (DM2)
CNBP
CCTG

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

risk of prostate cancer)

Neuronal intranuclear inclusion
NOTCH2NLC
GGC

disease (NIID)

Oculopharyngeal Muscular
PABPN1
GCG

Dystrophy (OPMD)

Spinal Muscular Bulbar Atrophy
AR
CAG

(SMBA)

Spinocerebellar ataxia type 1
ATXN1
CAG

(SCA1)

Spinocerebellar ataxia type 10
ATXN10
ATTCT

(SCA10)

Spinocerebellar ataxia type 12
PPP2R2B
CAG

(SCA12)

Spinocerebellar ataxia type 17
TBP/ATXN17
CAG

(SCA17)

Spinocerebellar ataxia type 2
ATXN2
CAG

(SCA2)

Spinocerebellar ataxia type 3
ATXN3
CAG

(SCA3)/Machado-Joseph Disease

Spinocerebellar ataxia type 45
FAT2
CAG

(SCA45)

Spinocerebellar ataxia type 6
CACNA1A
CAG

(SCA6)

Spinocerebellar ataxia type 7
ATXN7
CAG

(SCA7)

Spinocerebellar ataxia type 8
ATXN8
CTG

(SCA8)

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 nucleotide repeat expansion disorders (e.g., trinucleotide repeat expansion disorders) are typically selected based on an experimental association of the protein associated with a nucleotide repeat expansion disorders (e.g., trinucleotide repeat expansion disorder) to a nucleotide repeat expansion disorder. For example, the production rate or circulating concentration of a protein associated with a nucleotide repeat expansion disorder (e.g., trinucleotide repeat expansion disorder) can be elevated or depressed in a population having a nucleotide repeat expansion disorder (e.g., a trinucleotide repeat expansion disorder) relative to a population lacking the nucleotide repeat expansion disorder (e.g., a 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 nucleotide repeat expansion disorder (e.g., 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 Nucleotide Repeat Expansion

There is growing evidence that DNA repair pathways, particularly mismatch repair (MMR), are involved in the expansion of nucleotide repeats (e.g., 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 Mlh3 Modify CAG Instability in Huntington's Disease Mice: Genome-Wide and Candidate Approaches. PLoS Genet 9(10): e1003930). 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). MSH3, another component of the mismatch repair pathway, has been reported to be linked to somatic expansion: polymorphisms in Msh3 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 Msh3 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 e1003280). 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). Further evidence of Msh2 and Msh3's involvement in expansion repeats was reported in a study in which short hairpin RNA (shRNA) knockdown of either MSH2 or MSH3 slowed, and ectopic expression of either MSH2 or MSH3 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. dsRNA Agents

Agents described herein that reduce the level and/or activity of MSH3 in a cell can be, for example, a polynucleotide, e.g., a double stranded nucleotide, or pharmaceutically acceptable salt thereof. These agents reduce the level of an activity related to MSH3, or a related downstream effect, or reduce the level of MSH3 in a cell or subject.

In some aspects, the agent that reduces the level and/or activity of MSH3 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 MSH3. Inhibitory RNA molecules can be double stranded (dsRNA) molecules. For example, a dsRNA includes a short interfering RNA (siRNA) that targets full-length MSH3. 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 MSH3. 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 or pharmaceutically acceptable salt thereof decreases the level and/or activity of a positive regulator of function. In other aspects, the dsRNA or pharmaceutically acceptable salt thereof 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, or pharmaceutically acceptable salt thereof, decreases the level and/or activity or function of MSH3. In some aspects, the dsRNA, or pharmaceutically acceptable salt thereof, inhibits expression of MSH3. In other aspects, the dsRNA, or pharmaceutically acceptable salt thereof, increases degradation of MSH3 and/or decreases the stability (i.e., half-life) of MSH3. The dsRNA can be chemically synthesized or transcribed in vitro.

The dsRNA, or pharmaceutically acceptable salt thereof, 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 MSH3 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 MSH3 gene, the dsRNA, or pharmaceutically acceptable salt thereof, can reduce the expression of MSH3 (e.g., a human, a primate, a non-primate, or a bird MSH3) 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, or pharmaceutically acceptable salt thereof, 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 MSH3 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 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 or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 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 or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 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 MSH3 expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA, or pharmaceutically acceptable salt thereof, 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. 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. Various dsRNA overhangs are known in the art and can include, but are not limited to: dTdT, UU, or other nucleotides. The overhangs can include phosphorothioate linkages. The overhangs can be different between the sense and antisense oligonucleotides. In some aspects, the dsRNA sequences described herein can include any of the above mentioned overhangs.

A dsRNA, or pharmaceutically acceptable salt thereof, 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 can be prepared separately. Then, the component strands can be 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 3, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), 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 3, 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 3, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), 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 3, 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 4-10, 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 4-10, 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 11, 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 11, 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 11, 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 11, and the antisense strand consists of a nucleobase sequence complementary to the nucleobase sequence of the sense strand.

In these aspects, 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 3 or 11, and the second oligonucleotide is described as the corresponding antisense strand (guide strand) of the sense strand in Table 3 or 11, 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 MSH3 gene. In some aspects, the region of at least 15 contiguous that is complementary to an MSH3 gene corresponding to reference mRNA NM_002439.4 is one or more of positions 566-589, 678-701, 875-989, 1019-1088, 1370-1393, 1466-1569, 1721-1849, 1879-2038, 2086-2171, 2783-2806, 2847-2922, 3043-3119, 3241-3314, 3330-3353, or 3701-3792 of the MSH3 gene. In some aspects, the region of at least 15 contiguous that is complementary to an MSH3 gene corresponding to reference mRNA NM_002439.4 at one or more of positions 876-989, 1019-1088, 1370-1393, 1889-1938, or 3241-3314 of the MSH3 gene. In some aspects, the region of at least 15 contiguous that is complementary to an MSH3 gene corresponding to reference mRNA NM_002439.4 at one or more of positions 876-989, 1019-1088, 1370-1393, 1466-1569, 1756-1849, 1879-2038, 2086-2171, 2783-2806, 2847-2922, 3043-3119, 3241-3314, 3330-3353, or 3701-3792 of the MSH3 gene. In some aspects, the region of at least 15 contiguous that is complementary to an MSH3 gene corresponding to reference mRNA NM_002439.4 at one or more of positions 678-701, 875-989, 1019-1088, 1370-1393, 1466-1569, 1721-1849, 1879-2038, 2086-2171, 2783-2806, 2847-2922, 3043-3119, 3241-3314, 3330-3353, or 3701-3792 of the MSH3 gene. In some aspects, the region of at least 15 contiguous that is complementary to an MSH3 gene corresponding to reference mRNA NM_002439.4 at positions 879-921 of the MSH3 gene. In some aspects, the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at one or more of positions 875-989, 1019-1088, 1370-1393, 1466-1569, 1721-1849, 1879-1970, 2783-2806, 2847-2922, 3043-3119, 3241-3314, 3330-3353, or 3703-3792 of the MSH3 gene. In some aspects, the region of at least 15 contiguous that is complementary to an MSH3 gene corresponding to reference mRNA NM_002439.4 at one or more of positions 566-589, 678-701, 875-989, 1019-1088, 1370-1393, 1466-1569, 1721-1849, 1879-2038, 2086-2171, 2783-2806, 2847-2922, 3043-3119, 3241-3314, 3330-3353, or 3701-3792 of the MSH3 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: 6-2873, 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 comprises a nucleobase sequence of any one of SEQ ID NOs: 140, 156, 234, 240, 246, 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690. In some aspects, the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 2264, 2290, 2308, or 2318. In some aspects, the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690. In some aspects, the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 234, 240, 246, 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690. In some aspects, the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690. In some aspects, the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 140, 156, 234, 240, 246, 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.

In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 141, 157, 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G). In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 2265, 2291, 2309, or 2319, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G). In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G). In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G). In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G). In some aspects, the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 141, 157, 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).

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: 6-2873, 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: 140, 156, 234, 240, 246, 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690. In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 2264, 2290, 2308, or 2318. In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690. In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 234, 240, 246, 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690. In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690. In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 140, 156, 234, 240, 246, 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690. In some aspects, the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 141, 157, 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691.

In some aspects, the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 2265, 2291, 2309, or 2319, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G). In some aspects, the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G). In some aspects, the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G). In some aspects, the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G). In some aspects, the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 141, 157, 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).

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: 6-2873, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G). 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: 6-2873, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).

It will be understood that, although the sequences in SEQ ID NOs: 6-2873 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: 6-2873 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 MSH3 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 MSH3 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 MSH3 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. In one aspect, if the antisense strand of the dsRNA contains mismatches to a target sequence, 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 MSH3 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 a MSH3 gene. Consideration of the efficacy of dsRNAs with mismatches in reducing expression of MSH3 is important, especially if the particular region of complementarity in MSH3 is known to have polymorphic sequence variation within the population.

Construction of vectors for expression of polynucleotides for use in the methods described herein 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.

A. Alternative dsRNAs

In one aspect, one or more of the linked nucleosides or internucleosidic linkages of the 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 a 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, 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). 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 some aspects, substantially all of the nucleosides or internucleosidic linkages of a dsRNA are alternative nucleosides. In other aspects, all of the nucleosides or internucleosidic linkages of dsRNA are alternative nucleosides. dsRNA 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, 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 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, a 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; 564,562; 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 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 dsRNAs are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some aspects include dsRNAs with phosphorothioate backbones and oligonucleotides 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 dsRNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506. In other aspects, the 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 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, 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, 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 a dsRNA, or a group for improving the pharmacodynamic properties of a 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 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 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 a dsRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. 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.

A 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 oligomeric compounds described herein. 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.

A 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, a 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 dsRNAs has been shown to increase 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-C₁₂alkyl, or a 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 β-D-ribofuranose (see WO 99/14226).

A 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.”

A dsRNA described herein 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, a 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 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 a dsRNA include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic 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 dsRNAs comprise nucleosides with alternative sugar moieties and can comprise DNA or RNA nucleosides. In some aspects, the dsRNA comprises nucleosides comprising alternative sugar moieties and DNA nucleosides. Incorporation of alternative nucleosides into the dsRNA can enhance the affinity of the 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 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 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 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. In one aspect, the dsRNA comprises one or more nucleosides comprising alternative sugar moieties, e.g., 2′ sugar alternative nucleosides. In some aspects, the dsRNA comprise 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 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 oligonucleotide 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 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 dsRNA comprises at least one BNA unit and at least one 2′ substituted modified nucleoside. In some aspects, the dsRNA comprises both 2′ sugar modified nucleosides and DNA units.

B. dsRNAs Conjugated to Ligands

dsRNAs can be chemically linked to one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the 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 a 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, 03-(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 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 a 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. dsRNA that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides 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 dsRNAs can be synthesized by the use of a dsRNA that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the dsRNA (described below). This reactive 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 dsRNAs 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 dsRNA, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated dsRNA, 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 dsRNAs. In some aspects, the oligonucleotides or linked nucleosides described herein 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 as 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 dsRNA agents can affect pharmacokinetic distribution of the 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 a 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 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, a dsRNA further comprises a carbohydrate. 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 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 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, or 8-16 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 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 certain 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 can 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 is selected to be indicative of cleavage in a target cell and another 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 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 R^Bare the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

In one aspect, a dsRNA is conjugated to a carbohydrate through a linker. Linkers include bivalent and trivalent branched linker groups. Linkers for 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 oligonucleotide 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 a dsRNA. dsRNA compounds that are chimeric compounds are also contemplated. Chimeric dsRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the 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 dsRNA reduction of expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy 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 certain instances, the nucleosides of a dsRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution, or cellular uptake of the 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 dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of a 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 dsRNA still bound to the solid support or following cleavage of the dsRNA, in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.

IV. Pharmaceutical Uses

The dsRNA compositions described herein are useful in the methods 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 MutSβ heterodimer comprising MSH3, e.g., by reducing the activity or level of the MSH3 protein in a cell in a mammal.

Methods of treating disorders related to DNA mismatch repair such as nucleotide repeat expansion disorders (e.g., trinucleotide repeat expansion disorders) in a subject in need thereof are also contemplated. Another aspect includes reducing the level of MSH3 in a cell of a subject identified as having a nucleotide repeat expansion disorder (e.g., trinucleotide repeat expansion disorders). Still another aspect includes a method of reducing expression of MSH3 in a cell in a subject. Further aspects include methods of decreasing nucleotide repeat expansion in a cell (e.g., trinucleotide repeat expansion). The methods include contacting a cell with a dsRNA, in an amount effective to reduce expression of MSH3 in the cell, thereby reducing expression of MSH3 in the cell.

Based on the above methods, a dsRNA, or a composition comprising such a 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 MSH3 in a cell of a subject identified as having a trinucleotide repeat expansion disorder, or for use in reducing expression of MSH3 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 dsRNA, in an amount effective to reduce expression of MSH3 in the cell, thereby reducing expression of MSH3 in the cell. Aspects described below in relation to the methods are also applicable to these further aspects.

Contacting of a cell with a dsRNA, e.g., a double stranded dsRNA, can be done in vitro or in vivo. Contacting a cell in vivo with the dsRNA includes contacting a cell or group of cells within a subject, e.g., a human subject, with the 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 GalNAc₃ligand, or any other ligand that directs the dsRNA to a site of interest. Cells can include those of the central nervous system, or muscle cells.

Reducing expression of MSH3 includes any level of reduction of MSH3, e.g., at least partial suppression of the expression of a MSH3, such as a reduction by at least about 20%. In some aspects, the 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 MSH3 can be assessed based on the level of any variable associated with MSH3 expression, e.g., MSH3 mRNA level or MSH3 protein level.

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 reduction of MSH3. For example, effective treatment of a trinucleotide repeat expansion disorder, as demonstrated by acceptable diagnostic and monitoring criteria with an agent to reduce MSH3 expression can be understood to demonstrate a clinically relevant reduction in MSH3.

In some aspects of the methods, expression of a MSH3 is 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 reduction of expression of MSH3, e.g., as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of MSH3.

Reduction of the expression of MSH3 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 MSH3 is transcribed and which has or have been treated (e.g., by contacting the cell or cells with a dsRNA, or by administering a dsRNA to a subject in which the cells are or were present) such that the expression of MSH3 is 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 a dsRNA or not treated with a dsRNA targeted to the gene of interest). The degree of reduction can be expressed in terms of:

$\frac{(mRNA in control cells) - (mRNA in treated cells)}{(mRNA in control cells)} \times 100 %$

In other aspects, reduction of the expression of MSH3 can be assessed in terms of a reduction of a parameter that is functionally linked to MSH3 expression, e.g., MSH3 protein expression or MSH3 signaling pathways. MSH3 silencing can be determined in any cell expressing MSH3, either endogenous or heterologous from an expression construct, and by any assay known in the art.

Reduction of the expression of a MSH3 protein can be manifested by a reduction in the level of the MSH3 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 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 reduction of the expression of MSH3 includes a cell or group of cells that has not yet been contacted with a 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 a dsRNA.

The level of MSH3 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 MSH3 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the MSH3 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 MSH3 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 MSH3 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 MSH3 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 MSH3 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 MSH3 mRNA.

An alternative method for determining the level of expression of MSH3 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 particular aspects, the level of expression of MSH3 is determined by quantitative fluorogenic RT-PCR (i.e., the TAQMAN™ System) or the DUAL-GLO® Luciferase assay.

The expression levels of MSH3 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 MSH3 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 MSH3 nucleic acids.

The level of MSH3 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 MSH3 proteins.

In some aspects of the methods, the dsRNA is administered to a subject such that the dsRNA is delivered to a specific site within the subject. The reduction of expression of MSH3 can be assessed using measurements of the level or change in the level of MSH3 mRNA or MSH3 protein in a sample derived from a specific site within the subject. In some aspects, the methods include a clinically relevant reduction of expression of MSH3, e.g., as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of MSH3.

In other aspects, the 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 of): (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 nucleotide repeat expansion disorders (e.g., trinucleotide repeat expansion disorders) can result in an increase in average survival time of an individual or a population of subjects treated with the methods disclosed herein in comparison to a population of untreated subjects. For example, the survival time is of an individual or average survival time a of 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 nucleotide repeat expansion disorders (e.g., 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-MSH3 Agents

The delivery of a 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 nucleotide repeat expansion disorder (e.g., trinucleotide repeat expansion disorders) can be achieved in a number of different ways. For example, delivery can be performed by contacting a cell with a dsRNA either in vitro or in vivo. In vivo delivery can be performed directly by administering a composition comprising 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 a 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 in order to deliver a dsRNA molecule 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 a 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 dsRNA molecule to be administered.

For administering a dsRNA systemically for the treatment of a disease, the 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 dsRNA by endo- and exo-nucleases in vivo. Modification of the dsRNA or the pharmaceutical carrier can permit targeting of the dsRNA composition to the target tissue and avoid undesirable off-target effects. dsRNA molecules 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 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 a dsRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of a dsRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to a dsRNA, 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 a dsRNA. The formation of vesicles or micelles further prevents degradation of the 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 dsRNAs. Methods for making and administering cationic-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 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, a dsRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of 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 dsRNAs are delivered by polyplex or lipoplex nanoparticles. Methods for administration and pharmaceutical compositions of 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 MSH3 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 aspect, 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 aspects, the dsRNA agent that reduces the level and/or activity of MSH3 is delivered by a viral vector (e.g., a viral vector expressing an anti-MSH3 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

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 a 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 dsRNA are delivered into the cell where the dsRNA can specifically bind to a target RNA and can mediate RNAi. In some cases, the liposomes are also specifically targeted, e.g., to direct the dsRNA 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 a 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 dsRNA preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the dsRNA and condense around the dsRNA to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of 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 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 Gm′, 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 side 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 G_M1or 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 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 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 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 oligonucleotides 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 dsRNA into the skin. In some implementations, liposomes are used for delivering dsRNA to epidermal cells and also to enhance the penetration of 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 dsRNA 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 in order 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 dsRNA 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 dsRNA can be delivered, for example, subcutaneously by infection in order to deliver dsRNA to keratinocytes in the skin. In order 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 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 formulations that are amenable for use in the methods described herein.

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 dsRNA for use in the methods 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.

iii. Lipid Nanoparticle-Based Delivery Methods

dsRNAs described herein 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 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-Dilinoleyoxy-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.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 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)bu-tanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)ami-no)ethyl)piperazin-1-yeethylazanediyedidodecan-2-ol (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 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 (C₁₆), or a PEG-distearyloxypropyl (C₁₈). 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

A dsRNA can be used alone or in combination with at least one additional therapeutic agent, e.g., other agents that treat nucleotide repeat expansion disorders (e.g., 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 dsRNA agents described herein can be used in combination with at least one additional therapeutic agent to treat a nucleotide repeat expansion disorder (e.g., trinucleotide repeat expansion disorders) 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 additional therapeutic agent can be an oligonucleotide (e.g., an ASO) 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 inucleotide repeat expansion disorder (e.g., trinucleotide repeat expansion disorders) is Huntington's disease (HD). In some aspects, the gene associated with a nucleotide repeat expansion disorder (e.g., trinucleotide repeat expansion disorders) 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 other oligonucleotide (e.g., an ASO) 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. Hum. Gen. 2009, 84(3): 351-66 (herein incorporated by reference)). In some aspects, the other oligonucleotide (e.g., an ASO) hybridizes to an mRNA of the Huntingtin gene lacking any of the HD-associated SNPs. In some of the aspects, the other oligonucleotide (e.g., an ASO) 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 other oligonucleotide (e.g., an ASO) can be a modified oligonucleotide (e.g., an oligonucleotide including any of the modifications described herein). In some aspects, the modified oligonucleotides comprise one or more phosphorothioate internucleoside linkages. In some aspects, the modified oligonucleotide comprises one or more 2′-MOE moieties. In some aspects, the other oligonucleotide (e.g., an ASO) 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 NOs. 1-2432 of PCT Publication WO 2017/192679, the sequences of which are hereby incorporated by reference.

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

In any of the combination aspects described herein, the two or more therapeutic agents are 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 after one of more of the additional therapeutic agents.

V. Pharmaceutical Compositions

The 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, the 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 a 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 a dsRNA) is a prophylactically or a therapeutically effective amount.

VII. Kits

Kits including (a) a pharmaceutical composition including a dsRNA agent that reduces the level and/or activity of MSH3 in a cell or subject described herein, and (b) a package insert with instructions to perform any of the methods described herein are also contemplated. In some aspects, the kit includes (a) a pharmaceutical composition including a dsRNA agent that reduces the level and/or activity of MSH3 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 dsRNA Agents
Identification and Selection of Target Transcript

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 MSH3 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 MSH3 Transcripts

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

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

NM_002439.4
XM_005557283.2
NM_010829.2
NM_001191957.1

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).

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. 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 MSH3 gene, since seed mismatches govern specificity of dsRNA activity (Boudreau et al., (2011), Mol. Therapy 19: 2169-2177).

The sequences, positions in human transcript, and conservation in other species of each duplex are given in Table 3. In Table 3 below, the 5′ U of the antisense oligonucleotide can be any nucleotide (e.g., U, A, G, C, T). In some aspects, the 5′ U of the antisense oligonucleotide in Table 3 is U. Each sense and antisense oligonucleotides in Table 3 include a dTdT overhang on the 3′ end.

Additionally, every A and G in each sense oligonucleotide in Table 3 is a ribonucleotide. Every C and U in each sense oligonucleotide is a 2′-O-Methyl ribonucleotide.

Also, every A and G in each antisense oligonucleotide in Table 3 is a ribonucleotide. Every C and U preceding an A in the antisense oligonucleotide is a 2′-O-Methyl ribonucleotide, with one exception: U is the first nucleotide of the antisense strand, and it is a ribonucleotide.

Each nucleotide in the sense and antisense oligonucleotide in Table 3 is linked by a phosphate.

Furthermore, duplexes with sequence conservation in cynologous monkey, mouse, and rat are provided in Tables 4-10.

TABLE 3

Exemplary dsRNAs

SEQ

ID

NO/

SEQ ID NO/

Sense

Antisense

Oligo

Oligo

NO
Sense
Antisense
NO
Pos
Cyno
Mouse
Rat

6
CCGCCGCACAUAGCUACAA
UUGUAGCUAUGUGCGGCGG
7
306
No
No
No

8
CGCCGCACAUAGCUACAGA
UCUGUAGCUAUGUGCGGCG
9
307
No
No
No

10
GCCGCACAUAGCUACAGAA
UUCUGUAGCUAUGUGCGGC
11
308
No
No
No

12
CGCACAUAGCUACAGAAAA
UUUUCUGUAGCUAUGUGCG
13
310
No
No
No

14
GCACAUAGCUACAGAAAUA
UAUUUCUGUAGCUAUGUGC
15
311
No
No
No

16
CACAUAGCUACAGAAAUUA
UAAUUUCUGUAGCUAUGUG
17
312
No
No
No

18
AUAGCUACAGAAAUUGACA
UGUCAAUUUCUGUAGCUAU
19
315
No
No
No

20
GCUACAGAAAUUGACAGAA
UUCUGUCAAUUUCUGUAGC
21
318
No
No
No

22
UUGACAGAAGAAAGAAGAA
UUCUUCUUUCUUCUGUCAA
23
328
No
No
No

24
CAGAAGAAAGAAGAGACCA
UGGUCUCUUCUUUCUUCUG
25
332
No
No
No

26
AGAAGAAAGAAGAGACCAA
UUGGUCUCUUCUUUCUUCU
27
333
No
No
No

28
AGAAAGAAGAGACCAUUGA
UCAAUGGUCUCUUCUUUCU
29
336
No
No
No

30
GAGACCAUUGGAAAAUGAA
UUCAUUUUCCAAUGGUCUC
31
344
Yes
No
No

32
CCAUUGGAAAAUGAUGGGA
UCCCAUCAUUUUCCAAUGG
33
348
Yes
No
No

34
AAAAUGAUGGGCCUGUUAA
UUAACAGGCCCAUCAUUUU
35
355
Yes
No
No

36
AGAAAGUAAAGAAAGUCCA
UGGACUUUCUUUACUUUCU
37
376
No
No
No

38
GAAAGUAAAGAAAGUCCAA
UUGGACUUUCUUUACUUUC
39
377
No
No
No

40
AAGUAAAGAAAGUCCAACA
UGUUGGACUUUCUUUACUU
41
379
No
No
No

42
AGUAAAGAAAGUCCAACAA
UUGUUGGACUUUCUUUACU
43
380
No
No
No

44
GUAAAGAAAGUCCAACAAA
UUUGUUGGACUUUCUUUAC
45
381
No
No
No

46
UAAAGAAAGUCCAACAAAA
UUUUGUUGGACUUUCUUUA
47
382
No
No
No

48
AGAAAGUCCAACAAAAGGA
UCCUUUUGUUGGACUUUCU
49
385
No
No
No

50
GAAAGUCCAACAAAAGGAA
UUCCUUUUGUUGGACUUUC
51
386
No
No
No

52
AAAGUCCAACAAAAGGAAA
UUUCCUUUUGUUGGACUUU
53
387
No
No
No

54
AGUCCAACAAAAGGAAGGA
UCCUUCCUUUUGUUGGACU
55
389
No
No
No

56
AAGGAAGGAGGAAGUGAUA
UAUCACUUCCUCCUUCCUU
57
399
Yes
No
No

58
AGGAAGUGAUCUGGGAAUA
UAUUCCCAGAUCACUUCCU
59
407
Yes
No
No

60
AGUGAUCUGGGAAUGUCUA
UAGACAUUCCCAGAUCACU
61
411
Yes
No
No

62
UCUGGGAAUGUCUGGCAAA
UUUGCCAGACAUUCCCAGA
63
416
Yes
No
No

64
UGGGAAUGUCUGGCAACUA
UAGUUGCCAGACAUUCCCA
65
418
Yes
No
No

66
GAAUGUCUGGCAACUCUGA
UCAGAGUUGCCAGACAUUC
67
421
Yes
No
No

68
AAUGUCUGGCAACUCUGAA
UUCAGAGUUGCCAGACAUU
69
422
Yes
No
No

70
AUGUCUGGCAACUCUGAGA
UCUCAGAGUUGCCAGACAU
71
423
Yes
No
No

72
UGGCAACUCUGAGCCAAAA
UUUUGGCUCAGAGUUGCCA
73
428
Yes
No
No

74
GGCAACUCUGAGCCAAAGA
UCUUUGGCUCAGAGUUGCC
75
429
Yes
No
No

76
CAACUCUGAGCCAAAGAAA
UUUCUUUGGCUCAGAGUUG
77
431
Yes
No
No

78
ACUCUGAGCCAAAGAAAUA
UAUUUCUUUGGCUCAGAGU
79
433
Yes
No
No

80
UCUGAGCCAAAGAAAUGUA
UACAUUUCUUUGGCUCAGA
81
435
Yes
No
No

82
UGAGCCAAAGAAAUGUCUA
UAGACAUUUCUUUGGCUCA
83
437
Yes
No
Yes

84
GUCUGAGGACCAGGAAUGA
UCAUUCCUGGUCCUCAGAC
85
451
Yes
No
No

86
UCUGAGGACCAGGAAUGUA
UACAUUCCUGGUCCUCAGA
87
452
Yes
No
No

88
GAGGACCAGGAAUGUUUCA
UGAAACAUUCCUGGUCCUC
89
455
No
No
No

90
AGGACCAGGAAUGUUUCAA
UUGAAACAUUCCUGGUCCU
91
456
No
No
No

92
GGACCAGGAAUGUUUCAAA
UUUGAAACAUUCCUGGUCC
93
457
No
No
No

94
ACCAGGAAUGUUUCAAAGA
UCUUUGAAACAUUCCUGGU
95
459
No
No
No

96
GGAAUGUUUCAAAGUCUCA
UGAGACUUUGAAACAUUCC
97
463
No
No
No

98
UGUUUCAAAGUCUCUGGAA
UUCCAGAGACUUUGAAACA
99
467
No
No
No

100
AAUUCUGCUGCGAUUCUGA
UCAGAAUCGCAGCAGAAUU
101
496
No
No
No

102
GAUUCUGCCCUUCCUCAAA
UUUGAGGAAGGGCAGAAUC
103
507
Yes
No
No

104
CUGCCCUUCCUCAAAGUAA
UUACUUUGAGGAAGGGCAG
105
511
Yes
No
No

106
UGCCCUUCCUCAAAGUAGA
UCUACUUUGAGGAAGGGCA
107
512
Yes
No
No

108
CCCUUCCUCAAAGUAGAGA
UCUCUACUUUGAGGAAGGG
109
514
Yes
No
No

110
CCUUCCUCAAAGUAGAGUA
UACUCUACUUUGAGGAAGG
111
515
Yes
No
No

112
CUUCCUCAAAGUAGAGUCA
UGACUCUACUUUGAGGAAG
113
516
Yes
No
No

114
UUCCUCAAAGUAGAGUCCA
UGGACUCUACUUUGAGGAA
115
517
Yes
No
No

116
CUCAAAGUAGAGUCCAGAA
UUCUGGACUCUACUUUGAG
117
520
Yes
No
No

118
AAGUAGAGUCCAGACAGAA
UUCUGUCUGGACUCUACUU
119
524
Yes
No
No

120
AGUAGAGUCCAGACAGAAA
UUUCUGUCUGGACUCUACU
121
525
Yes
No
No

122
AGAGUCCAGACAGAAUCUA
UAGAUUCUGUCUGGACUCU
123
528
Yes
No
No

124
GAGUCCAGACAGAAUCUCA
UGAGAUUCUGUCUGGACUC
125
529
Yes
No
No

126
GUCCAGACAGAAUCUCUGA
UCAGAGAUUCUGUCUGGAC
127
531
Yes
No
No

128
UCCAGACAGAAUCUCUGCA
UGCAGAGAUUCUGUCUGGA
129
532
Yes
No
No

130
UCUCUGCAGGAGAGAUUUA
UAAAUCUCUCCUGCAGAGA
131
543
Yes
No
No

132
GCAGGAGAGAUUUGCAGUA
UACUGCAAAUCUCUCCUGC
133
548
Yes
No
No

134
CAGGAGAGAUUUGCAGUUA
UAACUGCAAAUCUCUCCUG
135
549
Yes
No
No

136
GAGAGAUUUGCAGUUCUGA
UCAGAACUGCAAAUCUCUC
137
552
Yes
No
No

138
AGAGAUUUGCAGUUCUGCA
UGCAGAACUGCAAAUCUCU
139
553
Yes
No
No

140
AGAUUUGCAGUUCUGCCAA
UUGGCAGAACUGCAAAUCU
141
555
Yes
No
No

142
GAUUUGCAGUUCUGCCAAA
UUUGGCAGAACUGCAAAUC
143
556
Yes
No
No

144
AUUUGCAGUUCUGCCAAAA
UUUUGGCAGAACUGCAAAU
145
557
Yes
No
No

146
UUGCAGUUCUGCCAAAAUA
UAUUUUGGCAGAACUGCAA
147
559
Yes
No
No

148
AGUUCUGCCAAAAUGUACA
UGUACAUUUUGGCAGAACU
149
563
Yes
No
No

150
GUUCUGCCAAAAUGUACUA
UAGUACAUUUUGGCAGAAC
151
564
Yes
No
No

152
UUCUGCCAAAAUGUACUGA
UCAGUACAUUUUGGCAGAA
153
565
Yes
No
No

154
CUGCCAAAAUGUACUGAUA
UAUCAGUACAUUUUGGCAG
155
567
Yes
No
No

156
GCCAAAAUGUACUGAUUUA
UAAAUCAGUACAUUUUGGC
157
569
Yes
No
No

158
GUACUGAUUUUGAUGAUAA
UUAUCAUCAAAAUCAGUAC
159
577
Yes
No
No

160
ACUGAUUUUGAUGAUAUCA
UGAUAUCAUCAAAAUCAGU
161
579
Yes
No
No

162
UGAUGAUAUCAGUCUUCUA
UAGAAGACUGAUAUCAUCA
163
587
Yes
No
No

164
AUGAUAUCAGUCUUCUACA
UGUAGAAGACUGAUAUCAU
165
589
Yes
No
No

166
UGAUAUCAGUCUUCUACAA
UUGUAGAAGACUGAUAUCA
167
590
No
No
No

168
AUAUCAGUCUUCUACACGA
UCGUGUAGAAGACUGAUAU
169
592
No
No
No

170
UCAGUCUUCUACACGCAAA
UUUGCGUGUAGAAGACUGA
171
595
No
No
No

172
CAGUCUUCUACACGCAAAA
UUUUGCGUGUAGAAGACUG
173
596
No
No
No

174
AGUCUUCUACACGCAAAGA
UCUUUGCGUGUAGAAGACU
175
597
No
No
No

176
UCUUCUACACGCAAAGAAA
UUUCUUUGCGUGUAGAAGA
177
599
No
No
No

178
CUUCUACACGCAAAGAAUA
UAUUCUUUGCGUGUAGAAG
179
600
No
No
No

180
UUCUACACGCAAAGAAUGA
UCAUUCUUUGCGUGUAGAA
181
601
No
No
No

182
UCUACACGCAAAGAAUGCA
UGCAUUCUUUGCGUGUAGA
183
602
No
No
No

184
CUACACGCAAAGAAUGCAA
UUGCAUUCUUUGCGUGUAG
185
603
No
No
No

186
ACACGCAAAGAAUGCAGUA
UACUGCAUUCUUUGCGUGU
187
605
No
No
No

188
CACGCAAAGAAUGCAGUUA
UAACUGCAUUCUUUGCGUG
189
606
No
No
No

190
AAGAAUGCAGUUUCUUCUA
UAGAAGAAACUGCAUUCUU
191
612
Yes
No
No

192
AGUUUCUUCUGAAGAUUCA
UGAAUCUUCAGAAGAAACU
193
620
Yes
No
No

194
UCUGAAGAUUCGAAACGUA
UACGUUUCGAAUCUUCAGA
195
627
No
No
No

196
UGAAGAUUCGAAACGUCAA
UUGACGUUUCGAAUCUUCA
197
629
No
No
No

198
GAAGAUUCGAAACGUCAAA
UUUGACGUUUCGAAUCUUC
199
630
No
No
No

200
AAGAUUCGAAACGUCAAAA
UUUUGACGUUUCGAAUCUU
201
631
No
No
No

202
AGAUUCGAAACGUCAAAUA
UAUUUGACGUUUCGAAUCU
203
632
No
No
No

204
GAUUCGAAACGUCAAAUUA
UAAUUUGACGUUUCGAAUC
205
633
No
No
No

206
UUCGAAACGUCAAAUUAAA
UUUAAUUUGACGUUUCGAA
207
635
No
No
No

208
GAAACGUCAAAUUAAUCAA
UUGAUUAAUUUGACGUUUC
209
638
No
No
No

210
AAACGUCAAAUUAAUCAAA
UUUGAUUAAUUUGACGUUU
211
639
No
No
No

212
AACGUCAAAUUAAUCAAAA
UUUUGAUUAAUUUGACGUU
213
640
No
No
No

214
CGUCAAAUUAAUCAAAAGA
UCUUUUGAUUAAUUUGACG
215
642
No
No
No

216
GUCAAAUUAAUCAAAAGGA
UCCUUUUGAUUAAUUUGAC
217
643
No
No
No

218
UUAAUCAAAAGGACACAAA
UUUGUGUCCUUUUGAUUAA
219
649
No
No
No

220
UAAUCAAAAGGACACAACA
UGUUGUGUCCUUUUGAUUA
221
650
No
No
No

222
CAAAAGGACACAACACUUA
UAAGUGUUGUGUCCUUUUG
223
654
No
No
No

224
AAAGGACACAACACUUUUA
UAAAAGUGUUGUGUCCUUU
225
656
No
No
No

226
UUUUGAUCUCAGUCAGUUA
UAACUGACUGAGAUCAAAA
227
671
No
No
No

228
UGAUCUCAGUCAGUUUGGA
UCCAAACUGACUGAGAUCA
229
674
No
No
No

230
AUCUCAGUCAGUUUGGAUA
UAUCCAAACUGACUGAGAU
231
676
No
No
No

232
CUCAGUCAGUUUGGAUCAA
UUGAUCCAAACUGACUGAG
233
678
No
No
No

234
UCAGUCAGUUUGGAUCAUA
UAUGAUCCAAACUGACUGA
235
679
Yes
No
No

236
CAGUCAGUUUGGAUCAUCA
UGAUGAUCCAAACUGACUG
237
680
Yes
No
No

238
AGUCAGUUUGGAUCAUCAA
UUGAUGAUCCAAACUGACU
239
681
Yes
No
No

240
CAGUUUGGAUCAUCAAAUA
UAUUUGAUGAUCCAAACUG
241
684
Yes
No
No

242
AGUUUGGAUCAUCAAAUAA
UUAUUUGAUGAUCCAAACU
243
685
Yes
No
No

244
GUUUGGAUCAUCAAAUACA
UGUAUUUGAUGAUCCAAAC
245
686
Yes
No
No

246
UUUGGAUCAUCAAAUACAA
UUGUAUUUGAUGAUCCAAA
247
687
Yes
No
No

248
UUGGAUCAUCAAAUACAAA
UUUGUAUUUGAUGAUCCAA
249
688
Yes
No
No

250
UGGAUCAUCAAAUACAAGA
UCUUGUAUUUGAUGAUCCA
251
689
Yes
No
No

252
GGAUCAUCAAAUACAAGUA
UACUUGUAUUUGAUGAUCC
253
690
Yes
No
No

254
CAUCAAAUACAAGUCAUGA
UCAUGACUUGUAUUUGAUG
255
694
Yes
No
No

256
UCAAAUACAAGUCAUGAAA
UUUCAUGACUUGUAUUUGA
257
696
Yes
No
No

258
AUACAAGUCAUGAAAAUUA
UAAUUUUCAUGACUUGUAU
259
700
Yes
No
No

260
UACAAGUCAUGAAAAUUUA
UAAAUUUUCAUGACUUGUA
261
701
Yes
No
No

262
UACAGAAAACUGCUUCCAA
UUGGAAGCAGUUUUCUGUA
263
718
No
No
No

264
AAAACUGCUUCCAAAUCAA
UUGAUUUGGAAGCAGUUUU
265
723
No
No
No

266
AACUGCUUCCAAAUCAGCA
UGCUGAUUUGGAAGCAGUU
267
725
No
No
No

268
ACUGCUUCCAAAUCAGCUA
UAGCUGAUUUGGAAGCAGU
269
726
No
No
No

270
CUGCUUCCAAAUCAGCUAA
UUAGCUGAUUUGGAAGCAG
271
727
No
No
No

272
GCUUCCAAAUCAGCUAACA
UGUUAGCUGAUUUGGAAGC
273
729
No
No
No

274
CUUCCAAAUCAGCUAACAA
UUGUUAGCUGAUUUGGAAG
275
730
No
No
No

276
CCAAAUCAGCUAACAAACA
UGUUUGUUAGCUGAUUUGG
277
733
No
No
No

278
AAAUCAGCUAACAAACGGA
UCCGUUUGUUAGCUGAUUU
279
735
No
No
No

280
AAUCAGCUAACAAACGGUA
UACCGUUUGUUAGCUGAUU
281
736
No
No
No

282
GCUAACAAACGGUCCAAAA
UUUUGGACCGUUUGUUAGC
283
741
No
No
No

284
UAACAAACGGUCCAAAAGA
UCUUUUGGACCGUUUGUUA
285
743
No
No
No

286
AACAAACGGUCCAAAAGCA
UGCUUUUGGACCGUUUGUU
287
744
No
No
No

288
AAACGGUCCAAAAGCAUCA
UGAUGCUUUUGGACCGUUU
289
747
No
No
No

290
AACGGUCCAAAAGCAUCUA
UAGAUGCUUUUGGACCGUU
291
748
No
No
No

292
CGGUCCAAAAGCAUCUAUA
UAUAGAUGCUUUUGGACCG
293
750
No
No
No

294
UCCAAAAGCAUCUAUACGA
UCGUAUAGAUGCUUUUGGA
295
753
No
No
No

296
CCAAAAGCAUCUAUACGCA
UGCGUAUAGAUGCUUUUGG
297
754
No
No
No

298
AUCUAUACGCCGCUAGAAA
UUUCUAGCGGCGUAUAGAU
299
762
No
No
No

300
CUAUACGCCGCUAGAAUUA
UAAUUCUAGCGGCGUAUAG
301
764
No
No
No

302
UAUACGCCGCUAGAAUUAA
UUAAUUCUAGCGGCGUAUA
303
765
Yes
No
No

304
AUACGCCGCUAGAAUUACA
UGUAAUUCUAGCGGCGUAU
305
766
Yes
No
No

306
ACGCCGCUAGAAUUACAAA
UUUGUAAUUCUAGCGGCGU
307
768
Yes
No
No

308
CCGCUAGAAUUACAAUACA
UGUAUUGUAAUUCUAGCGG
309
771
Yes
No
No

310
CGCUAGAAUUACAAUACAA
UUGUAUUGUAAUUCUAGCG
311
772
Yes
No
No

312
UAGAAUUACAAUACAUAGA
UCUAUGUAUUGUAAUUCUA
313
775
Yes
No
No

314
AGAAUUACAAUACAUAGAA
UUCUAUGUAUUGUAAUUCU
315
776
Yes
No
No

316
GAAUUACAAUACAUAGAAA
UUUCUAUGUAUUGUAAUUC
317
777
Yes
No
No

318
AAUUACAAUACAUAGAAAA
UUUUCUAUGUAUUGUAAUU
319
778
Yes
No
No

320
AUACAUAGAAAUGAAGCAA
UUGCUUCAUUUCUAUGUAU
321
785
Yes
No
No

322
UACAUAGAAAUGAAGCAGA
UCUGCUUCAUUUCUAUGUA
323
786
Yes
No
No

324
GCACAAAGAUGCAGUUUUA
UAAAACUGCAUCUUUGUGC
325
806
Yes
No
No

326
CACAAAGAUGCAGUUUUGA
UCAAAACUGCAUCUUUGUG
327
807
Yes
No
No

328
CAAAGAUGCAGUUUUGUGA
UCACAAAACUGCAUCUUUG
329
809
Yes
No
No

330
AAGAUGCAGUUUUGUGUGA
UCACACAAAACUGCAUCUU
331
811
Yes
No
No

332
AGAUGCAGUUUUGUGUGUA
UACACACAAAACUGCAUCU
333
812
Yes
No
No

334
GAUGCAGUUUUGUGUGUGA
UCACACACAAAACUGCAUC
335
813
Yes
No
No

336
UGCAGUUUUGUGUGUGGAA
UUCCACACACAAAACUGCA
337
815
Yes
No
No

338
GCAGUUUUGUGUGUGGAAA
UUUCCACACACAAAACUGC
339
816
Yes
No
No

340
AGUUUUGUGUGUGGAAUGA
UCAUUCCACACACAAAACU
341
818
Yes
No
No

342
GUUUUGUGUGUGGAAUGUA
UACAUUCCACACACAAAAC
343
819
Yes
No
Yes

344
UUUUGUGUGUGGAAUGUGA
UCACAUUCCACACACAAAA
345
820
Yes
No
Yes

346
UUUGUGUGUGGAAUGUGGA
UCCACAUUCCACACACAAA
347
821
Yes
No
Yes

348
UUGUGUGUGGAAUGUGGAA
UUCCACAUUCCACACACAA
349
822
Yes
No
No

350
UGUGUGUGGAAUGUGGAUA
UAUCCACAUUCCACACACA
351
823
Yes
No
No

352
GUGUGUGGAAUGUGGAUAA
UUAUCCACAUUCCACACAC
353
824
Yes
No
No

354
GUGUGGAAUGUGGAUAUAA
UUAUAUCCACAUUCCACAC
355
826
Yes
NO
No

356
UGUGGAAUGUGGAUAUAAA
UUUAUAUCCACAUUCCACA
357
827
Yes
No
No

358
GUGGAAUGUGGAUAUAAGA
UCUUAUAUCCACAUUCCAC
359
828
Yes
No
No

360
UGGAAUGUGGAUAUAAGUA
UACUUAUAUCCACAUUCCA
361
829
Yes
No
No

362
GGAAUGUGGAUAUAAGUAA
UUACUUAUAUCCACAUUCC
363
830
Yes
No
No

364
GAAUGUGGAUAUAAGUAUA
UAUACUUAUAUCCACAUUC
365
831
Yes
No
No

366
AAUGUGGAUAUAAGUAUAA
UUAUACUUAUAUCCACAUU
367
832
Yes
No
No

368
AUGUGGAUAUAAGUAUAGA
UCUAUACUUAUAUCCACAU
369
833
Yes
No
No

370
UGUGGAUAUAAGUAUAGAA
UUCUAUACUUAUAUCCACA
371
834
Yes
No
No

372
UGGAUAUAAGUAUAGAUUA
UAAUCUAUACUUAUAUCCA
373
836
Yes
No
No

374
GAUAUAAGUAUAGAUUCUA
UAGAAUCUAUACUUAUAUC
375
838
Yes
No
No

376
UAUAAGUAUAGAUUCUUUA
UAAAGAAUCUAUACUUAUA
377
840
Yes
No
No

378
UAAGUAUAGAUUCUUUGGA
UCCAAAGAAUCUAUACUUA
379
842
Yes
No
No

380
AAGUAUAGAUUCUUUGGGA
UCCCAAAGAAUCUAUACUU
381
843
Yes
No
No

382
UAUAGAUUCUUUGGGGAAA
UUUCCCCAAAGAAUCUAUA
383
846
Yes
No
No

384
AGAUUCUUUGGGGAAGAUA
UAUCUUCCCCAAAGAAUCU
385
849
Yes
Yes
No

386
UGCAGCCCGAGAGCUCAAA
UUUGAGCUCUCGGGCUGCA
387
875
Yes
No
No

388
AGCCCGAGAGCUCAAUAUA
UAUAUUGAGCUCUCGGGCU
389
878
Yes
No
No

390
GCCCGAGAGCUCAAUAUUA
UAAUAUUGAGCUCUCGGGC
391
879
Yes
No
No

392
CGAGAGCUCAAUAUUUAUA
UAUAAAUAUUGAGCUCUCG
393
882
Yes
No
No

394
GAGAGCUCAAUAUUUAUUA
UAAUAAAUAUUGAGCUCUC
395
883
Yes
No
No

396
GAGCUCAAUAUUUAUUGCA
UGCAAUAAAUAUUGAGCUC
397
885
Yes
No
No

398
CUCAAUAUUUAUUGCCAUA
UAUGGCAAUAAAUAUUGAG
399
888
Yes
No
No

400
UAUUUAUUGCCAUUUAGAA
UUCUAAAUGGCAAUAAAUA
401
893
Yes
No
No

402
UUUAUUGCCAUUUAGAUCA
UGAUCUAAAUGGCAAUAAA
403
895
Yes
No
No

404
UUAUUGCCAUUUAGAUCAA
UUGAUCUAAAUGGCAAUAA
405
896
Yes
No
No

406
UAUUGCCAUUUAGAUCACA
UGUGAUCUAAAUGGCAAUA
407
897
Yes
No
No

408
AUUGCCAUUUAGAUCACAA
UUGUGAUCUAAAUGGCAAU
409
898
Yes
No
No

410
UUGCCAUUUAGAUCACAAA
UUUGUGAUCUAAAUGGCAA
411
899
Yes
No
No

412
GCCAUUUAGAUCACAACUA
UAGUUGUGAUCUAAAUGGC
413
901
Yes
No
No

414
UUUAGAUCACAACUUUAUA
UAUAAAGUUGUGAUCUAAA
415
905
Yes
No
No

416
UUAGAUCACAACUUUAUGA
UCAUAAAGUUGUGAUCUAA
417
906
Yes
No
No

418
UAGAUCACAACUUUAUGAA
UUCAUAAAGUUGUGAUCUA
419
907
Yes
No
No

420
AGAUCACAACUUUAUGACA
UGUCAUAAAGUUGUGAUCU
421
908
Yes
No
No

422
AUCACAACUUUAUGACAGA
UCUGUCAUAAAGUUGUGAU
423
910
Yes
No
No

424
UCACAACUUUAUGACAGCA
UGCUGUCAUAAAGUUGUGA
425
911
Yes
No
No

426
CACAACUUUAUGACAGCAA
UUGCUGUCAUAAAGUUGUG
427
912
Yes
No
No

428
ACAACUUUAUGACAGCAAA
UUUGCUGUCAUAAAGUUGU
429
913
Yes
No
No

430
CAACUUUAUGACAGCAAGA
UCUUGCUGUCAUAAAGUUG
431
914
Yes
No
No

432
AACUUUAUGACAGCAAGUA
UACUUGCUGUCAUAAAGUU
433
915
Yes
No
No

434
CUUUAUGACAGCAAGUAUA
UAUACUUGCUGUCAUAAAG
435
917
Yes
No
No

436
AUGACAGCAAGUAUACCUA
UAGGUAUACUUGCUGUCAU
437
921
Yes
No
No

438
UGACAGCAAGUAUACCUAA
UUAGGUAUACUUGCUGUCA
439
922
Yes
No
No

440
GACAGCAAGUAUACCUACA
UGUAGGUAUACUUGCUGUC
441
923
Yes
No
No

442
CAGCAAGUAUACCUACUCA
UGAGUAGGUAUACUUGCUG
443
925
Yes
No
No

444
GCAAGUAUACCUACUCACA
UGUGAGUAGGUAUACUUGC
445
927
Yes
No
No

446
AGUAUACCUACUCACAGAA
UUCUGUGAGUAGGUAUACU
447
930
Yes
No
No

448
GUAUACCUACUCACAGACA
UGUCUGUGAGUAGGUAUAC
449
931
Yes
No
No

450
AUACCUACUCACAGACUGA
UCAGUCUGUGAGUAGGUAU
451
933
Yes
No
No

452
UACCUACUCACAGACUGUA
UACAGUCUGUGAGUAGGUA
453
934
Yes
No
No

454
ACCUACUCACAGACUGUUA
UAACAGUCUGUGAGUAGGU
455
935
Yes
No
No

456
ACUCACAGACUGUUUGUUA
UAACAAACAGUCUGUGAGU
457
939
Yes
No
No

458
CUCACAGACUGUUUGUUCA
UGAACAAACAGUCUGUGAG
459
940
Yes
No
No

460
CACAGACUGUUUGUUCAUA
UAUGAACAAACAGUCUGUG
461
942
Yes
No
No

462
ACAGACUGUUUGUUCAUGA
UCAUGAACAAACAGUCUGU
463
943
Yes
No
No

464
ACUGUUUGUUCAUGUACGA
UCGUACAUGAACAAACAGU
465
947
Yes
No
No

466
CUGUUUGUUCAUGUACGCA
UGCGUACAUGAACAAACAG
467
948
Yes
No
No

468
CGCCUGGUGGCAAAAGGAA
UUCCUUUUGCCACCAGGCG
469
966
Yes
No
No

470
CUGGUGGCAAAAGGAUAUA
UAUAUCCUUUUGCCACCAG
471
969
Yes
No
No

472
UGGUGGCAAAAGGAUAUAA
UUAUAUCCUUUUGCCACCA
473
970
Yes
No
No

474
GGUGGCAAAAGGAUAUAAA
UUUAUAUCCUUUUGCCACC
475
971
Yes
No
No

476
GUGGCAAAAGGAUAUAAGA
UCUUAUAUCCUUUUGCCAC
477
972
Yes
No
No

478
UGGCAAAAGGAUAUAAGGA
UCCUUAUAUCCUUUUGCCA
479
973
Yes
No
No

480
GGCAAAAGGAUAUAAGGUA
UACCUUAUAUCCUUUUGCC
481
974
Yes
No
No

482
GCAAAAGGAUAUAAGGUGA
UCACCUUAUAUCCUUUUGC
483
975
Yes
No
No

484
CAAAAGGAUAUAAGGUGGA
UCCACCUUAUAUCCUUUUG
485
976
Yes
No
No

486
GGAUAUAAGGUGGGAGUUA
UAACUCCCACCUUAUAUCC
487
981
Yes
No
No

488
UAUAAGGUGGGAGUUGUGA
UCACAACUCCCACCUUAUA
489
984
Yes
No
No

490
UAAGGUGGGAGUUGUGAAA
UUUCACAACUCCCACCUUA
491
986
Yes
No
No

492
AAGGUGGGAGUUGUGAAGA
UCUUCACAACUCCCACCUU
493
987
Yes
Yes
Yes

494
GUGGGAGUUGUGAAGCAAA
UUUGCUUCACAACUCCCAC
495
990
Yes
Yes
Yes

496
GGGAGUUGUGAAGCAAACA
UGUUUGCUUCACAACUCCC
497
992
Yes
Yes
Yes

498
GAGUUGUGAAGCAAACUGA
UCAGUUUGCUUCACAACUC
499
994
Yes
Yes
Yes

500
AGUUGUGAAGCAAACUGAA
UUCAGUUUGCUUCACAACU
501
995
Yes
Yes
Yes

502
GUUGUGAAGCAAACUGAAA
UUUCAGUUUGCUUCACAAC
503
996
Yes
Yes
Yes

504
UGUGAAGCAAACUGAAACA
UGUUUCAGUUUGCUUCACA
505
998
Yes
Yes
Yes

506
GAAGCAAACUGAAACUGCA
UGCAGUUUCAGUUUGCUUC
507
1001
Yes
Yes
Yes

508
AACUGAAACUGCAGCAUUA
UAAUGCUGCAGUUUCAGUU
509
1007
Yes
No
No

510
ACUGAAACUGCAGCAUUAA
UUAAUGCUGCAGUUUCAGU
511
1008
Yes
No
No

512
CUGAAACUGCAGCAUUAAA
UUUAAUGCUGCAGUUUCAG
513
1009
Yes
No
No

514
UGAAACUGCAGCAUUAAAA
UUUUAAUGCUGCAGUUUCA
515
1010
Yes
No
No

516
AACUGCAGCAUUAAAGGCA
UGCCUUUAAUGCUGCAGUU
517
1013
Yes
No
No

518
CUGCAGCAUUAAAGGCCAA
UUGGCCUUUAAUGCUGCAG
519
1015
Yes
No
No

520
GCAUUAAAGGCCAUUGGAA
UUCCAAUGGCCUUUAAUGC
521
1020
Yes
No
Yes

522
AUUAAAGGCCAUUGGAGAA
UUCUCCAAUGGCCUUUAAU
523
1022
Yes
No
Yes

524
UUAAAGGCCAUUGGAGACA
UGUCUCCAAUGGCCUUUAA
525
1023
Yes
No
Yes

526
UAAAGGCCAUUGGAGACAA
UUGUCUCCAAUGGCCUUUA
527
1024
Yes
No
Yes

528
AAAGGCCAUUGGAGACAAA
UUUGUCUCCAAUGGCCUUU
529
1025
Yes
No
Yes

530
AAGGCCAUUGGAGACAACA
UGUUGUCUCCAAUGGCCUU
531
1026
Yes
No
No

532
GGCCAUUGGAGACAACAGA
UCUGUUGUCUCCAAUGGCC
533
1028
Yes
No
No

534
GCCAUUGGAGACAACAGAA
UUCUGUUGUCUCCAAUGGC
535
1029
Yes
No
No

536
CCAUUGGAGACAACAGAAA
UUUCUGUUGUCUCCAAUGG
537
1030
Yes
No
No

538
CAUUGGAGACAACAGAAGA
UCUUCUGUUGUCUCCAAUG
539
1031
Yes
No
No

540
AUUGGAGACAACAGAAGUA
UACUUCUGUUGUCUCCAAU
541
1032
Yes
No
No

542
UUGGAGACAACAGAAGUUA
UAACUUCUGUUGUCUCCAA
543
1033
Yes
No
No

544
UGGAGACAACAGAAGUUCA
UGAACUUCUGUUGUCUCCA
545
1034
Yes
No
No

546
GAGACAACAGAAGUUCACA
UGUGAACUUCUGUUGUCUC
547
1036
Yes
No
No

548
ACAACAGAAGUUCACUCUA
UAGAGUGAACUUCUGUUGU
549
1039
Yes
No
No

550
CAACAGAAGUUCACUCUUA
UAAGAGUGAACUUCUGUUG
551
1040
Yes
No
No

552
ACAGAAGUUCACUCUUUUA
UAAAAGAGUGAACUUCUGU
553
1042
Yes
No
No

554
CAGAAGUUCACUCUUUUCA
UGAAAAGAGUGAACUUCUG
555
1043
Yes
No
No

556
GAAGUUCACUCUUUUCCCA
UGGGAAAAGAGUGAACUUC
557
1045
Yes
No
No

558
UCUUUUCCCGGAAAUUGAA
UUCAAUUUCCGGGAAAAGA
559
1054
Yes
No
Yes

560
CUUUUCCCGGAAAUUGACA
UGUCAAUUUCCGGGAAAAG
561
1055
Yes
No
Yes

562
UUUUCCCGGAAAUUGACUA
UAGUCAAUUUCCGGGAAAA
563
1056
Yes
No
Yes

564
UUCCCGGAAAUUGACUGCA
UGCAGUCAAUUUCCGGGAA
565
1058
Yes
No
Yes

566
GGAAAUUGACUGCCCUUUA
UAAAGGGCAGUCAAUUUCC
567
1063
Yes
No
No

568
GAAAUUGACUGCCCUUUAA
UUAAAGGGCAGUCAAUUUC
569
1064
Yes
No
No

570
AAAUUGACUGCCCUUUAUA
UAUAAAGGGCAGUCAAUUU
571
1065
Yes
No
No

572
UUGACUGCCCUUUAUACAA
UUGUAUAAAGGGCAGUCAA
573
1068
Yes
No
No

574
CUGCCCUUUAUACAAAAUA
UAUUUUGUAUAAAGGGCAG
575
1072
Yes
No
No

576
UGCCCUUUAUACAAAAUCA
UGAUUUUGUAUAAAGGGCA
577
1073
Yes
No
No

578
GCCCUUUAUACAAAAUCUA
UAGAUUUUGUAUAAAGGGC
579
1074
Yes
No
No

580
CCCUUUAUACAAAAUCUAA
UUAGAUUUUGUAUAAAGGG
581
1075
Yes
No
No

582
CCUUUAUACAAAAUCUACA
UGUAGAUUUUGUAUAAAGG
583
1076
Yes
No
No

584
UUAUACAAAAUCUACACUA
UAGUGUAGAUUUUGUAUAA
585
1079
Yes
No
No

586
UAUACAAAAUCUACACUUA
UAAGUGUAGAUUUUGUAUA
587
1080
No
No
No

588
AUACAAAAUCUACACUUAA
UUAAGUGUAGAUUUUGUAU
589
1081
No
No
No

590
CAAAAUCUACACUUAUUGA
UCAAUAAGUGUAGAUUUUG
591
1084
No
No
No

592
AAAAUCUACACUUAUUGGA
UCCAAUAAGUGUAGAUUUU
593
1085
No
No
No

594
AAAUCUACACUUAUUGGAA
UUCCAAUAAGUGUAGAUUU
595
1086
No
No
No

596
AUCUACACUUAUUGGAGAA
UUCUCCAAUAAGUGUAGAU
597
1088
No
No
No

598
UCUACACUUAUUGGAGAAA
UUUCUCCAAUAAGUGUAGA
599
1089
No
No
No

600
CACUUAUUGGAGAAGAUGA
UCAUCUUCUCCAAUAAGUG
601
1093
No
No
No

602
ACUUAUUGGAGAAGAUGUA
UACAUCUUCUCCAAUAAGU
603
1094
No
No
No

604
CUUAUUGGAGAAGAUGUGA
UCACAUCUUCUCCAAUAAG
605
1095
No
No
No

606
UUAUUGGAGAAGAUGUGAA
UUCACAUCUUCUCCAAUAA
607
1096
No
No
No

608
AGAAGAUGUGAAUCCCCUA
UAGGGGAUUCACAUCUUCU
609
1103
Yes
No
No

610
GAAGAUGUGAAUCCCCUAA
UUAGGGGAUUCACAUCUUC
611
1104
Yes
No
No

612
AGAUGUGAAUCCCCUAAUA
UAUUAGGGGAUUCACAUCU
613
1106
Yes
No
No

614
GAUGUGAAUCCCCUAAUCA
UGAUUAGGGGAUUCACAUC
615
1107
Yes
No
No

616
UGUGAAUCCCCUAAUCAAA
UUUGAUUAGGGGAUUCACA
617
1109
Yes
No
No

618
GUGAAUCCCCUAAUCAAGA
UCUUGAUUAGGGGAUUCAC
619
1110
Yes
No
No

620
UGAAUCCCCUAAUCAAGCA
UGCUUGAUUAGGGGAUUCA
621
1111
Yes
No
No

622
GAAUCCCCUAAUCAAGCUA
UAGCUUGAUUAGGGGAUUC
623
1112
Yes
No
No

624
CUAAUCAAGCUGGAUGAUA
UAUCAUCCAGCUUGAUUAG
625
1119
Yes
No
No

626
UAAUCAAGCUGGAUGAUGA
UCAUCAUCCAGCUUGAUUA
627
1120
Yes
No
No

628
AAUCAAGCUGGAUGAUGCA
UGCAUCAUCCAGCUUGAUU
629
1121
Yes
No
No

630
CAAGCUGGAUGAUGCUGUA
UACAGCAUCAUCCAGCUUG
631
1124
Yes
No
No

632
AGCUGGAUGAUGCUGUAAA
UUUACAGCAUCAUCCAGCU
633
1126
Yes
No
No

634
UGGAUGAUGCUGUAAAUGA
UCAUUUACAGCAUCAUCCA
635
1129
Yes
No
No

636
GAUGAUGCUGUAAAUGUUA
UAACAUUUACAGCAUCAUC
637
1131
Yes
No
No

638
UGUAAAUGUUGAUGAGAUA
UAUCUCAUCAACAUUUACA
639
1139
Yes
No
No

640
GUAAAUGUUGAUGAGAUAA
UUAUCUCAUCAACAUUUAC
641
1140
Yes
No
No

642
UAAAUGUUGAUGAGAUAAA
UUUAUCUCAUCAACAUUUA
643
1141
Yes
No
No

644
AUGUUGAUGAGAUAAUGAA
UUCAUUAUCUCAUCAACAU
645
1144
Yes
No
No

646
UGUUGAUGAGAUAAUGACA
UGUCAUUAUCUCAUCAACA
647
1145
Yes
No
No

648
GUUGAUGAGAUAAUGACUA
UAGUCAUUAUCUCAUCAAC
649
1146
Yes
No
No

650
UUGAUGAGAUAAUGACUGA
UCAGUCAUUAUCUCAUCAA
651
1147
Yes
No
No

652
UGAUGAGAUAAUGACUGAA
UUCAGUCAUUAUCUCAUCA
653
1148
Yes
No
No

654
GAUGAGAUAAUGACUGAUA
UAUCAGUCAUUAUCUCAUC
655
1149
Yes
No
No

656
AGAUAAUGACUGAUACUUA
UAAGUAUCAGUCAUUAUCU
657
1153
Yes
No
No

658
GAUAAUGACUGAUACUUCA
UGAAGUAUCAGUCAUUAUC
659
1154
Yes
No
No

660
AUAAUGACUGAUACUUCUA
UAGAAGUAUCAGUCAUUAU
661
1155
Yes
No
No

662
AAUGACUGAUACUUCUACA
UGUAGAAGUAUCAGUCAUU
663
1157
Yes
No
No

664
UGACUGAUACUUCUACCAA
UUGGUAGAAGUAUCAGUCA
665
1159
Yes
No
No

666
GACUGAUACUUCUACCAGA
UCUGGUAGAAGUAUCAGUC
667
1160
Yes
No
No

668
UGAUACUUCUACCAGCUAA
UUAGCUGGUAGAAGUAUCA
669
1163
Yes
No
No

670
GAUACUUCUACCAGCUAUA
UAUAGCUGGUAGAAGUAUC
671
1164
Yes
No
No

672
ACUUCUACCAGCUAUCUUA
UAAGAUAGCUGGUAGAAGU
673
1167
Yes
No
No

674
UUCUACCAGCUAUCUUCUA
UAGAAGAUAGCUGGUAGAA
675
1169
Yes
No
No

676
UCUACCAGCUAUCUUCUGA
UCAGAAGAUAGCUGGUAGA
677
1170
Yes
No
No

678
CUACCAGCUAUCUUCUGUA
UACAGAAGAUAGCUGGUAG
679
1171
Yes
No
No

680
UACCAGCUAUCUUCUGUGA
UCACAGAAGAUAGCUGGUA
681
1172
Yes
No
No

682
ACCAGCUAUCUUCUGUGCA
UGCACAGAAGAUAGCUGGU
683
1173
Yes
No
No

684
CCAGCUAUCUUCUGUGCAA
UUGCACAGAAGAUAGCUGG
685
1174
Yes
No
No

686
GCUAUCUUCUGUGCAUCUA
UAGAUGCACAGAAGAUAGC
687
1177
Yes
No
No

688
CUAUCUUCUGUGCAUCUCA
UGAGAUGCACAGAAGAUAG
689
1178
Yes
No
No

690
AUCUCUGAAAAUAAGGAAA
UUUCCUUAUUUUCAGAGAU
691
1191
Yes
No
No

692
GAAAAUAAGGAAAAUGUUA
UAACAUUUUCCUUAUUUUC
693
1197
Yes
No
No

694
AAAAUAAGGAAAAUGUUAA
UUAACAUUUUCCUUAUUUU
695
1198
Yes
No
No

696
AGGAAAAUGUUAGGGACAA
UUGUCCCUAACAUUUUCCU
697
1204
Yes
No
No

698
GGAAAAUGUUAGGGACAAA
UUUGUCCCUAACAUUUUCC
699
1205
Yes
No
No

700
UUUUAUUGGCAUUGUGGGA
UCCCACAAUGCCAAUAAAA
701
1238
Yes
No
No

702
UGCCACAGGCGAGGUUGUA
UACAACCUCGCCUGUGGCA
703
1265
Yes
No
No

704
CCACAGGCGAGGUUGUGUA
UACACAACCUCGCCUGUGG
705
1267
Yes
No
No

706
CACAGGCGAGGUUGUGUUA
UAACACAACCUCGCCUGUG
707
1268
Yes
No
No

708
CAGGCGAGGUUGUGUUUGA
UCAAACACAACCUCGCCUG
709
1270
Yes
No
No

710
AGGCGAGGUUGUGUUUGAA
UUCAAACACAACCUCGCCU
711
1271
Yes
No
No

712
GGCGAGGUUGUGUUUGAUA
UAUCAAACACAACCUCGCC
713
1272
Yes
No
No

714
GCGAGGUUGUGUUUGAUAA
UUAUCAAACACAACCUCGC
715
1273
Yes
No
No

716
CGAGGUUGUGUUUGAUAGA
UCUAUCAAACACAACCUCG
717
1274
Yes
No
No

718
GAGGUUGUGUUUGAUAGUA
UACUAUCAAACACAACCUC
719
1275
Yes
No
No

720
AGGUUGUGUUUGAUAGUUA
UAACUAUCAAACACAACCU
721
1276
Yes
No
No

722
GGUUGUGUUUGAUAGUUUA
UAAACUAUCAAACACAACC
723
1277
Yes
No
No

724
GUUGUGUUUGAUAGUUUCA
UGAAACUAUCAAACACAAC
725
1278
Yes
No
No

726
GUUUGAUAGUUUCCAGGAA
UUCCUGGAAACUAUCAAAC
727
1283
Yes
No
No

728
CAGGACUCUGCUUCUCGUA
UACGAGAAGCAGAGUCCUG
729
1296
Yes
No
No

730
AGGACUCUGCUUCUCGUUA
UAACGAGAAGCAGAGUCCU
731
1297
Yes
No
No

732
GGACUCUGCUUCUCGUUCA
UGAACGAGAAGCAGAGUCC
733
1298
Yes
No
No

734
GACUCUGCUUCUCGUUCAA
UUGAACGAGAAGCAGAGUC
735
1299
Yes
No
No

736
ACUCUGCUUCUCGUUCAGA
UCUGAACGAGAAGCAGAGU
737
1300
Yes
No
No

738
GCUUCUCGUUCAGAGCUAA
UUAGCUCUGAACGAGAAGC
739
1305
Yes
No
No

740
CUUCUCGUUCAGAGCUAGA
UCUAGCUCUGAACGAGAAG
741
1306
Yes
No
No

742
UUCUCGUUCAGAGCUAGAA
UUCUAGCUCUGAACGAGAA
743
1307
Yes
No
No

744
UCUCGUUCAGAGCUAGAAA
UUUCUAGCUCUGAACGAGA
745
1308
Yes
No
No

746
UCGUUCAGAGCUAGAAACA
UGUUUCUAGCUCUGAACGA
747
1310
Yes
No
No

748
UAGAAACCCGGAUGUCAAA
UUUGACAUCCGGGUUUCUA
749
1321
Yes
No
No

750
GAAACCCGGAUGUCAAGCA
UGCUUGACAUCCGGGUUUC
751
1323
No
No
No

752
AAGCCUGCAGCCAGUAGAA
UUCUACUGGCUGCAGGCUU
753
1337
No
No
No

754
CUGCAGCCAGUAGAGCUGA
UCAGCUCUACUGGCUGCAG
755
1341
Yes
No
No

756
UCCUUCGGCCUUGUCCGAA
UUCGGACAAGGCCGAAGGA
757
1364
Yes
No
No

758
CGGCCUUGUCCGAGCAAAA
UUUUGCUCGGACAAGGCCG
759
1369
Yes
No
No

760
CCUUGUCCGAGCAAACAGA
UCUGUUUGCUCGGACAAGG
761
1372
Yes
No
No

762
CUUGUCCGAGCAAACAGAA
UUCUGUUUGCUCGGACAAG
763
1373
Yes
No
No

764
UGUCCGAGCAAACAGAGGA
UCCUCUGUUUGCUCGGACA
765
1375
No
No
No

766
AACAGAGGCGCUCAUCCAA
UUGGAUGAGCGCCUCUGUU
767
1385
Vo
No
No

768
ACAGAGGCGCUCAUCCACA
UGUGGAUGAGCGCCUCUGU
769
1386
No
No
No

770
CAGAGGCGCUCAUCCACAA
UUGUGGAUGAGCGCCUCUG
771
1387
No
No
No

772
AGAGGCGCUCAUCCACAGA
UCUGUGGAUGAGCGCCUCU
773
1388
No
No
No

774
GAGGCGCUCAUCCACAGAA
UUCUGUGGAUGAGCGCCUC
775
1389
No
No
No

776
CACAGAGCCACAUCUGUUA
UAACAGAUGUGGCUCUGUG
777
1401
Yes
No
No

778
AGAGCCACAUCUGUUAGUA
UACUAACAGAUGUGGCUCU
779
1404
Yes
No
No

780
AGCCACAUCUGUUAGUGUA
UACACUAACAGAUGUGGCU
781
1406
Yes
No
No

782
GCCACAUCUGUUAGUGUGA
UCACACUAACAGAUGUGGC
783
1407
Yes
No
No

784
CCACAUCUGUUAGUGUGCA
UGCACACUAACAGAUGUGG
785
1408
Yes
No
No

786
UCUGUUAGUGUGCAGGAUA
UAUCCUGCACACUAACAGA
787
1413
Yes
No
No

788
GUUAGUGUGCAGGAUGACA
UGUCAUCCUGCACACUAAC
789
1416
Yes
No
No

790
UUAGUGUGCAGGAUGACAA
UUGUCAUCCUGCACACUAA
791
1417
Yes
No
No

792
GUGUGCAGGAUGACAGAAA
UUUCUGUCAUCCUGCACAC
793
1420
Yes
No
No

794
UGUGCAGGAUGACAGAAUA
UAUUCUGUCAUCCUGCACA
795
1421
Yes
No
No

796
GUGCAGGAUGACAGAAUUA
UAAUUCUGUCAUCCUGCAC
797
1422
Yes
No
No

798
AGGAUGACAGAAUUCGAGA
UCUCGAAUUCUGUCAUCCU
799
1426
Yes
No
No

800
ACAGAAUUCGAGUCGAAAA
UUUUCGACUCGAAUUCUGU
801
1432
No
No
No

802
CAGAAUUCGAGUCGAAAGA
UCUUUCGACUCGAAUUCUG
803
1433
No
No
No

804
AGAAUUCGAGUCGAAAGGA
UCCUUUCGACUCGAAUUCU
805
1434
No
No
No

806
UUCGAGUCGAAAGGAUGGA
UCCAUCCUUUCGACUCGAA
807
1438
No
No
No

808
CGAGUCGAAAGGAUGGAUA
UAUCCAUCCUUUCGACUCG
809
1440
No
No
No

810
GAGUCGAAAGGAUGGAUAA
UUAUCCAUCCUUUCGACUC
811
1441
No
No
No

812
AGUCGAAAGGAUGGAUAAA
UUUAUCCAUCCUUUCGACU
813
1442
No
No
No

814
GUCGAAAGGAUGGAUAACA
UGUUAUCCAUCCUUUCGAC
815
1443
No
No
No

816
UCGAAAGGAUGGAUAACAA
UUGUUAUCCAUCCUUUCGA
817
1444
No
No
No

818
CGAAAGGAUGGAUAACAUA
UAUGUUAUCCAUCCUUUCG
819
1445
No
No
No

820
GAAAGGAUGGAUAACAUUA
UAAUGUUAUCCAUCCUUUC
821
1446
Yes
No
No

822
AAAGGAUGGAUAACAUUUA
UAAAUGUUAUCCAUCCUUU
823
1447
Yes
No
No

824
AGGAUGGAUAACAUUUAUA
UAUAAAUGUUAUCCAUCCU
825
1449
Yes
No
No

826
GGAUAACAUUUAUUUUGAA
UUCAAAAUAAAUGUUAUCC
827
1454
Yes
No
No

828
GAUAACAUUUAUUUUGAAA
UUUCAAAAUAAAUGUUAUC
829
1455
Yes
No
No

830
AUAACAUUUAUUUUGAAUA
UAUUCAAAAUAAAUGUUAU
831
1456
Yes
No
No

832
UGAAUACAGCCAUGCUUUA
UAAAGCAUGGCUGUAUUCA
833
1469
Yes
Yes
Yes

834
CAUGCUUUCCAGGCAGUUA
UAACUGCCUGGAAAGCAUG
835
1479
Yes
No
No

836
AUGCUUUCCAGGCAGUUAA
UUAACUGCCUGGAAAGCAU
837
1480
Yes
No
No

838
UGCUUUCCAGGCAGUUACA
UGUAACUGCCUGGAAAGCA
839
1481
Yes
No
No

840
GCUUUCCAGGCAGUUACAA
UUGUAACUGCCUGGAAAGC
841
1482
Yes
No
No

842
UCCAGGCAGUUACAGAGUA
UACUCUGUAACUGCCUGGA
843
1486
Yes
No
No

844
CAGGCAGUUACAGAGUUUA
UAAACUCUGUAACUGCCUG
845
1488
Yes
No
No

846
GCAGUUACAGAGUUUUAUA
UAUAAAACUCUGUAACUGC
847
1491
Yes
No
No

848
CAGUUACAGAGUUUUAUGA
UCAUAAAACUCUGUAACUG
849
1492
Yes
No
No

850
GUUACAGAGUUUUAUGCAA
UUGCAUAAAACUCUGUAAC
851
1494
Yes
No
No

852
UUACAGAGUUUUAUGCAAA
UUUGCAUAAAACUCUGUAA
853
1495
Yes
No
No

854
UACAGAGUUUUAUGCAAAA
UUUUGCAUAAAACUCUGUA
855
1496
Yes
No
No

856
UUUUAUGCAAAAGAUACAA
UUGUAUCUUUUGCAUAAAA
857
1503
No
No
No

858
UAUGCAAAAGAUACAGUUA
UAACUGUAUCUUUUGCAUA
859
1506
No
No
No

860
UGCAAAAGAUACAGUUGAA
UUCAACUGUAUCUUUUGCA
861
1508
No
No
No

862
GCAAAAGAUACAGUUGACA
UGUCAACUGUAUCUUUUGC
863
1509
No
No
No

864
CAAAAGAUACAGUUGACAA
UUGUCAACUGUAUCUUUUG
865
1510
No
No
No

866
AAAAGAUACAGUUGACAUA
UAUGUCAACUGUAUCUUUU
867
1511
No
No
No

868
GAUACAGUUGACAUCAAAA
UUUUGAUGUCAACUGUAUC
869
1515
No
No
No

870
AUACAGUUGACAUCAAAGA
UCUUUGAUGUCAACUGUAU
871
1516
No
No
No

872
UACAGUUGACAUCAAAGGA
UCCUUUGAUGUCAACUGUA
873
1517
No
No
No

874
ACAGUUGACAUCAAAGGUA
UACCUUUGAUGUCAACUGU
875
1518
Yes
No
No

876
CAGUUGACAUCAAAGGUUA
UAACCUUUGAUGUCAACUG
877
1519
Yes
No
No

878
AGUUGACAUCAAAGGUUCA
UGAACCUUUGAUGUCAACU
879
1520
Yes
No
No

880
GUUGACAUCAAAGGUUCUA
UAGAACCUUUGAUGUCAAC
881
1521
Yes
No
No

882
UUGACAUCAAAGGUUCUCA
UGAGAACCUUUGAUGUCAA
883
1522
Yes
No
No

884
UGACAUCAAAGGUUCUCAA
UUGAGAACCUUUGAUGUCA
885
1523
Yes
No
No

886
GACAUCAAAGGUUCUCAAA
UUUGAGAACCUUUGAUGUC
887
1524
Yes
No
No

888
UCAAAGGUUCUCAAAUUAA
UUAAUUUGAGAACCUUUGA
889
1528
Yes
No
No

890
AAGGUUCUCAAAUUAUUUA
UAAAUAAUUUGAGAACCUU
891
1531
Yes
No
No

892
UCAAAUUAUUUCUGGCAUA
UAUGCCAGAAAUAAUUUGA
893
1538
Yes
No
No

894
CAAAUUAUUUCUGGCAUUA
UAAUGCCAGAAAUAAUUUG
895
1539
Yes
No
No

896
AAUUAUUUCUGGCAUUGUA
UACAAUGCCAGAAAUAAUU
897
1541
Yes
No
No

898
AUUAUUUCUGGCAUUGUUA
UAACAAUGCCAGAAAUAAU
899
1542
Yes
No
No

900
UUAUUUCUGGCAUUGUUAA
UUAACAAUGCCAGAAAUAA
901
1543
Yes
No
No

902
UUUCUGGCAUUGUUAACUA
UAGUUAACAAUGCCAGAAA
903
1546
Yes
No
No

904
UUCUGGCAUUGUUAACUUA
UAAGUUAACAAUGCCAGAA
905
1547
Yes
No
No

906
CUGGCAUUGUUAACUUAGA
UCUAAGUUAACAAUGCCAG
907
1549
Yes
No
No

908
GCAUUGUUAACUUAGAGAA
UUCUCUAAGUUAACAAUGC
909
1552
Yes
No
No

910
GUUAACUUAGAGAAGCCUA
UAGGCUUCUCUAAGUUAAC
911
1557
Yes
No
No

912
UAACUUAGAGAAGCCUGUA
UACAGGCUUCUCUAAGUUA
913
1559
Yes
No
No

914
UAGAGAAGCCUGUGAUUUA
UAAAUCACAGGCUUCUCUA
915
1564
Yes
No
No

916
AAGCCUGUGAUUUGCUCUA
UAGAGCAAAUCACAGGCUU
917
1569
Yes
No
No

918
GCCUGUGAUUUGCUCUUUA
UAAAGAGCAAAUCACAGGC
919
1571
Yes
No
No

920
UGUGAUUUGCUCUUUGGCA
UGCCAAAGAGCAAAUCACA
921
1574
Yes
No
No

922
GUGAUUUGCUCUUUGGCUA
UAGCCAAAGAGCAAAUCAC
923
1575
Yes
No
No

924
GCUCUUUGGCUGCCAUCAA
UUGAUGGCAGCCAAAGAGC
925
1582
Yes
No
No

926
UCUUUGGCUGCCAUCAUAA
UUAUGAUGGCAGCCAAAGA
927
1584
Yes
No
No

928
CUUUGGCUGCCAUCAUAAA
UUUAUGAUGGCAGCCAAAG
929
1585
Yes
No
No

930
UUUGGCUGCCAUCAUAAAA
UUUUAUGAUGGCAGCCAAA
931
1586
Yes
No
No

932
UGGCUGCCAUCAUAAAAUA
UAUUUUAUGAUGGCAGCCA
933
1588
Yes
No
No

934
GGCUGCCAUCAUAAAAUAA
UUAUUUUAUGAUGGCAGCC
935
1589
Yes
No
No

936
GCUGCCAUCAUAAAAUACA
UGUAUUUUAUGAUGGCAGC
937
1590
Yes
No
No

938
UGCCAUCAUAAAAUACCUA
UAGGUAUUUUAUGAUGGCA
939
1592
Yes
No
No

940
GCCAUCAUAAAAUACCUCA
UGAGGUAUUUUAUGAUGGC
941
1593
Yes
No
No

942
CAUCAUAAAAUACCUCAAA
UUUGAGGUAUUUUAUGAUG
943
1595
Yes
No
No

944
CAUAAAAUACCUCAAAGAA
UUCUUUGAGGUAUUUUAUG
945
1598
Yes
No
No

946
UACCUCAAAGAAUUCAACA
UGUUGAAUUCUUUGAGGUA
947
1605
Yes
No
No

948
CCUCAAAGAAUUCAACUUA
UAAGUUGAAUUCUUUGAGG
949
1607
Yes
No
No

950
UCAACUUGGAAAAGAUGCA
UGCAUCUUUUCCAAGUUGA
951
1618
No
No
No

952
CAACUUGGAAAAGAUGCUA
UAGCAUCUUUUCCAAGUUG
953
1619
No
No
No

954
GGAAAAGAUGCUCUCCAAA
UUUGGAGAGCAUCUUUUCC
955
1625
No
No
No

956
AAAGAUGCUCUCCAAACCA
UGGUUUGGAGAGCAUCUUU
957
1628
No
No
No

958
GAUGCUCUCCAAACCUGAA
UUCAGGUUUGGAGAGCAUC
959
1631
No
No
No

960
AUGCUCUCCAAACCUGAGA
UCUCAGGUUUGGAGAGCAU
961
1632
Yes
No
No

962
GCUCUCCAAACCUGAGAAA
UUUCUCAGGUUUGGAGAGC
963
1634
Yes
No
No

964
CUCUCCAAACCUGAGAAUA
UAUUCUCAGGUUUGGAGAG
965
1635
Yes
No
No

966
UCUCCAAACCUGAGAAUUA
UAAUUCUCAGGUUUGGAGA
967
1636
Yes
No
No

968
CUCCAAACCUGAGAAUUUA
UAAAUUCUCAGGUUUGGAG
969
1637
Yes
No
No

970
AACCUGAGAAUUUUAAACA
UGUUUAAAAUUCUCAGGUU
971
1642
Yes
No
No

972
CCUGAGAAUUUUAAACAGA
UCUGUUUAAAAUUCUCAGG
973
1644
Yes
No
No

974
CUGAGAAUUUUAAACAGCA
UGCUGUUUAAAAUUCUCAG
975
1645
Yes
No
No

976
GAGAAUUUUAAACAGCUAA
UUAGCUGUUUAAAAUUCUC
977
1647
Yes
No
No

978
AGAAUUUUAAACAGCUAUA
UAUAGCUGUUUAAAAUUCU
979
1648
Yes
No
No

980
GAAUUUUAAACAGCUAUCA
UGAUAGCUGUUUAAAAUUC
981
1649
Yes
No
No

982
UAAACAGCUAUCAAGUAAA
UUUACUUGAUAGCUGUUUA
983
1655
Yes
No
No

984
AAACAGCUAUCAAGUAAAA
UUUUACUUGAUAGCUGUUU
985
1656
Yes
No
No

986
CAGCUAUCAAGUAAAAUGA
UCAUUUUACUUGAUAGCUG
987
1659
Yes
No
No

988
GCUAUCAAGUAAAAUGGAA
UUCCAUUUUACUUGAUAGC
989
1661
Yes
No
No

990
CUAUCAAGUAAAAUGGAAA
UUUCCAUUUUACUUGAUAG
991
1662
Yes
No
No

992
UAUCAAGUAAAAUGGAAUA
UAUUCCAUUUUACUUGAUA
993
1663
Yes
No
No

994
UCAAGUAAAAUGGAAUUUA
UAAAUUCCAUUUUACUUGA
995
1665
Yes
No
No

996
CAAGUAAAAUGGAAUUUAA
UUAAAUUCCAUUUUACUUG
997
1666
Yes
No
No

998
AAAAUGGAAUUUAUGACAA
UUGUCAUAAAUUCCAUUUU
999
1671
Yes
No
No

1000
AAUGGAAUUUAUGACAAUA
UAUUGUCAUAAAUUCCAUU
1001
1673
Yes
No
No

1002
AUGGAAUUUAUGACAAUUA
UAAUUGUCAUAAAUUCCAU
1003
1674
Yes
No
No

1004
AAUUUAUGACAAUUAAUGA
UCAUUAAUUGUCAUAAAUU
1005
1678
Yes
No
No

1006
UUAUGACAAUUAAUGGAAA
UUUCCAUUAAUUGUCAUAA
1007
1681
Yes
No
No

1008
UAUGACAAUUAAUGGAACA
UGUUCCAUUAAUUGUCAUA
1009
1682
Yes
No
No

1010
AUGACAAUUAAUGGAACAA
UUGUUCCAUUAAUUGUCAU
1011
1683
Yes
No
No

1012
UGACAAUUAAUGGAACAAA
UUUGUUCCAUUAAUUGUCA
1013
1684
Yes
No
No

1014
GACAAUUAAUGGAACAACA
UGUUGUUCCAUUAAUUGUC
1015
1685
Yes
No
No

1016
ACAAUUAAUGGAACAACAA
UUGUUGUUCCAUUAAUUGU
1017
1686
Yes
No
No

1018
AAUGGAACAACAUUAAGGA
UCCUUAAUGUUGUUCCAUU
1019
1692
Yes
No
No

1020
GGAACAACAUUAAGGAAUA
UAUUCCUUAAUGUUGUUCC
1021
1695
Yes
No
No

1022
CAACAUUAAGGAAUCUGGA
UCCAGAUUCCUUAAUGUUG
1023
1699
Yes
No
No

1024
UAAGGAAUCUGGAAAUCCA
UGGAUUUCCAGAUUCCUUA
1025
1705
Yes
No
Yes

1026
GGAAUCUGGAAAUCCUACA
UGUAGGAUUUCCAGAUUCC
1027
1708
Yes
No
Yes

1028
GAAUCUGGAAAUCCUACAA
UUGUAGGAUUUCCAGAUUC
1029
1709
Yes
No
Yes

1030
UCUGGAAAUCCUACAGAAA
UUUCUGUAGGAUUUCCAGA
1031
1712
Yes
No
Yes

1032
CUGGAAAUCCUACAGAAUA
UAUUCUGUAGGAUUUCCAG
1033
1713
Yes
No
Yes

1034
UGGAAAUCCUACAGAAUCA
UGAUUCUGUAGGAUUUCCA
1035
1714
Yes
No
Yes

1036
GAAAUCCUACAGAAUCAGA
UCUGAUUCUGUAGGAUUUC
1037
1716
Yes
No
Yes

1038
AAUCCUACAGAAUCAGACA
UGUCUGAUUCUGUAGGAUU
1039
1718
Yes
No
Yes

1040
AUCCUACAGAAUCAGACUA
UAGUCUGAUUCUGUAGGAU
1041
1719
Yes
No
Yes

1042
UCCUACAGAAUCAGACUGA
UCAGUCUGAUUCUGUAGGA
1043
1720
Yes
No
Yes

1044
CCUACAGAAUCAGACUGAA
UUCAGUCUGAUUCUGUAGG
1045
1721
Yes
No
Yes

1046
CUACAGAAUCAGACUGAUA
UAUCAGUCUGAUUCUGUAG
1047
1722
Yes
No
Yes

1048
UACAGAAUCAGACUGAUAA
UUAUCAGUCUGAUUCUGUA
1049
1723
Yes
No
Yes

1050
ACAGAAUCAGACUGAUAUA
UAUAUCAGUCUGAUUCUGU
1051
1724
Yes
No
Yes

1052
CAGAAUCAGACUGAUAUGA
UCAUAUCAGUCUGAUUCUG
1053
1725
Yes
Yes
Yes

1054
UCAGACUGAUAUGAAAACA
UGUUUUCAUAUCAGUCUGA
1055
1730
Yes
No
No

1056
GACUGAUAUGAAAACCAAA
UUUGGUUUUCAUAUCAGUC
1057
1733
Yes
No
No

1058
ACUGAUAUGAAAACCAAAA
UUUUGGUUUUCAUAUCAGU
1059
1734
Yes
No
No

1060
CUGAUAUGAAAACCAAAGA
UCUUUGGUUUUCAUAUCAG
1061
1735
Yes
No
No

1062
GAUAUGAAAACCAAAGGAA
UUCCUUUGGUUUUCAUAUC
1063
1737
Yes
No
No

1064
GAAAACCAAAGGAAGUUUA
UAAACUUCCUUUGGUUUUC
1065
1742
Yes
No
No

1066
AAAACCAAAGGAAGUUUGA
UCAAACUUCCUUUGGUUUU
1067
1743
Yes
No
No

1068
AAACCAAAGGAAGUUUGCA
UGCAAACUUCCUUUGGUUU
1069
1744
Yes
No
No

1070
ACCAAAGGAAGUUUGCUGA
UCAGCAAACUUCCUUUGGU
1071
1746
Yes
No
No

1072
GGAAGUUUGCUGUGGGUUA
UAACCCACAGCAAACUUCC
1073
1752
Yes
No
No

1074
AGUUUGCUGUGGGUUUUAA
UUAAAACCCACAGCAAACU
1075
1755
Yes
No
No

1076
UUGCUGUGGGUUUUAGACA
UGUCUAAAACCCACAGCAA
1077
1758
Yes
No
No

1078
UGCUGUGGGUUUUAGACCA
UGGUCUAAAACCCACAGCA
1079
1759
Yes
No
No

1080
GCUGUGGGUUUUAGACCAA
UUGGUCUAAAACCCACAGC
1081
1760
Yes
No
No

1082
CUGUGGGUUUUAGACCACA
UGUGGUCUAAAACCCACAG
1083
1761
Yes
No
No

1084
UGGGUUUUAGACCACACUA
UAGUGUGGUCUAAAACCCA
1085
1764
Yes
Yes
Yes

1086
GGUUUUAGACCACACUAAA
UUUAGUGUGGUCUAAAACC
1087
1766
Yes
Yes
Yes

1088
GUUUUAGACCACACUAAAA
UUUUAGUGUGGUCUAAAAC
1089
1767
Yes
Yes
Yes

1090
UUUAGACCACACUAAAACA
UGUUUUAGUGUGGUCUAAA
1091
1769
Yes
Yes
Yes

1092
UUAGACCACACUAAAACUA
UAGUUUUAGUGUGGUCUAA
1093
1770
Yes
Yes
No

1094
UAGACCACACUAAAACUUA
UAAGUUUUAGUGUGGUCUA
1095
1771
Yes
Yes
No

1096
AGACCACACUAAAACUUCA
UGAAGUUUUAGUGUGGUCU
1097
1772
Yes
Yes
No

1098
CCACACUAAAACUUCAUUA
UAAUGAAGUUUUAGUGUGG
1099
1775
Yes
Yes
No

1100
CACUAAAACUUCAUUUGGA
UCCAAAUGAAGUUUUAGUG
1101
1778
Yes
Yes
No

1102
ACUAAAACUUCAUUUGGGA
UCCCAAAUGAAGUUUUAGU
1103
1779
Yes
Yes
No

1104
UAAAACUUCAUUUGGGAGA
UCUCCCAAAUGAAGUUUUA
1105
1781
Yes
Yes
No

1106
AUUUGGGAGACGGAAGUUA
UAACUUCCGUCUCCCAAAU
1107
1790
Yes
No
No

1108
UUGGGAGACGGAAGUUAAA
UUUAACUUCCGUCUCCCAA
1109
1792
Yes
No
No

1110
GGGAGACGGAAGUUAAAGA
UCUUUAACUUCCGUCUCCC
1111
1794
Yes
No
No

1112
GGAGACGGAAGUUAAAGAA
UUCUUUAACUUCCGUCUCC
1113
1795
Yes
No
No

1114
GACGGAAGUUAAAGAAGUA
UACUUCUUUAACUUCCGUC
1115
1798
Yes
No
No

1116
ACGGAAGUUAAAGAAGUGA
UCACUUCUUUAACUUCCGU
1117
1799
Yes
No
No

1118
CGGAAGUUAAAGAAGUGGA
UCCACUUCUUUAACUUCCG
1119
1800
Yes
No
No

1120
GUGACCCAGCCACUCCUUA
UAAGGAGUGGCUGGGUCAC
1121
1818
Yes
No
No

1122
UGACCCAGCCACUCCUUAA
UUAAGGAGUGGCUGGGUCA
1123
1819
Yes
No
No

1124
ACCCAGCCACUCCUUAAAA
UUUUAAGGAGUGGCUGGGU
1125
1821
Yes
No
No

1126
CCCAGCCACUCCUUAAAUA
UAUUUAAGGAGUGGCUGGG
1127
1822
Yes
No
No

1128
GCCACUCCUUAAAUUAAGA
UCUUAAUUUAAGGAGUGGC
1129
1826
Yes
No
No

1130
CACUCCUUAAAUUAAGGGA
UCCCUUAAUUUAAGGAGUG
1131
1828
Yes
No
No

1132
ACUCCUUAAAUUAAGGGAA
UUCCCUUAAUUUAAGGAGU
1133
1829
Yes
No
No

1134
CUCCUUAAAUUAAGGGAAA
UUUCCCUUAAUUUAAGGAG
1135
1830
Yes
No
No

1136
CCUUAAAUUAAGGGAAAUA
UAUUUCCCUUAAUUUAAGG
1137
1832
Yes
No
No

1138
UAAAUUAAGGGAAAUAAAA
UUUUAUUUCCCUUAAUUUA
1139
1835
Yes
Yes
No

1140
UUAAGGGAAAUAAAUGCCA
UGGCAUUUAUUUCCCUUAA
1141
1839
Yes
Yes
No

1142
AUAAAUGCCCGGCUUGAUA
UAUCAAGCCGGGCAUUUAU
1143
1848
Yes
No
No

1144
AAAUGCCCGGCUUGAUGCA
UGCAUCAAGCCGGGCAUUU
1145
1850
Yes
No
No

1146
AAUGCCCGGCUUGAUGCUA
UAGCAUCAAGCCGGGCAUU
1147
1851
Yes
No
No

1148
AUGCCCGGCUUGAUGCUGA
UCAGCAUCAAGCCGGGCAU
1149
1852
Yes
No
No

1150
UGCCCGGCUUGAUGCUGUA
UACAGCAUCAAGCCGGGCA
1151
1853
Yes
No
No

1152
GCCCGGCUUGAUGCUGUAA
UUACAGCAUCAAGCCGGGC
1153
1854
Yes
No
No

1154
CGGCUUGAUGCUGUAUCGA
UCGAUACAGCAUCAAGCCG
1155
1857
No
No
No

1156
GGCUUGAUGCUGUAUCGGA
UCCGAUACAGCAUCAAGCC
1157
1858
No
No
No

1158
GCUUGAUGCUGUAUCGGAA
UUCCGAUACAGCAUCAAGO
1159
1859
No
No
No

1160
UUGAUGCUGUAUCGGAAGA
UCUUCCGAUACAGCAUCAA
1161
1861
No
No
No

1162
UGAUGCUGUAUCGGAAGUA
UACUUCCGAUACAGCAUCA
1163
1862
No
No
No

1164
GAUGCUGUAUCGGAAGUUA
UAACUUCCGAUACAGCAUC
1165
1863
No
No
No

1166
AUGCUGUAUCGGAAGUUCA
UGAACUUCCGAUACAGCAU
1167
1864
No
No
No

1168
GCUGUAUCGGAAGUUCUCA
UGAGAACUUCCGAUACAGC
1169
1866
No
No
No

1170
CUGUAUCGGAAGUUCUCCA
UGGAGAACUUCCGAUACAG
1171
1867
No
No
No

1172
UGUAUCGGAAGUUCUCCAA
UUGGAGAACUUCCGAUACA
1173
1868
No
No
No

1174
GUAUCGGAAGUUCUCCAUA
UAUGGAGAACUUCCGAUAC
1175
1869
No
No
No

1176
AUCGGAAGUUCUCCAUUCA
UGAAUGGAGAACUUCCGAU
1177
1871
No
No
No

1178
UCGGAAGUUCUCCAUUCAA
UUGAAUGGAGAACUUCCGA
1179
1872
No
No
No

1180
CGGAAGUUCUCCAUUCAGA
UCUGAAUGGAGAACUUCCG
1181
1873
No
No
No

1182
AGUUCUCCAUUCAGAAUCA
UGAUUCUGAAUGGAGAACU
1183
1877
Yes
No
No

1184
GUUCUCCAUUCAGAAUCUA
UAGAUUCUGAAUGGAGAAC
1185
1878
Yes
No
No

1186
CUCCAUUCAGAAUCUAGUA
UACUAGAUUCUGAAUGGAG
1187
1881
Yes
No
No

1188
UCCAUUCAGAAUCUAGUGA
UCACUAGAUUCUGAAUGGA
1189
1882
Yes
No
No

1190
CCAUUCAGAAUCUAGUGUA
UACACUAGAUUCUGAAUGG
1191
1883
Yes
No
No

1192
CAUUCAGAAUCUAGUGUGA
UCACACUAGAUUCUGAAUG
1193
1884
Yes
No
NO

1194
AUUCAGAAUCUAGUGUGUA
UACACACUAGAUUCUGAAU
1195
1885
Yes
No
No

1196
UUCAGAAUCUAGUGUGUUA
UAACACACUAGAUUCUGAA
1197
1886
Yes
No
No

1198
AGAAUCUAGUGUGUUUGGA
UCCAAACACACUAGAUUCU
1199
1889
Yes
No
No

1200
GAAUCUAGUGUGUUUGGUA
UACCAAACACACUAGAUUC
1201
1890
Yes
No
No

1202
AAUCUAGUGUGUUUGGUCA
UGACCAAACACACUAGAUU
1203
1891
Yes
No
No

1204
AUCUAGUGUGUUUGGUCAA
UUGACCAAACACACUAGAU
1205
1892
Yes
No
No

1206
CUAGUGUGUUUGGUCAGAA
UUCUGACCAAACACACUAG
1207
1894
Yes
No
No

1208
UGUGUUUGGUCAGAUAGAA
UUCUAUCUGACCAAACACA
1209
1898
Yes
No
No

1210
GUGUUUGGUCAGAUAGAAA
UUUCUAUCUGACCAAACAC
1211
1899
Yes
No
No

1212
UGUUUGGUCAGAUAGAAAA
UUUUCUAUCUGACCAAACA
1213
1900
Yes
No
No

1214
UUUGGUCAGAUAGAAAAUA
UAUUUUCUAUCUGACCAAA
1215
1902
Yes
No
No

1216
UUGGUCAGAUAGAAAAUCA
UGAUUUUCUAUCUGACCAA
1217
1903
Yes
No
No

1218
GUCAGAUAGAAAAUCAUCA
UGAUGAUUUUCUAUCUGAC
1219
1906
Yes
No
No

1220
CAGAUAGAAAAUCAUCUAA
UUAGAUGAUUUUCUAUCUG
1221
1908
Yes
No
No

1222
AUAGAAAAUCAUCUACGUA
UACGUAGAUGAUUUUCUAU
1223
1911
Yes
No
No

1224
UAGAAAAUCAUCUACGUAA
UUACGUAGAUGAUUUUCUA
1225
1912
Yes
No
No

1226
AGAAAAUCAUCUACGUAAA
UUUACGUAGAUGAUUUUCU
1227
1913
Yes
No
No

1228
AAAAUCAUCUACGUAAAUA
UAUUUACGUAGAUGAUUUU
1229
1915
Yes
No
No

1230
AAUCAUCUACGUAAAUUGA
UCAAUUUACGUAGAUGAUU
1231
1917
Yes
No
No

1232
AUCAUCUACGUAAAUUGCA
UGCAAUUUACGUAGAUGAU
1233
1918
Yes
No
No

1234
CAUCUACGUAAAUUGCCCA
UGGGCAAUUUACGUAGAUG
1235
1920
No
No
No

1236
GUAAAUUGCCCGACAUAGA
UCUAUGUCGGGCAAUUUAC
1237
1927
No
No
No

1238
UAAAUUGCCCGACAUAGAA
UUCUAUGUCGGGCAAUUUA
1239
1928
No
No
No

1240
AAAUUGCCCGACAUAGAGA
UCUCUAUGUCGGGCAAUUU
1241
1929
No
No
No

1242
AUUGCCCGACAUAGAGAGA
UCUCUCUAUGUCGGGCAAU
1243
1931
No
No
No

1244
UUGCCCGACAUAGAGAGGA
UCCUCUCUAUGUCGGGCAA
1245
1932
No
No
No

1246
AUAGAGAGGGGACUCUGUA
UACAGAGUCCCCUCUCUAU
1247
1941
No
No
No

1248
AGAGGGGACUCUGUAGCAA
UUGCUACAGAGUCCCCUCU
1249
1945
No
No
No

1250
GGACUCUGUAGCAUUUAUA
UAUAAAUGCUACAGAGUCC
1251
1950
Yes
No
No

1252
GACUCUGUAGCAUUUAUCA
UGAUAAAUGCUACAGAGUC
1253
1951
Yes
No
No

1254
CUCUGUAGCAUUUAUCACA
UGUGAUAAAUGCUACAGAG
1255
1953
Yes
No
No

1256
AAAAUGUUCUACCCAAGAA
UUCUUGGGUAGAACAUUUU
1257
1973
Yes
No
No

1258
AAUGUUCUACCCAAGAGUA
UACUCUUGGGUAGAACAUU
1259
1975
Yes
No
No

1260
AUGUUCUACCCAAGAGUUA
UAACUCUUGGGUAGAACAU
1261
1976
Yes
No
No

1262
UGUUCUACCCAAGAGUUCA
UGAACUCUUGGGUAGAACA
1263
1977
Yes
No
No

1264
UUCUACCCAAGAGUUCUUA
UAAGAACUCUUGGGUAGAA
1265
1979
Yes
No
No

1266
UCUACCCAAGAGUUCUUCA
UGAAGAACUCUUGGGUAGA
1267
1980
Yes
Yes
No

1268
UACCCAAGAGUUCUUCUUA
UAAGAAGAACUCUUGGGUA
1269
1982
Yes
Yes
No

1270
ACCCAAGAGUUCUUCUUGA
UCAAGAAGAACUCUUGGGU
1271
1983
Yes
Yes
No

1272
GAGUUCUUCUUGAUUGUCA
UGACAAUCAAGAAGAACUC
1273
1989
Yes
No
No

1274
UCUUGAUUGUCAAAACUUA
UAAGUUUUGACAAUCAAGA
1275
1996
No
No
No

1276
UUGAUUGUCAAAACUUUAA
UUAAAGUUUUGACAAUCAA
1277
1998
No
No
No

1278
UGAUUGUCAAAACUUUAUA
UAUAAAGUUUUGACAAUCA
1279
1999
No
No
No

1280
UUGUCAAAACUUUAUAUCA
UGAUAUAAAGUUUUGACAA
1281
2002
No
No
No

1282
UGUCAAAACUUUAUAUCAA
UUGAUAUAAAGUUUUGACA
1283
2003
No
No
No

1284
CAAAACUUUAUAUCACCUA
UAGGUGAUAUAAAGUUUUG
1285
2006
No
No
No

1286
AAAACUUUAUAUCACCUAA
UUAGGUGAUAUAAAGUUUU
1287
2007
No
No
No

1288
AAACUUUAUAUCACCUAAA
UUUAGGUGAUAUAAAGUUU
1289
2008
No
No
No

1290
ACUUUAUAUCACCUAAAGA
UCUUUAGGUGAUAUAAAGU
1291
2010
No
No
No

1292
CUUUAUAUCACCUAAAGUA
UACUUUAGGUGAUAUAAAG
1293
2011
No
No
No

1294
UUAUAUCACCUAAAGUCAA
UUGACUUUAGGUGAUAUAA
1295
2013
Yes
No
No

1296
UAUAUCACCUAAAGUCAGA
UCUGACUUUAGGUGAUAUA
1297
2014
Yes
No
No

1298
UAUCACCUAAAGUCAGAAA
UUUCUGACUUUAGGUGAUA
1299
2016
Yes
No
No

1300
AUCACCUAAAGUCAGAAUA
UAUUCUGACUUUAGGUGAU
1301
2017
Yes
No
No

1302
UCACCUAAAGUCAGAAUUA
UAAUUCUGACUUUAGGUGA
1303
2018
Yes
No
No

1304
GUCAGAAUUUCAAGCAAUA
UAUUGCUUGAAAUUCUGAC
1305
2027
Yes
No
No

1306
AGAAUUUCAAGCAAUAAUA
UAUUAUUGCUUGAAAUUCU
1307
2030
Yes
No
No

1308
AUUUCAAGCAAUAAUACCA
UGGUAUUAUUGCUUGAAAU
1309
2033
Yes
No
No

1310
UUUCAAGCAAUAAUACCUA
UAGGUAUUAUUGCUUGAAA
1311
2034
Yes
No
No

1312
UCAAGCAAUAAUACCUGCA
UGCAGGUAUUAUUGCUUGA
1313
2036
Yes
No
No

1314
GCAAUAAUACCUGCUGUUA
UAACAGCAGGUAUUAUUGC
1315
2040
Yes
No
No

1316
UAAUACCUGCUGUUAAUUA
UAAUUAACAGCAGGUAUUA
1317
2044
Yes
No
No

1318
AUACCUGCUGUUAAUUCCA
UGGAAUUAACAGCAGGUAU
1319
2046
Yes
No
No

1320
UGCUGUUAAUUCCCACAUA
UAUGUGGGAAUUAACAGCA
1321
2051
No
No
No

1322
GCUGUUAAUUCCCACAUUA
UAAUGUGGGAAUUAACAGC
1323
2052
No
No
No

1324
CUGUUAAUUCCCACAUUCA
UGAAUGUGGGAAUUAACAG
1325
2053
No
No
No

1326
UGUUAAUUCCCACAUUCAA
UUGAAUGUGGGAAUUAACA
1327
2054
No
No
No

1328
UUAAUUCCCACAUUCAGUA
UACUGAAUGUGGGAAUUAA
1329
2056
No
No
No

1330
UAAUUCCCACAUUCAGUCA
UGACUGAAUGUGGGAAUUA
1331
2057
No
No
No

1332
UUCCCACAUUCAGUCAGAA
UUCUGACUGAAUGUGGGAA
1333
2060
No
No
No

1334
UCCCACAUUCAGUCAGACA
UGUCUGACUGAAUGUGGGA
1335
2061
No
No
No

1336
CCCACAUUCAGUCAGACUA
UAGUCUGACUGAAUGUGGG
1337
2062
No
No
No

1338
CACAUUCAGUCAGACUUGA
UCAAGUCUGACUGAAUGUG
1339
2064
No
No
No

1340
ACAUUCAGUCAGACUUGCA
UGCAAGUCUGACUGAAUGU
1341
2065
No
No
No

1342
CAUUCAGUCAGACUUGCUA
UAGCAAGUCUGACUGAAUG
1343
2066
No
No
No

1344
AUUCAGUCAGACUUGCUCA
UGAGCAAGUCUGACUGAAU
1345
2067
No
No
No

1346
ACUUGCUCCGGACCGUUAA
UUAACGGUCCGGAGCAAGU
1347
2077
No
No
No

1348
CUUGCUCCGGACCGUUAUA
UAUAACGGUCCGGAGCAAG
1349
2078
No
No
No

1350
UUGCUCCGGACCGUUAUUA
UAAUAACGGUCCGGAGCAA
1351
2079
No
No
No

1352
GCUCCGGACCGUUAUUUUA
UAAAAUAACGGUCCGGAGC
1353
2081
No
No
No

1354
CUCCGGACCGUUAUUUUAA
UUAAAAUAACGGUCCGGAG
1355
2082
No
No
No

1356
UCCGGACCGUUAUUUUAGA
UCUAAAAUAACGGUCCGGA
1357
2083
No
No
No

1358
GGACCGUUAUUUUAGAAAA
UUUUCUAAAAUAACGGUCC
1359
2086
No
No
No

1360
GACCGUUAUUUUAGAAAUA
UAUUUCUAAAAUAACGGUC
1361
2087
No
No
No

1362
ACCGUUAUUUUAGAAAUUA
UAAUUUCUAAAAUAACGGU
1363
2088
Yes
No
No

1364
CCGUUAUUUUAGAAAUUCA
UGAAUUUCUAAAAUAACGG
1365
2089
Yes
No
No

1366
CGUUAUUUUAGAAAUUCCA
UGGAAUUUCUAAAAUAACG
1367
2090
Yes
No
No

1368
UUUAGAAAUUCCUGAACUA
UAGUUCAGGAAUUUCUAAA
1369
2096
Yes
No
No

1370
UUAGAAAUUCCUGAACUCA
UGAGUUCAGGAAUUUCUAA
1371
2097
Yes
No
No

1372
GAAAUUCCUGAACUCCUCA
UGAGGAGUUCAGGAAUUUC
1373
2100
Yes
No
No

1374
AAAUUCCUGAACUCCUCAA
UUGAGGAGUUCAGGAAUUU
1375
2101
Yes
No
No

1376
CCUGAACUCCUCAGUCCAA
UUGGACUGAGGAGUUCAGG
1377
2106
Yes
No
No

1378
UGAACUCCUCAGUCCAGUA
UACUGGACUGAGGAGUUCA
1379
2108
Yes
No
No

1380
CUCAGUCCAGUGGAGCAUA
UAUGCUCCACUGGACUGAG
1381
2115
Yes
No
No

1382
UCAGUCCAGUGGAGCAUUA
UAAUGCUCCACUGGACUGA
1383
2116
Yes
No
No

1384
GUCCAGUGGAGCAUUACUA
UAGUAAUGCUCCACUGGAC
1385
2119
Yes
No
No

1386
UCCAGUGGAGCAUUACUUA
UAAGUAAUGCUCCACUGGA
1387
2120
Yes
No
No

1388
AGUGGAGCAUUACUUAAAA
UUUUAAGUAAUGCUCCACU
1389
2123
Yes
No
No

1390
GUGGAGCAUUACUUAAAGA
UCUUUAAGUAAUGCUCCAC
1391
2124
Yes
No
No

1392
GGAGCAUUACUUAAAGAUA
UAUCUUUAAGUAAUGCUCC
1393
2126
Yes
No
No

1394
GAGCAUUACUUAAAGAUAA
UUAUCUUUAAGUAAUGCUC
1395
2127
Yes
No
No

1396
AGCAUUACUUAAAGAUACA
UGUAUCUUUAAGUAAUGCU
1397
2128
Yes
No
No

1398
CAUUACUUAAAGAUACUCA
UGAGUAUCUUUAAGUAAUG
1399
2130
Yes
No
No

1400
UACUUAAAGAUACUCAAUA
UAUUGAGUAUCUUUAAGUA
1401
2133
Yes
No
No

1402
CUUAAAGAUACUCAAUGAA
UUCAUUGAGUAUCUUUAAG
1403
2135
Yes
No
No

1404
UUAAAGAUACUCAAUGAAA
UUUCAUUGAGUAUCUUUAA
1405
2136
Yes
No
No

1406
UAAAGAUACUCAAUGAACA
UGUUCAUUGAGUAUCUUUA
1407
2137
Yes
No
No

1408
GAUACUCAAUGAACAAGCA
UGCUUGUUCAUUGAGUAUC
1409
2141
Yes
No
No

1410
AUGAACAAGCUGCCAAAGA
UCUUUGGCAGCUUGUUCAU
1411
2149
Yes
No
Yes

1412
UGAACAAGCUGCCAAAGUA
UACUUUGGCAGCUUGUUCA
1413
2150
Yes
No
No

1414
GAACAAGCUGCCAAAGUUA
UAACUUUGGCAGCUUGUUC
1415
2151
Yes
No
No

1416
ACAAGCUGCCAAAGUUGGA
UCCAACUUUGGCAGCUUGU
1417
2153
Yes
No
No

1418
CUGCCAAAGUUGGGGAUAA
UUAUCCCCAACUUUGGCAG
1419
2158
Yes
V
No

1420
UGCCAAAGUUGGGGAUAAA
UUUAUCCCCAACUUUGGCA
1421
2159
Yes
No
No

1422
AAGUUGGGGAUAAAACUGA
UCAGUUUUAUCCCCAACUU
1423
2164
Yes
No
No

1424
GUUGGGGAUAAAACUGAAA
UUUCAGUUUUAUCCCCAAC
1425
2166
Yes
No
No

1426
UUGGGGAUAAAACUGAAUA
UAUUCAGUUUUAUCCCCAA
1427
2167
Yes
No
No

1428
GGGGAUAAAACUGAAUUAA
UUAAUUCAGUUUUAUCCCC
1429
2169
Yes
No
No

1430
GGGAUAAAACUGAAUUAUA
UAUAAUUCAGUUUUAUCCC
1431
2170
Yes
No
No

1432
GGAUAAAACUGAAUUAUUA
UAAUAAUUCAGUUUUAUCC
1433
2171
Yes
No
No

1434
CUGAAUUAUUUAAAGACCA
UGGUCUUUAAAUAAUUCAG
1435
2179
Yes
No
No

1436
UAUUUAAAGACCUUUCUGA
UCAGAAAGGUCUUUAAAUA
1437
2185
Yes
No
No

1438
AUUUAAAGACCUUUCUGAA
UUCAGAAAGGUCUUUAAAU
1439
2186
Yes
No
No

1440
UAAAGACCUUUCUGACUUA
UAAGUCAGAAAGGUCUUUA
1441
2189
Yes
No
No

1442
AAGACCUUUCUGACUUCCA
UGGAAGUCAGAAAGGUCUU
1443
2191
Yes
No
No

1444
GACCUUUCUGACUUCCCUA
UAGGGAAGUCAGAAAGGUC
1445
2193
Yes
No
No

1446
ACCUUUCUGACUUCCCUUA
UAAGGGAAGUCAGAAAGGU
1447
2194
Yes
No
No

1448
UUCUGACUUCCCUUUAAUA
UAUUAAAGGGAAGUCAGAA
1449
2198
Yes
No
No

1450
UCUGACUUCCCUUUAAUAA
UUAUUAAAGGGAAGUCAGA
1451
2199
Yes
Yes
Yes

1452
UGACUUCCCUUUAAUAAAA
UUUUAUUAAAGGGAAGUCA
1453
2201
Yes
Yes
Yes

1454
AGAGGAAGGAUGAAAUUCA
UGAAUUUCAUCCUUCCUCU
1455
2221
Yes
No
No

1456
GGAUGAAAUUCAAGGUGUA
UACACCUUGAAUUUCAUCC
1457
2228
Yes
No
No

1458
UGAAAUUCAAGGUGUUAUA
UAUAACACCUUGAAUUUCA
1459
2231
No
No
No

1460
GAAAUUCAAGGUGUUAUUA
UAAUAACACCUUGAAUUUC
1461
2232
No
No
No

1462
AAUUCAAGGUGUUAUUGAA
UUCAAUAACACCUUGAAUU
1463
2234
No
No
No

1464
AUUCAAGGUGUUAUUGACA
UGUCAAUAACACCUUGAAU
1465
2235
No
No
No

1466
UUCAAGGUGUUAUUGACGA
UCGUCAAUAACACCUUGAA
1467
2236
No
No
No

1468
CAAGGUGUUAUUGACGAGA
UCUCGUCAAUAACACCUUG
1469
2238
No
No
No

1470
AAGGUGUUAUUGACGAGAA
UUCUCGUCAAUAACACCUU
1471
2239
No
No
No

1472
GGUGUUAUUGACGAGAUCA
UGAUCUCGUCAAUAACACC
1473
2241
No
No
No

1474
GUUAUUGACGAGAUCCGAA
UUCGGAUCUCGUCAAUAAC
1475
2244
No
No
No

1476
UUAUUGACGAGAUCCGAAA
UUUCGGAUCUCGUCAAUAA
1477
2245
No
No
No

1478
UAUUGACGAGAUCCGAAUA
UAUUCGGAUCUCGUCAAUA
1479
2246
No
No
No

1480
AUUGACGAGAUCCGAAUGA
UCAUUCGGAUCUCGUCAAU
1481
2247
No
No
No

1482
UGACGAGAUCCGAAUGCAA
UUGCAUUCGGAUCUCGUCA
1483
2249
No
No
No

1484
GACGAGAUCCGAAUGCAUA
UAUGCAUUCGGAUCUCGUC
1485
2250
No
No
No

1486
ACGAGAUCCGAAUGCAUUA
UAAUGCAUUCGGAUCUCGU
1487
2251
No
No
No

1488
CGAGAUCCGAAUGCAUUUA
UAAAUGCAUUCGGAUCUCG
1489
2252
No
No
No

1490
GAGAUCCGAAUGCAUUUGA
UCAAAUGCAUUCGGAUCUC
1491
2253
No
No
No

1492
AGAUCCGAAUGCAUUUGCA
UGCAAAUGCAUUCGGAUCU
1493
2254
Yes
No
No

1494
GAUCCGAAUGCAUUUGCAA
UUGCAAAUGCAUUCGGAUC
1495
2255
Yes
No
No

1496
UCCGAAUGCAUUUGCAAGA
UCUUGCAAAUGCAUUCGGA
1497
2257
Yes
No
No

1498
CCGAAUGCAUUUGCAAGAA
UUCUUGCAAAUGCAUUCGG
1499
2258
Yes
No
No

1500
GCAUUUGCAAGAAAUACGA
UCGUAUUUCUUGCAAAUGC
1501
2264
Yes
No
No

1502
AAAAUCCUUCUGCACAAUA
UAUUGUGCAGAAGGAUUUU
1503
2293
Yes
No
No

1504
AAAUCCUUCUGCACAAUAA
UUAUUGUGCAGAAGGAUUU
1505
2294
Yes
No
No

1506
AUCCUUCUGCACAAUAUGA
UCAUAUUGUGCAGAAGGAU
1507
2296
Yes
No
No

1508
CUUCUGCACAAUAUGUGAA
UUCACAUAUUGUGCAGAAG
1509
2299
Yes
No
No

1510
UCUGCACAAUAUGUGACAA
UUGUCACAUAUUGUGCAGA
1511
2301
Yes
No
No

1512
UGCACAAUAUGUGACAGUA
UACUGUCACAUAUUGUGCA
1513
2303
Yes
No
No

1514
GCACAAUAUGUGACAGUAA
UUACUGUCACAUAUUGUGC
1515
2304
Yes
No
No

1516
UAUGUGACAGUAUCAGGAA
UUCCUGAUACUGUCACAUA
1517
2310
Yes
No
No

1518
AUGUGACAGUAUCAGGACA
UGUCCUGAUACUGUCACAU
1519
2311
Yes
No
No

1520
GUAUCAGGACAGGAGUUUA
UAAACUCCUGUCCUGAUAC
1521
2319
Yes
No
No

1522
UAUCAGGACAGGAGUUUAA
UUAAACUCCUGUCCUGAUA
1523
2320
Yes
No
No

1524
CAGGACAGGAGUUUAUGAA
UUCAUAAACUCCUGUCCUG
1525
2323
Yes
No
No

1526
AGGACAGGAGUUUAUGAUA
UAUCAUAAACUCCUGUCCU
1527
2324
Yes
No
No

1528
GGACAGGAGUUUAUGAUAA
UUAUCAUAAACUCCUGUCC
1529
2325
No
No
No

1530
UUAUGAUAGAAAUAAAGAA
UUCUUUAUUUCUAUCAUAA
1531
2335
No
No
No

1532
GAAAUAAAGAACUCUGCUA
UAGCAGAGUUCUUUAUUUC
1533
2343
Yes
No
No

1534
AAUAAAGAACUCUGCUGUA
UACAGCAGAGUUCUUUAUU
1535
2345
Yes
No
No

1536
AUAAAGAACUCUGCUGUAA
UUACAGCAGAGUUCUUUAU
1537
2346
Yes
No
No

1538
UAAAGAACUCUGCUGUAUA
UAUACAGCAGAGUUCUUUA
1539
2347
Yes
No
No

1540
AGAACUCUGCUGUAUCUUA
UAAGAUACAGCAGAGUUCU
1541
2350
Yes
No
No

1542
GAACUCUGCUGUAUCUUGA
UCAAGAUACAGCAGAGUUC
1543
2351
Yes
No
No

1544
ACUCUGCUGUAUCUUGUAA
UUACAAGAUACAGCAGAGU
1545
2353
Yes
No
No

1546
CUCUGCUGUAUCUUGUAUA
UAUACAAGAUACAGCAGAG
1547
2354
Yes
No
No

1548
UGCUGUAUCUUGUAUACCA
UGGUAUACAAGAUACAGCA
1549
2357
Yes
No
No

1550
GCUGUAUCUUGUAUACCAA
UUGGUAUACAAGAUACAGC
1551
2358
Yes
No
No

1552
GUAUCUUGUAUACCAACUA
UAGUUGGUAUACAAGAUAC
1553
2361
Yes
No
No

1554
UAUCUUGUAUACCAACUGA
UCAGUUGGUAUACAAGAUA
1555
2362
Yes
No
No

1556
UCUUGUAUACCAACUGAUA
UAUCAGUUGGUAUACAAGA
1557
2364
Yes
No
No

1558
UGUAUACCAACUGAUUGGA
UCCAAUCAGUUGGUAUACA
1559
2367
Yes
No
No

1560
UAUACCAACUGAUUGGGUA
UACCCAAUCAGUUGGUAUA
1561
2369
Yes
No
No

1562
AUACCAACUGAUUGGGUAA
UUACCCAAUCAGUUGGUAU
1563
2370
Yes
No
No

1564
ACCAACUGAUUGGGUAAAA
UUUUACCCAAUCAGUUGGU
1565
2372
Yes
No
No

1566
CCAACUGAUUGGGUAAAGA
UCUUUACCCAAUCAGUUGG
1567
2373
Yes
No
No

1568
CAACUGAUUGGGUAAAGGA
UCCUUUACCCAAUCAGUUG
1569
2374
Yes
No
No

1570
AACUGAUUGGGUAAAGGUA
UACCUUUACCCAAUCAGUU
1571
2375
Yes
No
No

1572
CUGAUUGGGUAAAGGUUGA
UCAACCUUUACCCAAUCAG
1573
2377
Yes
No
No

1574
UGAUUGGGUAAAGGUUGGA
UCCAACCUUUACCCAAUCA
1575
2378
Yes
No
No

1576
GAUUGGGUAAAGGUUGGAA
UUCCAACCUUUACCCAAUC
1577
2379
Yes
No
No

1578
AUUGGGUAAAGGUUGGAAA
UUUCCAACCUUUACCCAAU
1579
2380
Yes
No
No

1580
UGGGUAAAGGUUGGAAGCA
UGCUUCCAACCUUUACCCA
1581
2382
Yes
No
No

1582
GUAAAGGUUGGAAGCACAA
UUGUGCUUCCAACCUUUAC
1583
2385
Yes
No
No

1584
GUUGGAAGCACAAAAGCUA
UAGCUUUUGUGCUUCCAAC
1585
2391
Yes
Yes
Yes

1586
UUGGAAGCACAAAAGCUGA
UCAGCUUUUGUGCUUCCAA
1587
2392
Yes
Yes
Yes

1588
UGGAAGCACAAAAGCUGUA
UACAGCUUUUGUGCUUCCA
1589
2393
Yes
Yes
Yes

1590
GGAAGCACAAAAGCUGUGA
UCACAGCUUUUGUGCUUCC
1591
2394
Yes
Yes
Yes

1592
GAAGCACAAAAGCUGUGAA
UUCACAGCUUUUGUGCUUC
1593
2395
Yes
Yes
Yes

1594
AGCUGUGAGCCGCUUUCAA
UUGAAAGCGGCUCACAGCU
1595
2405
No
Yes
Yes

1596
UGUGAGCCGCUUUCACUCA
UGAGUGAAAGCGGCUCACA
1597
2408
No
No
Yes

1598
GUGAGCCGCUUUCACUCUA
UAGAGUGAAAGCGGCUCAC
1599
2409
No
No
Yes

1600
AGCCGCUUUCACUCUCCUA
UAGGAGAGUGAAAGCGGCU
1601
2412
No
No
Yes

1602
GCCGCUUUCACUCUCCUUA
UAAGGAGAGUGAAAGCGGC
1603
2413
No
No
Yes

1604
GCUUUCACUCUCCUUUUAA
UUAAAAGGAGAGUGAAAGC
1605
2416
No
No
No

1606
CUUUCACUCUCCUUUUAUA
UAUAAAAGGAGAGUGAAAG
1607
2417
No
No
No

1608
UUUCACUCUCCUUUUAUUA
UAAUAAAAGGAGAGUGAAA
1609
2418
No
No
No

1610
CACUCUCCUUUUAUUGUAA
UUACAAUAAAAGGAGAGUG
1611
2421
No
No
No

1612
ACUCUCCUUUUAUUGUAGA
UCUACAAUAAAAGGAGAGU
1613
2422
No
No
No

1614
CUUUUAUUGUAGAAAAUUA
UAAUUUUCUACAAUAAAAG
1615
2428
No
No
No

1616
UUUAUUGUAGAAAAUUACA
UGUAAUUUUCUACAAUAAA
1617
2430
No
No
No

1618
AUUGUAGAAAAUUACAGAA
UUCUGUAAUUUUCUACAAU
1619
2433
No
No
No

1620
GUAGAAAAUUACAGACAUA
UAUGUCUGUAAUUUUCUAC
1621
2436
Yes
No
No

1622
AGAAAAUUACAGACAUCUA
UAGAUGUCUGUAAUUUUCU
1623
2438
Yes
No
No

1624
GAAAAUUACAGACAUCUGA
UCAGAUGUCUGUAAUUUUC
1625
2439
Yes
No
No

1626
UUACAGACAUCUGAAUCAA
UUGAUUCAGAUGUCUGUAA
1627
2444
Yes
No
No

1628
AGACAUCUGAAUCAGCUCA
UGAGCUGAUUCAGAUGUCU
1629
2448
No
No
No

1630
CGGGAGCAGCUAGUCCUUA
UAAGGACUAGCUGCUCCCG
1631
2466
Yes
Yes
Yes

1632
GGGAGCAGCUAGUCCUUGA
UCAAGGACUAGCUGCUCCC
1633
2467
Yes
Yes
Yes

1634
GGAGCAGCUAGUCCUUGAA
UUCAAGGACUAGCUGCUCC
1635
2468
Yes
Yes
Yes

1636
GAGCAGCUAGUCCUUGACA
UGUCAAGGACUAGCUGCUC
1637
2469
Yes
Yes
Yes

1638
AGCAGCUAGUCCUUGACUA
UAGUCAAGGACUAGCUGCU
1639
2470
Yes
Yes
Yes

1640
CAGCUAGUCCUUGACUGCA
UGCAGUCAAGGACUAGCUG
1641
2472
Yes
Yes
Yes

1642
AGCUAGUCCUUGACUGCAA
UUGCAGUCAAGGACUAGCU
1643
2473
Yes
Yes
Yes

1644
CCUUGACUGCAGUGCUGAA
UUCAGCACUGCAGUCAAGG
1645
2480
Yes
No
No

1646
UGACUGCAGUGCUGAAUGA
UCAUUCAGCACUGCAGUCA
1647
2483
Yes
No
No

1648
ACUGCAGUGCUGAAUGGCA
UGCCAUUCAGCACUGCAGU
1649
2485
Yes
No
No

1650
CUGCAGUGCUGAAUGGCUA
UAGCCAUUCAGCACUGCAG
1651
2486
Yes
No
No

1652
UGCAGUGCUGAAUGGCUUA
UAAGCCAUUCAGCACUGCA
1653
2487
Yes
No
No

1654
GCAGUGCUGAAUGGCUUGA
UCAAGCCAUUCAGCACUGC
1655
2488
Yes
No
No

1656
GAAUGGCUUGAUUUUCUAA
UUAGAAAAUCAAGCCAUUC
1657
2496
Yes
No
No

1658
AAUGGCUUGAUUUUCUAGA
UCUAGAAAAUCAAGCCAUU
1659
2497
Yes
No
No

1660
AUGGCUUGAUUUUCUAGAA
UUCUAGAAAAUCAAGCCAU
1661
2498
Yes
No
No

1662
UGGCUUGAUUUUCUAGAGA
UCUCUAGAAAAUCAAGCCA
1663
2499
Yes
No
No

1664
GAUUUUCUAGAGAAAUUCA
UGAAUUUCUCUAGAAAAUC
1665
2505
Yes
No
No

1666
UUUCUAGAGAAAUUCAGUA
UACUGAAUUUCUCUAGAAA
1667
2508
Yes
No
No

1668
UUCUAGAGAAAUUCAGUGA
UCACUGAAUUUCUCUAGAA
1669
2509
Yes
No
No

1670
UCUAGAGAAAUUCAGUGAA
UUCACUGAAUUUCUCUAGA
1671
2510
Yes
No
No

1672
UAGAGAAAUUCAGUGAACA
UGUUCACUGAAUUUCUCUA
1673
2512
Yes
No
No

1674
AAAUUCAGUGAACAUUAUA
UAUAAUGUUCACUGAAUUU
1675
2517
Yes
No
No

1676
AAUUCAGUGAACAUUAUCA
UGAUAAUGUUCACUGAAUU
1677
2518
Yes
No
No

1678
AUUCAGUGAACAUUAUCAA
UUGAUAAUGUUCACUGAAU
1679
2519
Yes
No
No

1680
GUGAACAUUAUCACUCCUA
UAGGAGUGAUAAUGUUCAC
1681
2524
No
No
No

1682
UGAACAUUAUCACUCCUUA
UAAGGAGUGAUAAUGUUCA
1683
2525
No
No
No

1684
GAACAUUAUCACUCCUUGA
UCAAGGAGUGAUAAUGUUC
1685
2526
No
No
No

1686
ACAUUAUCACUCCUUGUGA
UCACAAGGAGUGAUAAUGU
1687
2528
No
No
No

1688
AUCACUCCUUGUGUAAAGA
UCUUUACACAAGGAGUGAU
1689
2533
No
No
No

1690
ACUCCUUGUGUAAAGCAGA
UCUGCUUUACACAAGGAGU
1691
2536
No
No
No

1692
UUGUGUAAAGCAGUGCAUA
UAUGCACUGCUUUACACAA
1693
2541
Yes
No
No

1694
UAAAGCAGUGCAUCACCUA
UAGGUGAUGCACUGCUUUA
1695
2546
Yes
No
No

1696
AAAGCAGUGCAUCACCUAA
UUAGGUGAUGCACUGCUUU
1697
2547
Yes
No
No

1698
GUGCAUCACCUAGCAACUA
UAGUUGCUAGGUGAUGCAC
1699
2553
Yes
No
No

1700
GCAUCACCUAGCAACUGUA
UACAGUUGCUAGGUGAUGC
1701
2555
Yes
No
No

1702
UCACCUAGCAACUGUUGAA
UUCAACAGUUGCUAGGUGA
1703
2558
Yes
Yes
No

1704
ACCUAGCAACUGUUGACUA
UAGUCAACAGUUGCUAGGU
1705
2560
Yes
Yes
No

1706
CCUAGCAACUGUUGACUGA
UCAGUCAACAGUUGCUAGG
1707
2561
Yes
Yes
No

1708
CUAGCAACUGUUGACUGCA
UGCAGUCAACAGUUGCUAG
1709
2562
Yes
No
No

1710
UAGCAACUGUUGACUGCAA
UUGCAGUCAACAGUUGCUA
1711
2563
Yes
No
No

1712
AGCAACUGUUGACUGCAUA
UAUGCAGUCAACAGUUGCU
1713
2564
Yes
No
No

1714
GCAACUGUUGACUGCAUUA
UAAUGCAGUCAACAGUUGC
1715
2565
Yes
No
No

1716
AACUGUUGACUGCAUUUUA
UAAAAUGCAGUCAACAGUU
1717
2567
Yes
No
No

1718
ACUGUUGACUGCAUUUUCA
UGAAAAUGCAGUCAACAGU
1719
2568
Yes
No
No

1720
CUGUUGACUGCAUUUUCUA
UAGAAAAUGCAGUCAACAG
1721
2569
Yes
No
No

1722
UGUUGACUGCAUUUUCUCA
UGAGAAAAUGCAGUCAACA
1723
2570
Yes
No
No

1724
CCUGGCCAAGGUCGCUAAA
UUUAGCGACCUUGGCCAGG
1725
2588
Yes
Yes
No

1726
CCAAGGUCGCUAAGCAAGA
UCUUGCUUAGCGACCUUGG
1727
2593
Yes
No
No

1728
CAAGGUCGCUAAGCAAGGA
UCCUUGCUUAGCGACCUUG
1729
2594
Yes
No
No

1730
AAGGUCGCUAAGCAAGGAA
UUCCUUGCUUAGCGACCUU
1731
2595
Yes
No
No

1732
GCUAAGCAAGGAGAUUACA
UGUAAUCUCCUUGCUUAGC
1733
2601
No
No
No

1734
CUAAGCAAGGAGAUUACUA
UAGUAAUCUCCUUGCUUAG
1735
2602
No
No
No

1736
UAAGCAAGGAGAUUACUGA
UCAGUAAUCUCCUUGCUUA
1737
2603
No
No
No

1738
AAGCAAGGAGAUUACUGCA
UGCAGUAAUCUCCUUGCUU
1739
2604
No
No
No

1740
GCAAGGAGAUUACUGCAGA
UCUGCAGUAAUCUCCUUGC
1741
2606
No
No
No

1742
CAAGGAGAUUACUGCAGAA
UUCUGCAGUAAUCUCCUUG
1743
2607
No
No
No

1744
GAGAUUACUGCAGACCAAA
UUUGGUCUGCAGUAAUCUC
1745
2611
No
No
No

1746
AGAUUACUGCAGACCAACA
UGUUGGUCUGCAGUAAUCU
1747
2612
No
No
No

1748
GAUUACUGCAGACCAACUA
UAGUUGGUCUGCAGUAAUC
1749
2613
No
No
No

1750
AUUACUGCAGACCAACUGA
UCAGUUGGUCUGCAGUAAU
1751
2614
Yes
No
No

1752
UACUGCAGACCAACUGUAA
UUACAGUUGGUCUGCAGUA
1753
2616
Yes
No
No

1754
CUGCAGACCAACUGUACAA
UUGUACAGUUGGUCUGCAG
1755
2618
Yes
No
No

1756
UGCAGACCAACUGUACAAA
UUUGUACAGUUGGUCUGCA
1757
2619
Yes
No
No

1758
GCAGACCAACUGUACAAGA
UCUUGUACAGUUGGUCUGC
1759
2620
Yes
No
No

1760
CAGACCAACUGUACAAGAA
UUCUUGUACAGUUGGUCUG
1761
2621
Yes
No
No

1762
AGACCAACUGUACAAGAAA
UUUCUUGUACAGUUGGUCU
1763
2622
Yes
No
No

1764
CAACUGUACAAGAAGAAAA
UUUUCUUCUUGUACAGUUG
1765
2626
Yes
No
No

1766
ACUGUACAAGAAGAAAGAA
UUCUUUCUUCUUGUACAGU
1767
2628
Yes
No
No

1768
CUGUACAAGAAGAAAGAAA
UUUCUUUCUUCUUGUACAG
1769
2629
Yes
No
No

1770
UGUACAAGAAGAAAGAAAA
UUUUCUUUCUUCUUGUACA
1771
2630
Yes
No
No

1772
UGGAAGGCACCCUGUGAUA
UAUCACAGGGUGCCUUCCA
1773
2663
No
No
No

1774
GGAAGGCACCCUGUGAUUA
UAAUCACAGGGUGCCUUCC
1775
2664
No
No
No

1776
AAGGCACCCUGUGAUUGAA
UUCAAUCACAGGGUGCCUU
1777
2666
No
No
No

1778
AGGCACCCUGUGAUUGAUA
UAUCAAUCACAGGGUGCCU
1779
2667
No
No
No

1780
GGCACCCUGUGAUUGAUGA
UCAUCAAUCACAGGGUGCC
1781
2668
No
No
No

1782
GCACCCUGUGAUUGAUGUA
UACAUCAAUCACAGGGUGC
1783
2669
No
No
No

1784
CACCCUGUGAUUGAUGUGA
UCACAUCAAUCACAGGGUG
1785
2670
No
No
No

1786
CUGUGAUUGAUGUGUUGCA
UGCAACACAUCAAUCACAG
1787
2674
Yes
No
No

1788
UGUGAUUGAUGUGUUGCUA
UAGCAACACAUCAAUCACA
1789
2675
Yes
No
No

1790
GUGAUUGAUGUGUUGCUGA
UCAGCAACACAUCAAUCAC
1791
2676
Yes
No
No

1792
UGUGUUGCUGGGAGAACAA
UUGUUCUCCCAGCAACACA
1793
2684
Yes
No
No

1794
CUGGGAGAACAGGAUCAAA
UUUGAUCCUGUUCUCCCAG
1795
2691
Yes
No
No

1796
UGGGAGAACAGGAUCAAUA
UAUUGAUCCUGUUCUCCCA
1797
2692
Yes
No
No

1798
GGGAGAACAGGAUCAAUAA
UUAUUGAUCCUGUUCUCCC
1799
2693
Yes
No
No

1800
GAGAACAGGAUCAAUAUGA
UCAUAUUGAUCCUGUUCUC
1801
2695
Yes
No
No

1802
AGAACAGGAUCAAUAUGUA
UACAUAUUGAUCCUGUUCU
1803
2696
Yes
No
No

1804
AGGAUCAAUAUGUCCCAAA
UUUGGGACAUAUUGAUCCU
1805
2701
No
No
No

1806
GGAUCAAUAUGUCCCAAAA
UUUUGGGACAUAUUGAUCC
1807
2702
No
No
No

1808
GAUCAAUAUGUCCCAAAUA
UAUUUGGGACAUAUUGAUC
1809
2703
No
No
No

1810
AUCAAUAUGUCCCAAAUAA
UUAUUUGGGACAUAUUGAU
1811
2704
No
No
No

1812
CAAUAUGUCCCAAAUAAUA
UAUUAUUUGGGACAUAUUG
1813
2706
No
No
No

1814
AUAUGUCCCAAAUAAUACA
UGUAUUAUUUGGGACAUAU
1815
2708
No
No
No

1816
GUCCCAAAUAAUACAGAUA
UAUCUGUAUUAUUUGGGAC
1817
2712
No
No
No

1818
CCCAAAUAAUACAGAUUUA
UAAAUCUGUAUUAUUUGGG
1819
2714
No
No
No

1820
AUAAUACAGAUUUAUCAGA
UCUGAUAAAUCUGUAUUAU
1821
2719
No
No
No

1822
UAAUACAGAUUUAUCAGAA
UUCUGAUAAAUCUGUAUUA
1823
2720
No
No
No

1824
AAUACAGAUUUAUCAGAGA
UCUCUGAUAAAUCUGUAUU
1825
2721
No
No
No

1826
UACAGAUUUAUCAGAGGAA
UUCCUCUGAUAAAUCUGUA
1827
2723
Yes
No
No

1828
GAUUUAUCAGAGGACUCAA
UUGAGUCCUCUGAUAAAUC
1829
2727
Yes
No
No

1830
UUAUCAGAGGACUCAGAGA
UCUCUGAGUCCUCUGAUAA
1831
2730
Yes
No
No

1832
GGACUCAGAGAGAGUAAUA
UAUUACUCUCUCUGAGUCC
1833
2738
Yes
No
No

1834
GACUCAGAGAGAGUAAUGA
UCAUUACUCUCUCUGAGUC
1835
2739
Yes
No
No

1836
ACUCAGAGAGAGUAAUGAA
UUCAUUACUCUCUCUGAGU
1837
2740
Yes
No
No

1838
UCAGAGAGAGUAAUGAUAA
UUAUCAUUACUCUCUCUGA
1839
2742
Yes
No
No

1840
CAGAGAGAGUAAUGAUAAA
UUUAUCAUUACUCUCUCUG
1841
2743
Yes
No
No

1842
AGAGAGAGUAAUGAUAAUA
UAUUAUCAUUACUCUCUCU
1843
2744
Yes
No
No

1844
AGAGUAAUGAUAAUUACCA
UGGUAAUUAUCAUUACUCU
1845
2748
No
No
No

1846
AGUAAUGAUAAUUACCGGA
UCCGGUAAUUAUCAUUACU
1847
2750
No
No
No

1848
AUAAUUACCGGACCAAACA
UGUUUGGUCCGGUAAUUAU
1849
2757
No
No
No

1850
UAAUUACCGGACCAAACAA
UUGUUUGGUCCGGUAAUUA
1851
2758
No
No
No

1852
AAUUACCGGACCAAACAUA
UAUGUUUGGUCCGGUAAUU
1853
2759
No
No
No

1854
AUUACCGGACCAAACAUGA
UCAUGUUUGGUCCGGUAAU
1855
2760
No
No
No

1856
UUACCGGACCAAACAUGGA
UCCAUGUUUGGUCCGGUAA
1857
2761
No
No
No

1858
ACCAAACAUGGGUGGAAAA
UUUUCCACCCAUGUUUGGU
1859
2768
Yes
No
No

1860
CAAACAUGGGUGGAAAGAA
UUCUUUCCACCCAUGUUUG
1861
2770
Yes
No
No

1862
AAACAUGGGUGGAAAGAGA
UCUCUUUCCACCCAUGUUU
1863
2771
Yes
No
No

1864
AACAUGGGUGGAAAGAGCA
UGCUCUUUCCACCCAUGUU
1865
2772
Yes
No
No

1866
UGGAAAGAGCUCCUACAUA
UAUGUAGGAGCUCUUUCCA
1867
2780
Yes
No
No

1868
GAAAGAGCUCCUACAUAAA
UUUAUGUAGGAGCUCUUUC
1869
2782
Yes
No
No

1870
GAGCUCCUACAUAAAACAA
UUGUUUUAUGUAGGAGCUC
1871
2786
Yes
No
Yes

1872
AGCUCCUACAUAAAACAAA
UUUGUUUUAUGUAGGAGCU
1873
2787
Yes
No
No

1874
GCUCCUACAUAAAACAAGA
UCUUGUUUUAUGUAGGAGC
1875
2788
Yes
No
No

1876
UCCUACAUAAAACAAGUUA
UAACUUGUUUUAUGUAGGA
1877
2790
Yes
No
No

1878
CUACAUAAAACAAGUUGCA
UGCAACUUGUUUUAUGUAG
1879
2792
Yes
No
No

1880
UACAUAAAACAAGUUGCAA
UUGCAACUUGUUUUAUGUA
1881
2793
Yes
No
No

1882
UAAAACAAGUUGCAUUGAA
UUCAAUGCAACUUGUUUUA
1883
2797
Yes
No
No

1884
AAAACAAGUUGCAUUGAUA
UAUCAAUGCAACUUGUUUU
1885
2798
Yes
No
No

1886
AAACAAGUUGCAUUGAUUA
UAAUCAAUGCAACUUGUUU
1887
2799
Yes
No
No

1888
AACAAGUUGCAUUGAUUAA
UUAAUCAAUGCAACUUGUU
1889
2800
Yes
No
No

1890
ACAAGUUGCAUUGAUUACA
UGUAAUCAAUGCAACUUGU
1891
2801
Yes
No
No

1892
CAAGUUGCAUUGAUUACCA
UGGUAAUCAAUGCAACUUG
1893
2802
Yes
No
No

1894
AGUUGCAUUGAUUACCAUA
UAUGGUAAUCAAUGCAACU
1895
2804
Yes
No
No

1896
GUUGCAUUGAUUACCAUCA
UGAUGGUAAUCAAUGCAAC
1897
2805
Yes
No
No

1898
UUGCAUUGAUUACCAUCAA
UUGAUGGUAAUCAAUGCAA
1899
2806
Yes
No
No

1900
UGCAUUGAUUACCAUCAUA
UAUGAUGGUAAUCAAUGCA
1901
2807
Yes
No
No

1902
CAUUGAUUACCAUCAUGGA
UCCAUGAUGGUAAUCAAUG
1903
2809
Yes
No
No

1904
AUUGAUUACCAUCAUGGCA
UGCCAUGAUGGUAAUCAAU
1905
2810
Yes
No
No

1906
UACCAUCAUGGCUCAGAUA
UAUCUGAGCCAUGAUGGUA
1907
2816
Yes
Yes
No

1908
ACCAUCAUGGCUCAGAUUA
UAAUCUGAGCCAUGAUGGU
1909
2817
Yes
Yes
No

1910
CCAUCAUGGCUCAGAUUGA
UCAAUCUGAGCCAUGAUGG
1911
2818
Yes
Yes
No

1912
CAUCAUGGCUCAGAUUGGA
UCCAAUCUGAGCCAUGAUG
1913
2819
Yes
Yes
No

1914
AUCAUGGCUCAGAUUGGCA
UGCCAAUCUGAGCCAUGAU
1915
2820
Yes
Yes
No

1916
CAUGGCUCAGAUUGGCUCA
UGAGCCAAUCUGAGCCAUG
1917
2822
Yes
Yes
No

1918
CUCAGAUUGGCUCCUAUGA
UCAUAGGAGCCAAUCUGAG
1919
2827
Yes
No
No

1920
CAGAUUGGCUCCUAUGUUA
UAACAUAGGAGCCAAUCUG
1921
2829
Yes
No
No

1922
AUUGGCUCCUAUGUUCCUA
UAGGAACAUAGGAGCCAAU
1923
2832
Yes
No
No

1924
UUGGCUCCUAUGUUCCUGA
UCAGGAACAUAGGAGCCAA
1925
2833
Yes
No
No

1926
GGCUCCUAUGUUCCUGCAA
UUGCAGGAACAUAGGAGCC
1927
2835
Yes
No
No

1928
UCCUAUGUUCCUGCAGAAA
UUUCUGCAGGAACAUAGGA
1929
2838
Yes
No
No

1930
CCUAUGUUCCUGCAGAAGA
UCUUCUGCAGGAACAUAGG
1931
2839
Yes
No
No

1932
CUAUGUUCCUGCAGAAGAA
UUCUUCUGCAGGAACAUAG
1933
2840
Yes
No
No

1934
CUGCAGAAGAAGCGACAAA
UUUGUCGCUUCUUCUGCAG
1935
2848
Yes
No
No

1936
UGCAGAAGAAGCGACAAUA
UAUUGUCGCUUCUUCUGCA
1937
2849
Yes
No
No

1938
GCAGAAGAAGCGACAAUUA
UAAUUGUCGCUUCUUCUGC
1939
2850
Yes
No
No

1940
AGAAGAAGCGACAAUUGGA
UCCAAUUGUCGCUUCUUCU
1941
2852
Yes
No
No

1942
AAGAAGCGACAAUUGGGAA
UUCCCAAUUGUCGCUUCUU
1943
2854
Yes
No
No

1944
AGAAGCGACAAUUGGGAUA
UAUCCCAAUUGUCGCUUCU
1945
2855
Yes
No
No

1946
GAAGCGACAAUUGGGAUUA
UAAUCCCAAUUGUCGCUUC
1947
2856
Yes
No
No

1948
AGCGACAAUUGGGAUUGUA
UACAAUCCCAAUUGUCGCU
1949
2858
Yes
No
No

1950
GCGACAAUUGGGAUUGUGA
UCACAAUCCCAAUUGUCGC
1951
2859
Yes
No
No

1952
CAAUUGGGAUUGUGGAUGA
UCAUCCACAAUCCCAAUUG
1953
2863
Yes
Yes
No

1954
UGGGAUUGUGGAUGGCAUA
UAUGCCAUCCACAAUCCCA
1955
2867
Yes
Yes
No

1956
GGGAUUGUGGAUGGCAUUA
UAAUGCCAUCCACAAUCCC
1957
2868
Yes
Yes
No

1958
GGAUUGUGGAUGGCAUUUA
UAAAUGCCAUCCACAAUCC
1959
2869
Yes
Yes
No

1960
GAUUGUGGAUGGCAUUUUA
UAAAAUGCCAUCCACAAUC
1961
2870
Yes
Yes
No

1962
UGUGGAUGGCAUUUUCACA
UGUGAAAAUGCCAUCCACA
1963
2873
Yes
Yes
No

1964
GUGGAUGGCAUUUUCACAA
UUGUGAAAAUGCCAUCCAC
1965
2874
Yes
Yes
No

1966
GGAUGGCAUUUUCACAAGA
UCUUGUGAAAAUGCCAUCC
1967
2876
Yes
Yes
No

1968
GAUGGCAUUUUCACAAGGA
UCCUUGUGAAAAUGCCAUC
1969
2877
Yes
Yes
No

1970
UGGCAUUUUCACAAGGAUA
UAUCCUUGUGAAAAUGCCA
1971
2879
Yes
Yes
No

1972
GGCAUUUUCACAAGGAUGA
UCAUCCUUGUGAAAAUGCC
1973
2880
Yes
Yes
No

1974
GGAUGGGUGCUGCAGACAA
UUGUCUGCAGCACCCAUCC
1975
2893
Yes
Yes
No

1976
GAUGGGUGCUGCAGACAAA
UUUGUCUGCAGCACCCAUC
1977
2894
Yes
Yes
No

1978
AUGGGUGCUGCAGACAAUA
UAUUGUCUGCAGCACCCAU
1979
2895
Yes
Yes
No

1980
UGGGUGCUGCAGACAAUAA
UUAUUGUCUGCAGCACCCA
1981
2896
Yes
Yes
No

1982
GGGUGCUGCAGACAAUAUA
UAUAUUGUCUGCAGCACCC
1983
2897
Yes
Yes
Yes

1984
GUGCUGCAGACAAUAUAUA
UAUAUAUUGUCUGCAGCAC
1985
2899
Yes
Yes
Yes

1986
GCUGCAGACAAUAUAUAUA
UAUAUAUAUUGUCUGCAGC
1987
2901
Yes
No
No

1988
CUGCAGACAAUAUAUAUAA
UUAUAUAUAUUGUCUGCAG
1989
2902
Yes
No
No

1990
UGCAGACAAUAUAUAUAAA
UUUAUAUAUAUUGUCUGCA
1991
2903
Yes
No
No

1992
GACAAUAUAUAUAAAGGAA
UUCCUUUAUAUAUAUUGUC
1993
2907
Yes
No
No

1994
ACAAUAUAUAUAAAGGACA
UGUCCUUUAUAUAUAUUGU
1995
2908
Yes
No
No

1996
CAAUAUAUAUAAAGGACAA
UUGUCCUUUAUAUAUAUUG
1997
2909
No
No
No

1998
AUAUAUAAAGGACAGAGUA
UACUCUGUCCUUUAUAUAU
1999
2913
No
No
No

2000
UAUAUAAAGGACAGAGUAA
UUACUCUGUCCUUUAUAUA
2001
2914
No
No
No

2002
UAUAAAGGACAGAGUACAA
UUGUACUCUGUCCUUUAUA
2003
2916
No
No
No

2004
AUAAAGGACAGAGUACAUA
UAUGUACUCUGUCCUUUAU
2005
2917
No
No
No

2006
UAAAGGACAGAGUACAUUA
UAAUGUACUCUGUCCUUUA
2007
2918
No
No
No

2008
AAAGGACAGAGUACAUUUA
UAAAUGUACUCUGUCCUUU
2009
2919
No
No
No

2010
CAGAGUACAUUUAUGGAAA
UUUCCAUAAAUGUACUCUG
2011
2925
No
No
No

2012
AGAGUACAUUUAUGGAAGA
UCUUCCAUAAAUGUACUCU
2013
2926
No
No
No

2014
GUACAUUUAUGGAAGAACA
UGUUCUUCCAUAAAUGUAC
2015
2929
Yes
No
No

2016
UACAUUUAUGGAAGAACUA
UAGUUCUUCCAUAAAUGUA
2017
2930
Yes
No
No

2018
AUUUAUGGAAGAACUGACA
UGUCAGUUCUUCCAUAAAU
2019
2933
Yes
No
No

2020
UUUAUGGAAGAACUGACUA
UAGUCAGUUCUUCCAUAAA
2021
2934
Yes
No
No

2022
UUAUGGAAGAACUGACUGA
UCAGUCAGUUCUUCCAUAA
2023
2935
Yes
No
No

2024
GGAAGAACUGACUGACACA
UGUGUCAGUCAGUUCUUCC
2025
2939
Yes
No
No

2026
GAACUGACUGACACAGCAA
UUGCUGUGUCAGUCAGUUC
2027
2943
Yes
No
No

2028
UGACACAGCAGAAAUAAUA
UAUUAUUUCUGCUGUGUCA
2029
2951
Yes
No
No

2030
GACACAGCAGAAAUAAUCA
UGAUUAUUUCUGCUGUGUC
2031
2952
Yes
No
No

2032
CAGAAAAGCAACAUCACAA
UUGUGAUGUUGCUUUUCUG
2033
2969
Yes
No
No

2034
AGAAAAGCAACAUCACAGA
UCUGUGAUGUUGCUUUUCU
2035
2970
Yes
No
No

2036
GCAACAUCACAGUCCUUGA
UCAAGGACUGUGAUGUUGC
2037
2976
Yes
No
No

2038
CAUCACAGUCCUUGGUUAA
UUAACCAAGGACUGUGAUG
2039
2980
Yes
No
No

2040
UCACAGUCCUUGGUUAUCA
UGAUAACCAAGGACUGUGA
2041
2982
Yes
No
No

2042
CACAGUCCUUGGUUAUCUA
UAGAUAACCAAGGACUGUG
2043
2983
Yes
No
No

2044
CAGUCCUUGGUUAUCUUGA
UCAAGAUAACCAAGGACUG
2045
2985
Yes
No
No

2046
UCCUUGGUUAUCUUGGAUA
UAUCCAAGAUAACCAAGGA
2047
2988
Yes
No
No

2048
CCUUGGUUAUCUUGGAUGA
UCAUCCAAGAUAACCAAGG
2049
2989
Yes
No
No

2050
GGUUAUCUUGGAUGAACUA
UAGUUCAUCCAAGAUAACC
2051
2993
No
No
No

2052
GUUAUCUUGGAUGAACUAA
UUAGUUCAUCCAAGAUAAC
2053
2994
No
No
No

2054
AUCUUGGAUGAACUAGGAA
UUCCUAGUUCAUCCAAGAU
2055
2997
No
No
No

2056
UCUUGGAUGAACUAGGAAA
UUUCCUAGUUCAUCCAAGA
2057
2998
No
No
No

2058
CUUGGAUGAACUAGGAAGA
UCUUCCUAGUUCAUCCAAG
2059
2999
No
No
No

2060
UUGGAUGAACUAGGAAGAA
UUCUUCCUAGUUCAUCCAA
2061
3000
No
No
No

2062
AAGAGGGACGAGCACUCAA
UUGAGUGCUCGUCCCUCUU
2063
3014
No
No
No

2064
AGAGGGACGAGCACUCAUA
UAUGAGUGCUCGUCCCUCU
2065
3015
No
No
No

2066
AGGGACGAGCACUCAUGAA
UUCAUGAGUGCUCGUCCCU
2067
3017
No
No
No

2068
GGGACGAGCACUCAUGAUA
UAUCAUGAGUGCUCGUCCC
2069
3018
No
No
No

2070
GGACGAGCACUCAUGAUGA
UCAUCAUGAGUGCUCGUCC
2071
3019
No
No
No

2072
ACGAGCACUCAUGAUGGAA
UUCCAUCAUGAGUGCUCGU
2073
3021
No
No
No

2074
CGAGCACUCAUGAUGGAAA
UUUCCAUCAUGAGUGCUCG
2075
3022
No
No
No

2076
GAGCACUCAUGAUGGAAUA
UAUUCCAUCAUGAGUGCUC
2077
3023
No
No
No

2078
CACUCAUGAUGGAAUUGCA
UGCAAUUCCAUCAUGAGUG
2079
3026
No
No
No

2080
ACUCAUGAUGGAAUUGCCA
UGGCAAUUCCAUCAUGAGU
2081
3027
No
No
No

2082
CUCAUGAUGGAAUUGCCAA
UUGGCAAUUCCAUCAUGAG
2083
3028
No
No
No

2084
UCAUGAUGGAAUUGCCAUA
UAUGGCAAUUCCAUCAUGA
2085
3029
No
No
No

2086
GAAUUGCCAUUGCCUAUGA
UCAUAGGCAAUGGCAAUUC
2087
3037
Yes
No
No

2088
AUUGCCAUUGCCUAUGCUA
UAGCAUAGGCAAUGGCAAU
2089
3039
Yes
No
No

2090
UUGCCAUUGCCUAUGCUAA
UUAGCAUAGGCAAUGGCAA
2091
3040
Yes
No
No

2092
UGCCAUUGCCUAUGCUACA
UGUAGCAUAGGCAAUGGCA
2093
3041
Yes
No
No

2094
CAUUGCCUAUGCUACACUA
UAGUGUAGCAUAGGCAAUG
2095
3044
Yes
No
No

2096
AUUGCCUAUGCUACACUUA
UAAGUGUAGCAUAGGCAAU
2097
3045
Yes
No
No

2098
UUGCCUAUGCUACACUUGA
UCAAGUGUAGCAUAGGCAA
2099
3046
Yes
No
No

2100
GCCUAUGCUACACUUGAGA
UCUCAAGUGUAGCAUAGGC
2101
3048
Yes
No
No

2102
CCUAUGCUACACUUGAGUA
UACUCAAGUGUAGCAUAGG
2103
3049
Yes
No
No

2104
CUAUGCUACACUUGAGUAA
UUACUCAAGUGUAGCAUAG
2105
3050
Yes
No
No

2106
UAUGCUACACUUGAGUAUA
UAUACUCAAGUGUAGCAUA
2107
3051
Yes
No
No

2108
AUGCUACACUUGAGUAUUA
UAAUACUCAAGUGUAGCAU
2109
3052
Yes
No
No

2110
GCUACACUUGAGUAUUUCA
UGAAAUACUCAAGUGUAGC
2111
3054
Yes
No
No

2112
CUACACUUGAGUAUUUCAA
UUGAAAUACUCAAGUGUAG
2113
3055
Yes
No
No

2114
CUUGAGUAUUUCAUCAGAA
UUCUGAUGAAAUACUCAAG
2115
3060
Yes
No
No

2116
UUGAGUAUUUCAUCAGAGA
UCUCUGAUGAAAUACUCAA
2117
3061
Yes
No
No

2118
UGAGUAUUUCAUCAGAGAA
UUCUCUGAUGAAAUACUCA
2119
3062
Yes
No
No

2120
GAGUAUUUCAUCAGAGAUA
UAUCUCUGAUGAAAUACUC
2121
3063
Yes
No
No

2122
UUCAUCAGAGAUGUGAAAA
UUUUCACAUCUCUGAUGAA
2123
3069
Yes
No
No

2124
CAUCAGAGAUGUGAAAUCA
UGAUUUCACAUCUCUGAUG
2125
3071
Yes
No
No

2126
AGAGAUGUGAAAUCCUUAA
UUAAGGAUUUCACAUCUCU
2127
3075
Yes
No
No

2128
GAGAUGUGAAAUCCUUAAA
UUUAAGGAUUUCACAUCUC
2129
3076
Yes
No
No

2130
AGAUGUGAAAUCCUUAACA
UGUUAAGGAUUUCACAUCU
2131
3077
Yes
No
No

2132
GAUGUGAAAUCCUUAACCA
UGGUUAAGGAUUUCACAUC
2133
3078
Yes
No
No

2134
UGUGAAAUCCUUAACCCUA
UAGGGUUAAGGAUUUCACA
2135
3080
Yes
No
No

2136
AUCCUUAACCCUGUUUGUA
UACAAACAGGGUUAAGGAU
2137
3086
Yes
No
No

2138
CCUUAACCCUGUUUGUCAA
UUGACAAACAGGGUUAAGG
2139
3088
Yes
No
No

2140
CUUAACCCUGUUUGUCACA
UGUGACAAACAGGGUUAAG
2141
3089
Yes
No
No

2142
ACCCUGUUUGUCACCCAUA
UAUGGGUGACAAACAGGGU
2143
3093
Yes
No
No

2144
CCCUGUUUGUCACCCAUUA
UAAUGGGUGACAAACAGGG
2145
3094
Yes
No
No

2146
CUGUUUGUCACCCAUUAUA
UAUAAUGGGUGACAAACAG
2147
3096
Yes
No
No

2148
GUUUGUCACCCAUUAUCCA
UGGAUAAUGGGUGACAAAC
2149
3098
Yes
No
No

2150
UUUGUCACCCAUUAUCCGA
UCGGAUAAUGGGUGACAAA
2151
3099
Yes
No
No

2152
CCCAUUAUCCGCCAGUUUA
UAAACUGGCGGAUAAUGGG
2153
3106
Yes
No
No

2154
CCAUUAUCCGCCAGUUUGA
UCAAACUGGCGGAUAAUGG
2155
3107
Yes
No
No

2156
UUAUCCGCCAGUUUGUGAA
UUCACAAACUGGCGGAUAA
2157
3110
Yes
No
No

2158
UAUCCGCCAGUUUGUGAAA
UUUCACAAACUGGCGGAUA
2159
3111
No
No
No

2160
AUCCGCCAGUUUGUGAACA
UGUUCACAAACUGGCGGAU
2161
3112
No
No
No

2162
CCGCCAGUUUGUGAACUAA
UUAGUUCACAAACUGGCGG
2163
3114
No
No
No

2164
CGCCAGUUUGUGAACUAGA
UCUAGUUCACAAACUGGCG
2165
3115
No
No
No

2166
GCCAGUUUGUGAACUAGAA
UUCUAGUUCACAAACUGGC
2167
3116
No
No
No

2168
CCAGUUUGUGAACUAGAAA
UUUCUAGUUCACAAACUGG
2169
3117
No
No
No

2170
CAGUUUGUGAACUAGAAAA
UUUUCUAGUUCACAAACUG
2171
3118
No
No
No

2172
UCACACCAGGUGGGGAAUA
UAUUCCCCACCUGGUGUGA
2173
3144
Yes
No
No

2174
CACACCAGGUGGGGAAUUA
UAAUUCCCCACCUGGUGUG
2175
3145
Yes
No
No

2176
ACACCAGGUGGGGAAUUAA
UUAAUUCCCCACCUGGUGU
2177
3146
Yes
No
No

2178
CACCAGGUGGGGAAUUACA
UGUAAUUCCCCACCUGGUG
2179
3147
Yes
No
No

2180
ACCAGGUGGGGAAUUACCA
UGGUAAUUCCCCACCUGGU
2181
3148
Yes
No
No

2182
CCAGGUGGGGAAUUACCAA
UUGGUAAUUCCCCACCUGG
2183
3149
Yes
No
No

2184
AGGUGGGGAAUUACCACAA
UUGUGGUAAUUCCCCACCU
2185
3151
Yes
No
Yes

2186
GGUGGGGAAUUACCACAUA
UAUGUGGUAAUUCCCCACC
2187
3152
Yes
No
Yes

2188
GUGGGGAAUUACCACAUGA
UCAUGUGGUAAUUCCCCAC
2189
3153
Yes
Yes
Yes

2190
GGGAAUUACCACAUGGGAA
UUCCCAUGUGGUAAUUCCC
2191
3156
Yes
Yes
No

2192
GGAAUUACCACAUGGGAUA
UAUCCCAUGUGGUAAUUCC
2193
3157
Yes
Yes
No

2194
GAAUUACCACAUGGGAUUA
UAAUCCCAUGUGGUAAUUC
2195
3158
Yes
Yes
No

2196
AAUUACCACAUGGGAUUCA
UGAAUCCCAUGUGGUAAUU
2197
3159
Yes
Yes
No

2198
AUUACCACAUGGGAUUCUA
UAGAAUCCCAUGUGGUAAU
2199
3160
Yes
Yes
No

2200
UACCACAUGGGAUUCUUGA
UCAAGAAUCCCAUGUGGUA
2201
3162
Yes
Yes
No

2202
CCACAUGGGAUUCUUGGUA
UACCAAGAAUCCCAUGUGG
2203
3164
Yes
Yes
No

2204
CAUGGGAUUCUUGGUCAGA
UCUGACCAAGAAUCCCAUG
2205
3167
Yes
No
No

2206
GGGAUUCUUGGUCAGUGAA
UUCACUGACCAAGAAUCCC
2207
3170
No
No
No

2208
GGAUUCUUGGUCAGUGAGA
UCUCACUGACCAAGAAUCC
2209
3171
No
No
No

2210
GUCAGUGAGGAUGAAAGCA
UGCUUUCAUCCUCACUGAC
2211
3180
No
No
No

2212
CAGUGAGGAUGAAAGCAAA
UUUGCUUUCAUCCUCACUG
2213
3182
No
No
No

2214
GAUGAAAGCAAACUGGAUA
UAUCCAGUUUGCUUUCAUC
2215
3189
No
No
No

2216
GAAAGCAAACUGGAUCCAA
UUGGAUCCAGUUUGCUUUC
2217
3192
No
No
No

2218
CCAGGCGCAGCAGAACAAA
UUUGUUCUGCUGCGCCUGG
2219
3207
No
No
No

2220
CAGGCGCAGCAGAACAAGA
UCUUGUUCUGCUGCGCCUG
2221
3208
No
No
No

2222
GGCGCAGCAGAACAAGUCA
UGACUUGUUCUGCUGCGCC
2223
3210
No
No
No

2224
GCGCAGCAGAACAAGUCCA
UGGACUUGUUCUGCUGCGC
2225
3211
No
No
No

2226
GCAGCAGAACAAGUCCCUA
UAGGGACUUGUUCUGCUGC
2227
3213
No
No
No

2228
AGAACAAGUCCCUGAUUUA
UAAAUCAGGGACUUGUUCU
2229
3218
Yes
No
No

2230
AACAAGUCCCUGAUUUUGA
UCAAAAUCAGGGACUUGUU
2231
3220
Yes
No
No

2232
GUCCCUGAUUUUGUCACCA
UGGUGACAAAAUCAGGGAC
2233
3225
Yes
No
No

2234
CCCUGAUUUUGUCACCUUA
UAAGGUGACAAAAUCAGGG
2235
3227
Yes
No
No

2236
CCUGAUUUUGUCACCUUCA
UGAAGGUGACAAAAUCAGG
2237
3228
Yes
No
No

2238
UGAUUUUGUCACCUUCCUA
UAGGAAGGUGACAAAAUCA
2239
3230
Yes
No
No

2240
AUUUUGUCACCUUCCUUUA
UAAAGGAAGGUGACAAAAU
2241
3232
Yes
No
No

2242
UUUUGUCACCUUCCUUUAA
UUAAAGGAAGGUGACAAAA
2243
3233
Yes
No
No

2244
UGUCACCUUCCUUUACCAA
UUGGUAAAGGAAGGUGACA
2245
3236
Yes
No
No

2246
UCACCUUCCUUUACCAAAA
UUUUGGUAAAGGAAGGUGA
2247
3238
Yes
No
No

2248
UUCCUUUACCAAAUAACUA
UAGUUAUUUGGUAAAGGAA
2249
3243
Yes
No
No

2250
UCCUUUACCAAAUAACUAA
UUAGUUAUUUGGUAAAGGA
2251
3244
Yes
No
No

2252
CCUUUACCAAAUAACUAGA
UCUAGUUAUUUGGUAAAGG
2253
3245
Yes
No
No

2254
CUUUACCAAAUAACUAGAA
UUCUAGUUAUUUGGUAAAG
2255
3246
Yes
No
No

2256
UACCAAAUAACUAGAGGAA
UUCCUCUAGUUAUUUGGUA
2257
3249
Yes
No
No

2258
CAAAUAACUAGAGGAAUUA
UAAUUCCUCUAGUUAUUUG
2259
3252
Yes
No
No

2260
UAACUAGAGGAAUUGCAGA
UCUGCAAUUCCUCUAGUUA
2261
3256
Yes
No
No

2262
CUAGAGGAAUUGCAGCAAA
UUUGCUGCAAUUCCUCUAG
2263
3259
Yes
No
No

2264
GGAAUUGCAGCAAGGAGUA
UACUCCUUGCUGCAAUUCC
2265
3264
Yes
No
No

2266
GAAUUGCAGCAAGGAGUUA
UAACUCCUUGCUGCAAUUC
2267
3265
Yes
No
No

2268
AUUGCAGCAAGGAGUUAUA
UAUAACUCCUUGCUGCAAU
2269
3267
Yes
No
No

2270
UUGCAGCAAGGAGUUAUGA
UCAUAACUCCUUGCUGCAA
2271
3268
Yes
No
No

2272
GCAAGGAGUUAUGGAUUAA
UUAAUCCAUAACUCCUUGC
2273
3273
Yes
No
No

2274
AAGGAGUUAUGGAUUAAAA
UUUUAAUCCAUAACUCCUU
2275
3275
Yes
No
No

2276
AGGAGUUAUGGAUUAAAUA
UAUUUAAUCCAUAACUCCU
2277
3276
Yes
No
No

2278
GUUAUGGAUUAAAUGUGGA
UCCACAUUUAAUCCAUAAC
2279
3280
Yes
No
No

2280
UUAUGGAUUAAAUGUGGCA
UGCCACAUUUAAUCCAUAA
2281
3281
Yes
No
No

2282
UAUGGAUUAAAUGUGGCUA
UAGCCACAUUUAAUCCAUA
2283
3282
Yes
No
No

2284
AUGGAUUAAAUGUGGCUAA
UUAGCCACAUUUAAUCCAU
2285
3283
Yes
No
No

2286
UGGAUUAAAUGUGGCUAAA
UUUAGCCACAUUUAAUCCA
2287
3284
Yes
No
No

2288
GAUUAAAUGUGGCUAAACA
UGUUUAGCCACAUUUAAUC
2289
3286
Yes
No
No

2290
AUUAAAUGUGGCUAAACUA
UAGUUUAGCCACAUUUAAU
2291
3287
Yes
No
No

2292
UUAAAUGUGGCUAAACUAA
UUAGUUUAGCCACAUUUAA
2293
3288
Yes
No
No

2294
UAAAUGUGGCUAAACUAGA
UCUAGUUUAGCCACAUUUA
2295
3289
Yes
No
No

2296
AAUGUGGCUAAACUAGCAA
UUGCUAGUUUAGCCACAUU
2297
3291
Yes
No
No

2298
AUGUGGCUAAACUAGCAGA
UCUGCUAGUUUAGCCACAU
2299
3292
Yes
No
No

2300
UGUGGCUAAACUAGCAGAA
UUCUGCUAGUUUAGCCACA
2301
3293
Yes
No
No

2302
GUGGCUAAACUAGCAGAUA
UAUCUGCUAGUUUAGCCAC
2303
3294
Yes
No
No

2304
UGGCUAAACUAGCAGAUGA
UCAUCUGCUAGUUUAGCCA
2305
3295
No
No
No

2306
GCUAAACUAGCAGAUGUUA
UAACAUCUGCUAGUUUAGC
2307
3297
No
No
No

2308
CUAAACUAGCAGAUGUUCA
UGAACAUCUGCUAGUUUAG
2309
3298
No
No
No

2310
UAAACUAGCAGAUGUUCCA
UGGAACAUCUGCUAGUUUA
2311
3299
No
No
No

2312
AAACUAGCAGAUGUUCCUA
UAGGAACAUCUGCUAGUUU
2313
3300
No
No
No

2314
CAGAUGUUCCUGGAGAAAA
UUUUCUCCAGGAACAUCUG
2315
3307
No
No
No

2316
AUGUUCCUGGAGAAAUUUA
UAAAUUUCUCCAGGAACAU
2317
3310
No
No
No

2318
AGAAAGCAGCUCACAAGUA
UACUUGUGAGCUGCUUUCU
2319
3331
Yes
No
No

2320
GAAAGCAGCUCACAAGUCA
UGACUUGUGAGCUGCUUUC
2321
3332
Yes
No
No

2322
AAAGCAGCUCACAAGUCAA
UUGACUUGUGAGCUGCUUU
2323
3333
Yes
No
No

2324
AGCUCACAAGUCAAAAGAA
UUCUUUUGACUUGUGAGCU
2325
3338
Yes
No
No

2326
CUCACAAGUCAAAAGAGCA
UGCUCUUUUGACUUGUGAG
2327
3340
Yes
No
No

2328
UCACAAGUCAAAAGAGCUA
UAGCUCUUUUGACUUGUGA
2329
3341
Yes
No
No

2330
GAGCUGGAAGGAUUAAUAA
UUAUUAAUCCUUCCAGCUC
2331
3354
Yes
No
No

2332
GAAGGAUUAAUAAAUACGA
UCGUAUUUAUUAAUCCUUC
2333
3360
Yes
No
No

2334
GGAUUAAUAAAUACGAAAA
UUUUCGUAUUUAUUAAUCC
2335
3363
Yes
No
No

2336
AUUAAUAAAUACGAAAAGA
UCUUUUCGUAUUUAUUAAU
2337
3365
Yes
No
No

2338
AUACGAAAAGAAAGAGACA
UGUCUCUUUCUUUUCGUAU
2339
3373
No
No
No

2340
UACGAAAAGAAAGAGACUA
UAGUCUCUUUCUUUUCGUA
2341
3374
No
No
No

2342
AAAGAAAGAGACUCAAGUA
UACUUGAGUCUCUUUCUUU
2343
3379
No
No
No

2344
AAGAAAGAGACUCAAGUAA
UUACUUGAGUCUCUUUCUU
2345
3380
No
No
No

2346
AGAAAGAGACUCAAGUAUA
UAUACUUGAGUCUCUUUCU
2347
3381
No
No
No

2348
GAAAGAGACUCAAGUAUUA
UAAUACUUGAGUCUCUUUC
2349
3382
No
No
No

2350
AAAGAGACUCAAGUAUUUA
UAAAUACUUGAGUCUCUUU
2351
3383
No
No
No

2352
AGAGACUCAAGUAUUUUGA
UCAAAAUACUUGAGUCUCU
2353
3385
Yes
No
No

2354
AGACUCAAGUAUUUUGCAA
UUGCAAAAUACUUGAGUCU
2355
3387
Yes
No
No

2356
ACUCAAGUAUUUUGCAAAA
UUUUGCAAAAUACUUGAGU
2357
3389
Yes
No
No

2358
CUCAAGUAUUUUGCAAAGA
UCUUUGCAAAAUACUUGAG
2359
3390
Yes
No
No

2360
AAGUAUUUUGCAAAGUUAA
UUAACUUUGCAAAAUACUU
2361
3393
Yes
No
No

2362
GUAUUUUGCAAAGUUAUGA
UCAUAACUUUGCAAAAUAC
2363
3395
Yes
No
No

2364
AUUUUGCAAAGUUAUGGAA
UUCCAUAACUUUGCAAAAU
2365
3397
Yes
No
No

2366
UUUUGCAAAGUUAUGGACA
UGUCCAUAACUUUGCAAAA
2367
3398
Yes
No
No

2368
UUUGCAAAGUUAUGGACGA
UCGUCCAUAACUUUGCAAA
2369
3399
Yes
No
No

2370
UUGCAAAGUUAUGGACGAA
UUCGUCCAUAACUUUGCAA
2371
3400
Yes
No
No

2372
UGCAAAGUUAUGGACGAUA
UAUCGUCCAUAACUUUGCA
2373
3401
Yes
No
No

2374
AAGUUAUGGACGAUGCAUA
UAUGCAUCGUCCAUAACUU
2375
3405
Yes
No
No

2376
AGUUAUGGACGAUGCAUAA
UUAUGCAUCGUCCAUAACU
2377
3406
Yes
No
No

2378
GUUAUGGACGAUGCAUAAA
UUUAUGCAUCGUCCAUAAC
2379
3407
Yes
No
No

2380
UAUGGACGAUGCAUAAUGA
UCAUUAUGCAUCGUCCAUA
2381
3409
Yes
No
No

2382
AUGGACGAUGCAUAAUGCA
UGCAUUAUGCAUCGUCCAU
2383
3410
Yes
No
No

2384
UGGACGAUGCAUAAUGCAA
UUGCAUUAUGCAUCGUCCA
2385
3411
Yes
No
No

2386
GGACGAUGCAUAAUGCACA
UGUGCAUUAUGCAUCGUCC
2387
3412
Yes
No
No

2388
GACGAUGCAUAAUGCACAA
UUGUGCAUUAUGCAUCGUC
2389
3413
Yes
No
No

2390
CGAUGCAUAAUGCACAAGA
UCUUGUGCAUUAUGCAUCG
2391
3415
Yes
No
No

2392
GAUGCAUAAUGCACAAGAA
UUCUUGUGCAUUAUGCAUC
2393
3416
Yes
No
No

2394
AUGCAUAAUGCACAAGACA
UGUCUUGUGCAUUAUGCAU
2395
3417
Yes
No
No

2396
UGCAUAAUGCACAAGACCA
UGGUCUUGUGCAUUAUGCA
2397
3418
Yes
No
No

2398
UGCACAAGACCUGCAGAAA
UUUCUGCAGGUCUUGUGCA
2399
3425
Yes
No
No

2400
GCACAAGACCUGCAGAAGA
UCUUCUGCAGGUCUUGUGC
2401
3426
Yes
No
No

2402
ACAAGACCUGCAGAAGUGA
UCACUUCUGCAGGUCUUGU
2403
3428
Yes
No
No

2404
AGACCUGCAGAAGUGGACA
UGUCCACUUCUGCAGGUCU
2405
3431
Yes
No
No

2406
GACCUGCAGAAGUGGACAA
UUGUCCACUUCUGCAGGUC
2407
3432
Yes
No
No

2408
AAGUGGACAGAGGAGUUCA
UGAACUCCUCUGUCCACUU
2409
3441
Yes
No
No

2410
AGUGGACAGAGGAGUUCAA
UUGAACUCCUCUGUCCACU
2411
3442
No
No
No

2412
GGACAGAGGAGUUCAACAA
UUGUUGAACUCCUCUGUCC
2413
3445
No
No
No

2414
GACAGAGGAGUUCAACAUA
UAUGUUGAACUCCUCUGUC
2415
3446
No
No
No

2416
ACAGAGGAGUUCAACAUGA
UCAUGUUGAACUCCUCUGU
2417
3447
No
No
No

2418
AGAGGAGUUCAACAUGGAA
UUCCAUGUUGAACUCCUCU
2419
3449
No
No
No

2420
GAGGAGUUCAACAUGGAAA
UUUCCAUGUUGAACUCCUC
2421
3450
No
No
No

2422
GGAGUUCAACAUGGAAGAA
UUCUUCCAUGUUGAACUCC
2423
3452
No
No
No

2424
AUGGAAGAAACACAGACUA
UAGUCUGUGUUUCUUCCAU
2425
3462
Yes
No
No

2426
GAAGAAACACAGACUUCUA
UAGAAGUCUGUGUUUCUUC
2427
3465
Yes
No
No

2428
AAGAAACACAGACUUCUCA
UGAGAAGUCUGUGUUUCUU
2429
3466
Yes
No
No

2430
AGAAACACAGACUUCUCUA
UAGAGAAGUCUGUGUUUCU
2431
3467
Yes
No
No

2432
ACACAGACUUCUCUUCUUA
UAAGAAGAGAAGUCUGUGU
2433
3471
No
No
No

2434
UCUCUUCUUCAUUAAAAUA
UAUUUUAAUGAAGAAGAGA
2435
3480
No
No
No

2436
UUAAAAUGAAGACUACAUA
UAUGUAGUCUUCAUUUUAA
2437
3491
No
No
No

2438
UGAAGACUACAUUUGUGAA
UUCACAAAUGUAGUCUUCA
2439
3497
No
No
No

2440
GAAGACUACAUUUGUGAAA
UUUCACAAAUGUAGUCUUC
2441
3498
No
No
No

2442
AAGACUACAUUUGUGAACA
UGUUCACAAAUGUAGUCUU
2443
3499
No
No
No

2444
AGACUACAUUUGUGAACAA
UUGUUCACAAAUGUAGUCU
2445
3500
No
No
No

2446
AAAAUACCAACUGUACAAA
UUUGUACAGUUGGUAUUUU
2447
3533
Yes
No
No

2448
CAACUGUACAAAAUAACUA
UAGUUAUUUUGUACAGUUG
2449
3540
Yes
No
No

2450
ACUGUACAAAAUAACUCUA
UAGAGUUAUUUUGUACAGU
2451
3542
Yes
No
No

2452
UGUACAAAAUAACUCUCCA
UGGAGAGUUAUUUUGUACA
2453
3544
Yes
No
No

2454
CAAAAUAACUCUCCAGUAA
UUACUGGAGAGUUAUUUUG
2455
3548
Yes
No
No

2456
AAAAUAACUCUCCAGUAAA
UUUACUGGAGAGUUAUUUU
2457
3549
Yes
No
No

2458
AAAUAACUCUCCAGUAACA
UGUUACUGGAGAGUUAUUU
2459
3550
Yes
No
No

2460
AAUAACUCUCCAGUAACAA
UUGUUACUGGAGAGUUAUU
2461
3551
Yes
No
No

2462
AUAACUCUCCAGUAACAGA
UCUGUUACUGGAGAGUUAU
2463
3552
Yes
No
No

2464
UAACUCUCCAGUAACAGCA
UGCUGUUACUGGAGAGUUA
2465
3553
Yes
No
No

2466
AACUCUCCAGUAACAGCCA
UGGCUGUUACUGGAGAGUU
2467
3554
Yes
No
No

2468
CUCUCCAGUAACAGCCUAA
UUAGGCUGUUACUGGAGAG
2469
3556
Yes
No
No

2470
CUCCAGUAACAGCCUAUCA
UGAUAGGCUGUUACUGGAG
2471
3558
No
No
No

2472
CCAGUAACAGCCUAUCUUA
UAAGAUAGGCUGUUACUGG
2473
3560
No
No
No

2474
CAGUAACAGCCUAUCUUUA
UAAAGAUAGGCUGUUACUG
2475
3561
No
No
No

2476
AGUAACAGCCUAUCUUUGA
UCAAAGAUAGGCUGUUACU
2477
3562
No
No
No

2478
GUAACAGCCUAUCUUUGUA
UACAAAGAUAGGCUGUUAC
2479
3563
No
No
No

2480
UAACAGCCUAUCUUUGUGA
UCACAAAGAUAGGCUGUUA
2481
3564
No
No
No

2482
ACAGCCUAUCUUUGUGUGA
UCACACAAAGAUAGGCUGU
2483
3566
No
No
No

2484
CAGCCUAUCUUUGUGUGAA
UUCACACAAAGAUAGGCUG
2485
3567
No
No
No

2486
AGCCUAUCUUUGUGUGACA
UGUCACACAAAGAUAGGCU
2487
3568
No
No
No

2488
GCCUAUCUUUGUGUGACAA
UUGUCACACAAAGAUAGGC
2489
3569
No
No
No

2490
UAUCUUUGUGUGACAUGUA
UACAUGUCACACAAAGAUA
2491
3572
No
No
No

2492
AUCUUUGUGUGACAUGUGA
UCACAUGUCACACAAAGAU
2493
3573
No
No
No

2494
UCUUUGUGUGACAUGUGAA
UUCACAUGUCACACAAAGA
2495
3574
No
No
No

2496
CUUUGUGUGACAUGUGAGA
UCUCACAUGUCACACAAAG
2497
3575
No
No
No

2498
UGUGACAUGUGAGCAUAAA
UUUAUGCUCACAUGUCACA
2499
3580
Yes
No
No

2500
GACAUGUGAGCAUAAAAUA
UAUUUUAUGCUCACAUGUC
2501
3583
Yes
No
No

2502
ACAUGUGAGCAUAAAAUUA
UAAUUUUAUGCUCACAUGU
2503
3584
Yes
No
No

2504
CAUGUGAGCAUAAAAUUAA
UUAAUUUUAUGCUCACAUG
2505
3585
Yes
No
No

2506
UGUGAGCAUAAAAUUAUGA
UCAUAAUUUUAUGCUCACA
2507
3587
Yes
No
No

2508
GUGAGCAUAAAAUUAUGAA
UUCAUAAUUUUAUGCUCAC
2509
3588
Yes
No
No

2510
UGAGCAUAAAAUUAUGACA
UGUCAUAAUUUUAUGCUCA
2511
3589
Yes
No
No

2512
UAAAAUUAUGACCAUGGUA
UACCAUGGUCAUAAUUUUA
2513
3595
Yes
No
No

2514
AAAAUUAUGACCAUGGUAA
UUACCAUGGUCAUAAUUUU
2515
3596
Yes
No
No

2516
AAUUAUGACCAUGGUAUAA
UUAUACCAUGGUCAUAAUU
2517
3598
Yes
No
No

2518
AUUAUGACCAUGGUAUAUA
UAUAUACCAUGGUCAUAAU
2519
3599
Yes
No
No

2520
UGACCAUGGUAUAUUCCUA
UAGGAAUAUACCAUGGUCA
2521
3603
Yes
No
No

2522
GACCAUGGUAUAUUCCUAA
UUAGGAAUAUACCAUGGUC
2523
3604
Yes
No
No

2524
ACCAUGGUAUAUUCCUAUA
UAUAGGAAUAUACCAUGGU
2525
3605
No
No
No

2526
AUGGUAUAUUCCUAUUGGA
UCCAAUAGGAAUAUACCAU
2527
3608
No
No
No

2528
UGGUAUAUUCCUAUUGGAA
UUCCAAUAGGAAUAUACCA
2529
3609
No
No
No

2530
GGUAUAUUCCUAUUGGAAA
UUUCCAAUAGGAAUAUACC
2531
3610
No
No
No

2532
AUUCCUAUUGGAAACAGAA
UUCUGUUUCCAAUAGGAAU
2533
3615
No
No
No

2534
UUCCUAUUGGAAACAGAGA
UCUCUGUUUCCAAUAGGAA
2535
3616
No
No
No

2536
UCCUAUUGGAAACAGAGAA
UUCUCUGUUUCCAAUAGGA
2537
3617
No
No
No

2538
CCUAUUGGAAACAGAGAGA
UCUCUCUGUUUCCAAUAGG
2539
3618
No
No
No

2540
AUUGGAAACAGAGAGGUUA
UAACCUCUCUGUUUCCAAU
2541
3621
No
No
No

2542
UUGGAAACAGAGAGGUUUA
UAAACCUCUCUGUUUCCAA
2543
3622
No
No
No

2544
UGGAAACAGAGAGGUUUUA
UAAAACCUCUCUGUUUCCA
2545
3623
Yes
No
No

2546
GUUUCUGUCUUCCUAACUA
UAGUUAGGAAGACAGAAAC
2547
3662
No
No
No

2548
UCUGUCUUCCUAACUUUUA
UAAAAGUUAGGAAGACAGA
2549
3665
No
No
No

2550
CUGUCUUCCUAACUUUUCA
UGAAAAGUUAGGAAGACAG
2551
3666
Yes
No
No

2552
GUCUUCCUAACUUUUCUAA
UUAGAAAAGUUAGGAAGAC
2553
3668
Yes
No
No

2554
UCUUCCUAACUUUUCUACA
UGUAGAAAAGUUAGGAAGA
2555
3669
No
No
No

2556
CCUAACUUUUCUACGUAUA
UAUACGUAGAAAAGUUAGG
2557
3673
No
No
No

2558
CUAACUUUUCUACGUAUAA
UUAUACGUAGAAAAGUUAG
2559
3674
No
No
No

2560
UAACUUUUCUACGUAUAAA
UUUAUACGUAGAAAAGUUA
2561
3675
No
No
No

2562
AACUUUUCUACGUAUAAAA
UUUUAUACGUAGAAAAGUU
2563
3676
No
No
No

2564
ACUUUUCUACGUAUAAACA
UGUUUAUACGUAGAAAAGU
2565
3677
No
No
No

2566
UUUUCUACGUAUAAACACA
UGUGUUUAUACGUAGAAAA
2567
3679
No
No
No

2568
UUUCUACGUAUAAACACUA
UAGUGUUUAUACGUAGAAA
2569
3680
No
No
No

2570
UCUACGUAUAAACACUCUA
UAGAGUGUUUAUACGUAGA
2571
3682
No
No
No

2572
UACGUAUAAACACUCUUGA
UCAAGAGUGUUUAUACGUA
2573
3684
No
No
No

2574
ACGUAUAAACACUCUUGAA
UUCAAGAGUGUUUAUACGU
2575
3685
No
No
No

2576
CGUAUAAACACUCUUGAAA
UUUCAAGAGUGUUUAUACG
2577
3686
No
No
No

2578
AUAAACACUCUUGAAUAGA
UCUAUUCAAGAGUGUUUAU
2579
3689
Yes
No
No

2580
UAAACACUCUUGAAUAGAA
UUCUAUUCAAGAGUGUUUA
2581
3690
Yes
No
No

2582
ACACUCUUGAAUAGACUUA
UAAGUCUAUUCAAGAGUGU
2583
3693
Yes
No
No

2584
ACUCUUGAAUAGACUUCCA
UGGAAGUCUAUUCAAGAGU
2585
3695
Yes
No
No

2586
CUCUUGAAUAGACUUCCAA
UUGGAAGUCUAUUCAAGAG
2587
3696
Yes
No
No

2588
GAAUAGACUUCCACUUUGA
UCAAAGUGGAAGUCUAUUC
2589
3701
Yes
No
No

2590
AAUAGACUUCCACUUUGUA
UACAAAGUGGAAGUCUAUU
2591
3702
Yes
No
No

2592
AUAGACUUCCACUUUGUAA
UUACAAAGUGGAAGUCUAU
2593
3703
Yes
No
No

2594
UAGACUUCCACUUUGUAAA
UUUACAAAGUGGAAGUCUA
2595
3704
Yes
No
No

2596
AGACUUCCACUUUGUAAUA
UAUUACAAAGUGGAAGUCU
2597
3705
Yes
No
No

2598
GACUUCCACUUUGUAAUUA
UAAUUACAAAGUGGAAGUC
2599
3706
Yes
No
No

2600
ACUUCCACUUUGUAAUUAA
UUAAUUACAAAGUGGAAGU
2601
3707
Yes
No
No

2602
CUUCCACUUUGUAAUUAGA
UCUAAUUACAAAGUGGAAG
2603
3708
Yes
No
No

2604
UCCACUUUGUAAUUAGAAA
UUUCUAAUUACAAAGUGGA
2605
3710
Yes
No
No

2606
ACUUUGUAAUUAGAAAAUA
UAUUUUCUAAUUACAAAGU
2607
3713
Yes
No
No

2608
AGAAAAUUUUAUGGACAGA
UCUGUCCAUAAAAUUUUCU
2609
3724
No
No
No

2610
GAAAAUUUUAUGGACAGUA
UACUGUCCAUAAAAUUUUC
2611
3725
No
No
No

2612
AAUUUUAUGGACAGUAAGA
UCUUACUGUCCAUAAAAUU
2613
3728
No
No
No

2614
AUUUUAUGGACAGUAAGUA
UACUUACUGUCCAUAAAAU
2615
3729
No
No
No

2616
UUUAUGGACAGUAAGUCCA
UGGACUUACUGUCCAUAAA
2617
3731
No
No
No

2618
UUAUGGACAGUAAGUCCAA
UUGGACUUACUGUCCAUAA
2619
3732
No
No
No

2620
UGGACAGUAAGUCCAGUAA
UUACUGGACUUACUGUCCA
2621
3735
Yes
No
No

2622
GACAGUAAGUCCAGUAAAA
UUUUACUGGACUUACUGUC
2623
3737
Yes
No
No

2624
ACAGUAAGUCCAGUAAAGA
UCUUUACUGGACUUACUGU
2625
3738
Yes
No
No

2626
CAGUAAGUCCAGUAAAGCA
UGCUUUACUGGACUUACUG
2627
3739
Yes
No
No

2628
AGUAAGUCCAGUAAAGCCA
UGGCUUUACUGGACUUACU
2629
3740
Yes
No
No

2630
GUAAGUCCAGUAAAGCCUA
UAGGCUUUACUGGACUUAC
2631
3741
Yes
No
No

2632
UAAGUCCAGUAAAGCCUUA
UAAGGCUUUACUGGACUUA
2633
3742
Yes
No
No

2634
AAGUCCAGUAAAGCCUUAA
UUAAGGCUUUACUGGACUU
2635
3743
Yes
No
No

2636
AGUCCAGUAAAGCCUUAAA
UUUAAGGCUUUACUGGACU
2637
3744
Yes
No
No

2638
GUCCAGUAAAGCCUUAAGA
UCUUAAGGCUUUACUGGAC
2639
3745
Yes
No
No

2640
UCCAGUAAAGCCUUAAGUA
UACUUAAGGCUUUACUGGA
2641
3746
Yes
No
No

2642
CCAGUAAAGCCUUAAGUGA
UCACUUAAGGCUUUACUGG
2643
3747
Yes
No
No

2644
CAGUAAAGCCUUAAGUGGA
UCCACUUAAGGCUUUACUG
2645
3748
Yes
No
No

2646
GUAAAGCCUUAAGUGGCAA
UUGCCACUUAAGGCUUUAC
2647
3750
Yes
No
No

2648
AAGCCUUAAGUGGCAGAAA
UUUCUGCCACUUAAGGCUU
2649
3753
Yes
No
No

2650
AGCCUUAAGUGGCAGAAUA
UAUUCUGCCACUUAAGGCU
2651
3754
Yes
No
No

2652
CCUUAAGUGGCAGAAUAUA
UAUAUUCUGCCACUUAAGG
2653
3756
Yes
No
No

2654
CUUAAGUGGCAGAAUAUAA
UUAUAUUCUGCCACUUAAG
2655
3757
Yes
No
No

2656
UUAAGUGGCAGAAUAUAAA
UUUAUAUUCUGCCACUUAA
2657
3758
Yes
No
No

2658
UAAGUGGCAGAAUAUAAUA
UAUUAUAUUCUGCCACUUA
2659
3759
Yes
No
No

2660
GUGGCAGAAUAUAAUUCCA
UGGAAUUAUAUUCUGCCAC
2661
3762
Yes
No
No

2662
UGGCAGAAUAUAAUUCCCA
UGGGAAUUAUAUUCUGCCA
2663
3763
Yes
No
No

2664
GCAGAAUAUAAUUCCCAAA
UUUGGGAAUUAUAUUCUGC
2665
3765
Yes
No
No

2666
AAUAUAAUUCCCAAGCUUA
UAAGCUUGGGAAUUAUAUU
2667
3769
Yes
No
No

2668
UAUAAUUCCCAAGCUUUUA
UAAAAGCUUGGGAAUUAUA
2669
3771
Yes
No
No

2670
AUAAUUCCCAAGCUUUUGA
UCAAAAGCUUGGGAAUUAU
2671
3772
Yes
No
No

2672
UAAUUCCCAAGCUUUUGGA
UCCAAAAGCUUGGGAAUUA
2673
3773
Yes
No
No

2674
AAUUCCCAAGCUUUUGGAA
UUCCAAAAGCUUGGGAAUU
2675
3774
Yes
No
No

2676
AUUCCCAAGCUUUUGGAGA
UCUCCAAAAGCUUGGGAAU
2677
3775
Yes
No
No

2678
AAGCUUUUGGAGGGUGAUA
UAUCACCCUCCAAAAGCUU
2679
3781
Yes
No
No

2680
AGCUUUUGGAGGGUGAUAA
UUAUCACCCUCCAAAAGCU
2681
3782
Yes
No
No

2682
GCUUUUGGAGGGUGAUAUA
UAUAUCACCCUCCAAAAGC
2683
3783
Yes
No
No

2684
CUUUUGGAGGGUGAUAUAA
UUAUAUCACCCUCCAAAAG
2685
3784
Yes
No
No

2686
UUUUGGAGGGUGAUAUAAA
UUUAUAUCACCCUCCAAAA
2687
3785
Yes
No
No

2688
UUUGUUUCAGUUCAGAUAA
UUAUCUGAACUGAAACAAA
2689
3823
Yes
No
No

2690
UCAGUUCAGAUAAUUGGCA
UGCCAAUUAUCUGAACUGA
2691
3829
Yes
No
No

2692
UUGGCAACUGGGUGAAUCA
UGAUUCACCCAGUUGCCAA
2693
3842
No
No
No

2694
GGCAACUGGGUGAAUCUGA
UCAGAUUCACCCAGUUGCC
2695
3844
No
No
No

2696
GCAACUGGGUGAAUCUGGA
UCCAGAUUCACCCAGUUGC
2697
3845
No
No
No

2698
CAACUGGGUGAAUCUGGCA
UGCCAGAUUCACCCAGUUG
2699
3846
No
No
No

2700
GUGAAUCUGGCAGGAAUCA
UGAUUCCUGCCAGAUUCAC
2701
3853
Yes
No
No

2702
UGAAUCUGGCAGGAAUCUA
UAGAUUCCUGCCAGAUUCA
2703
3854
Yes
No
No

2704
GAAUCUGGCAGGAAUCUAA
UUAGAUUCCUGCCAGAUUC
2705
3855
No
No
No

2706
AAUCUGGCAGGAAUCUAUA
UAUAGAUUCCUGCCAGAUU
2707
3856
No
No
No

2708
AUCUGGCAGGAAUCUAUCA
UGAUAGAUUCCUGCCAGAU
2709
3857
No
No
No

2710
CUGGCAGGAAUCUAUCCAA
UUGGAUAGAUUCCUGCCAG
2711
3859
No
No
No

2712
UGGCAGGAAUCUAUCCAUA
UAUGGAUAGAUUCCUGCCA
2713
3860
No
No
No

2714
GGCAGGAAUCUAUCCAUUA
UAAUGGAUAGAUUCCUGCC
2715
3861
No
No
No

2716
GCAGGAAUCUAUCCAUUGA
UCAAUGGAUAGAUUCCUGC
2717
3862
No
No
No

2718
CAGGAAUCUAUCCAUUGAA
UUCAAUGGAUAGAUUCCUG
2719
3863
No
No
No

2720
AGGAAUCUAUCCAUUGAAA
UUUCAAUGGAUAGAUUCCU
2721
3864
No
No
No

2722
GGAAUCUAUCCAUUGAACA
UGUUCAAUGGAUAGAUUCC
2723
3865
No
No
No

2724
GAAUCUAUCCAUUGAACUA
UAGUUCAAUGGAUAGAUUC
2725
3866
No
No
No

2726
AAUCUAUCCAUUGAACUAA
UUAGUUCAAUGGAUAGAUU
2727
3867
No
No
No

2728
AUCUAUCCAUUGAACUAAA
UUUAGUUCAAUGGAUAGAU
2729
3868
No
No
No

2730
UCUAUCCAUUGAACUAAAA
UUUUAGUUCAAUGGAUAGA
2731
3869
No
No
No

2732
UAUCCAUUGAACUAAAAUA
UAUUUUAGUUCAAUGGAUA
2733
3871
No
No
No

2734
CCAUUGAACUAAAAUAAUA
UAUUAUUUUAGUUCAAUGG
2735
3874
No
No
No

2736
UGAACUAAAAUAAUUUUAA
UUAAAAUUAUUUUAGUUCA
2737
3878
Yes
No
No

2738
ACUAAAAUAAUUUUAUUAA
UUAAUAAAAUUAUUUUAGU
2739
3881
Yes
No
No

2740
UUUAUUAUGCAACCAGUUA
UAACUGGUUGCAUAAUAAA
2741
3892
No
No
No

2742
UUAUUAUGCAACCAGUUUA
UAAACUGGUUGCAUAAUAA
2743
3893
No
No
No

2744
UAUUAUGCAACCAGUUUAA
UUAAACUGGUUGCAUAAUA
2745
3894
No
No
No

2746
AUUAUGCAACCAGUUUAUA
UAUAAACUGGUUGCAUAAU
2747
3895
No
No
No

2748
UAUGCAACCAGUUUAUCCA
UGGAUAAACUGGUUGCAUA
2749
3897
No
No
No

2750
AUGCAACCAGUUUAUCCAA
UUGGAUAAACUGGUUGCAU
2751
3898
No
No
No

2752
CCAGUUUAUCCACCAAGAA
UUCUUGGUGGAUAAACUGG
2753
3904
Yes
No
No

2754
AGUUUAUCCACCAAGAACA
UGUUCUUGGUGGAUAAACU
2755
3906
Yes
No
No

2756
UUAUCCACCAAGAACAUAA
UUAUGUUCUUGGUGGAUAA
2757
3909
Yes
No
No

2758
UAUCCACCAAGAACAUAAA
UUUAUGUUCUUGGUGGAUA
2759
3910
Yes
No
No

2760
AUCCACCAAGAACAUAAGA
UCUUAUGUUCUUGGUGGAU
2761
3911
Yes
No
No

2762
UCCACCAAGAACAUAAGAA
UUCUUAUGUUCUUGGUGGA
2763
3912
Yes
No
No

2764
CCACCAAGAACAUAAGAAA
UUUCUUAUGUUCUUGGUGG
2765
3913
Yes
No
No

2766
CACCAAGAACAUAAGAAUA
UAUUCUUAUGUUCUUGGUG
2767
3914
Yes
No
No

2768
ACCAAGAACAUAAGAAUUA
UAAUUCUUAUGUUCUUGGU
2769
3915
Yes
No
No

2770
UAAGUAGAAAGAAUUGGCA
UGCCAAUUCUUUCUACUUA
2771
3938
No
No
No

2772
AGUAGAAAGAAUUGGCCAA
UUGGCCAAUUCUUUCUACU
2773
3940
No
No
No

2774
AUAAAGUACAUCUCUACUA
UAGUAGAGAUGUACUUUAU
2775
4079
Yes
No
No

2776
AAUGAGCCGAGAUCACGUA
UACGUGAUCUCGGCUCAUU
2777
4202
No
No
No

2778
GAAAUAGAAUUAUCAAGCA
UGCUUGAUAAUUCUAUUUC
2779
4286
Yes
No
No

2780
AAUAGAAUUAUCAAGCUUA
UAAGCUUGAUAAUUCUAUU
2781
4288
Yes
No
No

2782
UAGAAUUAUCAAGCUUUUA
UAAAAGCUUGAUAAUUCUA
2783
4290
Yes
No
No

2784
AGAAUUAUCAAGCUUUUAA
UUAAAAGCUUGAUAAUUCU
2785
4291
Yes
No
No

2786
GAAUUAUCAAGCUUUUAAA
UUUAAAAGCUUGAUAAUUC
2787
4292
Yes
No
No

2788
AAUUAUCAAGCUUUUAAAA
UUUUAAAAGCUUGAUAAUU
2789
4293
Yes
No
No

2790
UAGAGCACAGAAGGAAUAA
UUAUUCCUUCUGUGCUCUA
2791
4314
No
No
No

2792
GCACAGAAGGAAUAAGGUA
UACCUUAUUCCUUCUGUGC
2793
4318
No
No
No

2794
CACAGAAGGAAUAAGGUCA
UGACCUUAUUCCUUCUGUG
2795
4319
No
No
No

2796
ACAGAAGGAAUAAGGUCAA
UUGACCUUAUUCCUUCUGU
2797
4320
No
No
No

2798
CAGAAGGAAUAAGGUCAUA
UAUGACCUUAUUCCUUCUG
2799
4321
No
No
No

2800
AGAAGGAAUAAGGUCAUGA
UCAUGACCUUAUUCCUUCU
2801
4322
No
No
No

2802
GAAGGAAUAAGGUCAUGAA
UUCAUGACCUUAUUCCUUC
2803
4323
No
No
No

2804
AAGGAAUAAGGUCAUGAAA
UUUCAUGACCUUAUUCCUU
2805
4324
No
No
No

2806
AGGAAUAAGGUCAUGAAAA
UUUUCAUGACCUUAUUCCU
2807
4325
No
No
No

2808
GGAAUAAGGUCAUGAAAUA
UAUUUCAUGACCUUAUUCC
2809
4326
No
No
No

2810
AAUAAGGUCAUGAAAUUUA
UAAAUUUCAUGACCUUAUU
2811
4328
Yes
No
No

2812
AUAAGGUCAUGAAAUUUAA
UUAAAUUUCAUGACCUUAU
2813
4329
Yes
No
No

2814
GAAAUUUAAAAGGUUAAAA
UUUUAACCUUUUAAAUUUC
2815
4339
No
No
No

2816
GGUUAAAUAUUGUCAUAGA
UCUAUGACAAUAUUUAACC
2817
4350
No
No
No

2818
UUAAAUAUUGUCAUAGGAA
UUCCUAUGACAAUAUUUAA
2819
4352
No
No
No

2820
AAUAUUGUCAUAGGAUUAA
UUAAUCCUAUGACAAUAUU
2821
4355
No
No
No

2822
UAUUGUCAUAGGAUUAAGA
UCUUAAUCCUAUGACAAUA
2823
4357
No
No
No

2824
UUGUCAUAGGAUUAAGCAA
UUGCUUAAUCCUAUGACAA
2825
4359
No
No
No

2826
GUCAUAGGAUUAAGCAGUA
UACUGCUUAAUCCUAUGAC
2827
4361
No
No
No

2828
UAGGAUUAAGCAGUUUAAA
UUUAAACUGCUUAAUCCUA
2829
4365
No
No
No

2830
UUAAGCAGUUUAAAGAUUA
UAAUCUUUAAACUGCUUAA
2831
4370
No
No
No

2832
AGCAGUUUAAAGAUUGUUA
UAACAAUCUUUAAACUGCU
2833
4373
No
No
No

2834
GCAGUUUAAAGAUUGUUGA
UCAACAAUCUUUAAACUGC
2835
4374
No
No
No

2836
CAGUUUAAAGAUUGUUGGA
UCCAACAAUCUUUAAACUG
2837
4375
No
No
No

2838
GUUUAAAGAUUGUUGGAUA
UAUCCAACAAUCUUUAAAC
2839
4377
No
No
No

2840
UUAAAGAUUGUUGGAUGAA
UUCAUCCAACAAUCUUUAA
2841
4379
No
No
No

2842
GAUUGUUGGAUGAAAUUAA
UUAAUUUCAUCCAACAAUC
2843
4384
No
No
No

2844
UUGGAUGAAAUUAUUUGUA
UACAAAUAAUUUCAUCCAA
2845
4389
Yes
No
No

2846
GAUGAAAUUAUUUGUCAUA
UAUGACAAAUAAUUUCAUC
2847
4392
Yes
No
No

2848
UAUUUGUCAUUCAUUCAAA
UUUGAAUGAAUGACAAAUA
2849
4400
No
No
No

2850
UGUCAUUCAUUCAAGUAAA
UUUACUUGAAUGAAUGACA
2851
4404
No
No
No

2852
GUCAUUCAUUCAAGUAAUA
UAUUACUUGAAUGAAUGAC
2853
4405
No
No
No

2854
AUUCAUUCAAGUAAUAAAA
UUUUAUUACUUGAAUGAAU
2855
4408
No
No
No

2856
UUCAUUCAAGUAAUAAAUA
UAUUUAUUACUUGAAUGAA
2857
4409
No
No
No

2858
UCAAGUAAUAAAUAUUUAA
UUAAAUAUUUAUUACUUGA
2859
4414
No
No
No

2860
CAAGUAAUAAAUAUUUAAA
UUUAAAUAUUUAUUACUUG
2861
4415
No
No
No

2862
AGUAAUAAAUAUUUAAUGA
UCAUUAAAUAUUUAUUACU
2863
4417
Yes
No
No

2864
GUAAUAAAUAUUUAAUGAA
UUCAUUAAAUAUUUAUUAC
2865
4418
Yes
No
No

2866
AAAUAUUUAAUGAAUACUA
UAGUAUUCAUUAAAUAUUU
2867
4423
Yes
No
No

2868
UAAUGAAUACUUGCUAUAA
UUAUAGCAAGUAUUCAUUA
2869
4430
Yes
No
No

2870
AAUGAAUACUUGCUAUAAA
UUUAUAGCAAGUAUUCAUU
2871
4431
No
No
No

2872
AUGAAUACUUGCUAUAAAA
UUUUAUAGCAAGUAUUCAU
2873
4432
No
No
No

TABLE 4

Sense strands with cross-species compatibility with Human and Cyno MSH3

SENSE STRAND SEQ ID NOS/SENSE OLIGO NOS

30, 32, 34, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 102, 104, 106, 108,

110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,

148, 150, 152, 154, 156, 158, 160, 162, 164, 190, 192, 234, 236, 238, 240, 242, 244, 246, 248,

250, 252, 254, 256, 258, 260, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326,

328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,

366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402,

404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440,

442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478,

480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516,

518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554,

556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 608, 610, 612, 614,

616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652,

654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690,

692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728,

730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 754, 756, 758, 760, 762, 776, 778, 780, 782,

784, 786, 788, 790, 792, 794, 796, 798, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840,

842, 844, 846, 848, 850, 852, 854, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896,

898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934,

936, 938, 940, 942, 944, 946, 948, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982,

984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014,

1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044,

1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074,

1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104,

1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134,

1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1182, 1184, 1186, 1188, 1190, 1192,

1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222,

1224, 1226, 1228, 1230, 1232, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268,

1270, 1272, 1294, 1296, 1298, 1300, 1302, 1304, 1306, 1308, 1310, 1312, 1314, 1316, 1318,

1362, 1364, 1366, 1368, 1370, 1372, 1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390,

1392, 1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420,

1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450,

1452, 1454, 1456, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514,

1516, 1518, 1520, 1522, 1524, 1526, 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, 1620, 1622, 1624, 1626, 1630, 1632, 1634, 1636,

1638, 1640, 1642, 1644, 1646, 1648, 1650, 1652, 1654, 1656, 1658, 1660, 1662, 1664, 1666,

1668, 1670, 1672, 1674, 1676, 1678, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1706, 1708,

1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1750, 1752, 1754, 1756,

1758, 1760, 1762, 1764, 1766, 1768, 1770, 1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800,

1802, 1826, 1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1858, 1860, 1862, 1864, 1866,

1868, 1870, 1872, 1874, 1876, 1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896,

1898, 1900, 1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926,

1928, 1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944, 1946, 1948, 1950, 1952, 1954, 1956,

1958, 1960, 1962, 1964, 1966, 1968, 1970, 1972, 1974, 1976, 1978, 1980, 1982, 1984, 1986,

1988, 1990, 1992, 1994, 2014, 2016, 2018, 2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034,

2036, 2038, 2040, 2042, 2044, 2046, 2048, 2086, 2088, 2090, 2092, 2094, 2096, 2098, 2100,

2102, 2104, 2106, 2108, 2110, 2112, 2114, 2116, 2118, 2120, 2122, 2124, 2126, 2128, 2130,

2132, 2134, 2136, 2138, 2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2172, 2174,

2176, 2178, 2180, 2182, 2184, 2186, 2188, 2190, 2192, 2194, 2196, 2198, 2200, 2202, 2204,

2214, 2216, 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, 2318, 2320, 2322, 2324, 2326,

2328, 2330, 2332, 2334, 2336, 2352, 2354, 2356, 2358, 2360, 2362, 2364, 2366, 2368, 2370,

2372, 2374, 2376, 2378, 2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400,

2402, 2404, 2406, 2408, 2424, 2426, 2428, 2430, 2446, 2448, 2450, 2452, 2454, 2456, 2458,

2460, 2462, 2464, 2466, 2468, 2498, 2500, 2502, 2504, 2506, 2508, 2510, 2512, 2514, 2516,

2518, 2520, 2522, 2544, 2550, 2552, 2578, 2580, 2582, 2584, 2586, 2588, 2590, 2592, 2594,

2596, 2598, 2600, 2602, 2604, 2606, 2620, 2622, 2624, 2626, 2628, 2630, 2632, 2634, 2636,

2638, 2640, 2642, 2644, 2646, 2648, 2650, 2652, 2654, 2656, 2658, 2660, 2662, 2664, 2666,

2668, 2670, 2672, 2674, 2676, 2678, 2680, 2682, 2684, 2686, 2688, 2690, 2700, 2702, 2736,

2738, 2752, 2754, 2756, 2758, 2760, 2762, 2764, 2766, 2768, 2774, 2778, 2780, 2782, 2784,

2786, 2788, 2810, 2812, 2844, 2846, 2862, 2864, 2866, 2868,

TABLE 5

Sense strands with cross-species

compatibility with Human and Mouse MSH3

SENSE STRAND SEQ ID NOS/SENSE OLIGO NOS

384, 492, 494, 496, 498, 500, 502, 504, 506, 832, 1052, 1084, 1086,

1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1138, 1140,

1266, 1268, 1270, 1450, 1452, 1584, 1586, 1588, 1590, 1592, 1594,

1630, 1632, 1634, 1636, 1638, 1640, 1642, 1702, 1704, 1706, 1724,

1906, 1908, 1910, 1912, 1914, 1916, 1952, 1954, 1956, 1958, 1960,

1962, 1964, 1966, 1968, 1970, 1972, 1974, 1976, 1978, 1980, 1982,

1984, 2188, 2190, 2192, 2194, 2196, 2198, 2200, 2202

TABLE 6

Sense strands with cross-species

compatibility with Human and Rat MSH3

SENSE STRAND SEQ ID NOS/SENSE OLIGO NOS

82, 342, 344, 346, 492, 494, 496, 498, 500, 502, 504, 506, 520, 522,

524, 526, 528, 558, 560, 562, 564, 832, 1024, 1026, 1028, 1030, 1032,

1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1084, 1086,

1088, 1090, 1410, 1450, 1452, 1584, 1586, 1588, 1590, 1592, 1594, 1596,

1598, 1600, 1602, 1630, 1632, 1634, 1636, 1638, 1640, 1642, 1870, 1982,

1984, 2184, 2186, 2188

TABLE 7

Sense strands with cross-species

compatibility with Human, Cyno, and Mouse MSH3

SENSE STRAND SEQ ID NOS/SENSE OLIGO NOS

384, 492, 494, 496, 498, 500, 502, 504, 506, 832, 1052, 1084, 1086,

1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1138, 1140, 1266,

1268, 1270, 1450, 1452, 1584, 1586, 1588, 1590, 1592, 1630, 1632, 1634,

1636, 1638, 1640, 1642, 1702, 1704, 1706, 1724, 1906, 1908, 1910, 1912,

1914, 1916, 1952, 1954, 1956, 1958, 1960, 1962, 1964, 1966, 1968, 1970,

1972, 1974, 1976, 1978, 1980, 1982, 1984, 2188, 2190, 2192, 2194, 2196,

2198, 2200, 2202

TABLE 8

Sense strands with cross-species

compatibility with Human, Cyno, and Rat MSH3

SENSE STRAND SEQ ID NOS/SENSE OLIGO NOS

82, 342, 344, 346, 492, 494, 496, 498, 500, 502, 504, 506, 520, 522,

524, 526, 528, 558, 560, 562, 564, 832, 1024, 1026, 1028, 1030, 1032,

1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1084, 1086,

1088, 1090, 1410, 1450, 1452, 1584, 1586, 1588, 1590, 1592, 1630, 1632,

1634, 1636, 1638, 1640, 1642, 1870, 1982, 1984, 2184, 2186, 2188

TABLE 9

Sense strands with cross-species

compatibility with Human, Mouse, and Rat MSH3

SENSE STRAND SEQ ID NOS/SENSE OLIGO NOS

492, 494, 496, 498, 500, 502, 504, 506, 832, 1052, 1084, 1086, 1088,

1090, 1450, 1452, 1584, 1586, 1588, 1590, 1592, 1594, 1630, 1632, 1634,

1636, 1638, 1640, 1642, 1982, 1984, 2188

TABLE 10

Sense strands with cross-species

compatibility with Human, Cyno, Mouse, and Rat MSH3

SENSE STRAND SEQ ID NOS/SENSE OLIGO NOS

492, 494, 496, 498, 500, 502, 504, 506, 832, 1052, 1084, 1086, 1088,

1090, 1450, 1452, 1584, 1586, 1588, 1590, 1592, 1630, 1632, 1634, 1636,

1638, 1640, 1642, 1982, 1984, 2188

Example 2. In Vitro Screening of MSH3 Knockdown

Inhibition or knockdown of MSH3 can be demonstrated using a cell-based assay. For example, HEK293, NIH3T3, or Hela or another available mammalian cell line with dsRNA agents targeting MSH3 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 MSH3 mRNA and protein at the different dsRNA levels are compared with a mock oligonucleotide control. The most potent dsRNA agents (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.

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 11 below, the 5′ U of the antisense oligonucleotide can be any nucleotide (e.g., U, A, G, C, T). In some aspects, the 5′ U of the antisense oligonucleotide in Table 11 is U. The sense and antisense oligonucleotides in Table 11 each include a dTdT overhang on the 3′ end.

Additionally, every A and G in each sense oligonucleotide in Table 11 is a ribonucleotide. Every C and U in each sense oligonucleotide is a 2′-O-Methyl ribonucleotide.

Also, every A and G in each antisense oligonucleotide in Table 11 is a ribonucleotide. Every C and U preceding an A in the antisense oligonucleotide is a 2′-O-Methyl ribonucleotide, with one exception: U is the first nucleotide of the antisense strand, and it is a ribonucleotide.

Each nucleotide in the sense and antisense oligonucleotide in Table 11 is linked by a phosphate.

TABLE 11

SEQ ID

NO/

mean %

SENSE

SEQ ID NO/

mRNA

OLIGO

ANTISENSE

remaining

NO
Sense
Antisense
OLIGO NO
Pos
Cyno
Mouse
Rat
0.5 nM
10 nM

118
GUCCAGACAGAAUCUCUGA
UCAGAGAUUCUGUCUGGAC
119
531
Yes
No
No
67.25
44.74

140
GCCAAAAUGUACUGAUUUA
UAAAUCAGUACAUUUUGGC
141
569
Yes
No
No
27.44
23.29

156
AUGAUAUCAGUCUUCUACA
UGUAGAAGACUGAUAUCAU
157
589
Yes
No
No
71.33
45.58

234
AGUCAGUUUGGAUCAUCAA
UUGAUGAUCCAAACUGACU
235
681
Yes
No
No
66.41
40.47

240
UUGGAUCAUCAAAUACAAA
UUUGUAUUUGAUGAUCCAA
241
688
Yes
No
No
66.56
37.87

246
AUACAAGUCAUGAAAAUUA
UAAUUUUCAUGACUUGUAU
247
700
Yes
No
No
66.57
42.32

304
CGCUAGAAUUACAAUACAA
UUGUAUUGUAAUUCUAGCG
305
772
Yes
No
No
67.39
41.15

308
UAGAAUUACAAUACAUAGA
UCUAUGUAUUGUAAUUCUA
309
775
Yes
No
No
65.28
35.66

350
GUGGAAUGUGGAUAUAAGA
UCUUAUAUCCACAUUCCAC
351
828
Yes
No
No
52.23
36.69

364
GAUAUAAGUAUAGAUUCUA
UAGAAUCUAUACUUAUAUC
365
838
Yes
No
No
66.91
35.40

380
AGCCCGAGAGCUCAAUAUA
UAUAUUGAGCUCUCGGGCU
381
878
Yes
No
No
63.05
35.36

382
GCCCGAGAGCUCAAUAUUA
UAAUAUUGAGCUCUCGGGC
383
879
Yes
No
No
40.56
30.67

386
CGAGAGCUCAAUAUUUAUA
UAUAAAUAUUGAGCUCUCG
387
882
Yes
No
No
40.50
26.99

388
GAGAGCUCAAUAUUUAUUA
UAAUAAAUAUUGAGCUCUC
389
883
Yes
No
No
48.94
28.07

396
UUUAUUGCCAUUUAGAUCA
UGAUCUAAAUGGCAAUAAA
397
895
Yes
No
No
45.93
26.42

406
GCCAUUUAGAUCACAACUA
UAGUUGUGAUCUAAAUGGC
407
901
Yes
No
No
36.86
16.73

418
AGAUCACAACUUUAUGACA
UGUCAUAAAGUUGUGAUCU
419
908
Yes
No
No
49.30
30.61

464
CACAGACUGUUUGUUCAUA
UAUGAACAAACAGUCUGUG
465
942
Yes
No
No
48.64
31.30

478
CUGGUGGCAAAAGGAUAUA
UAUAUCCUUUUGCCACCAG
479
969
Yes
No
No
46.40
29.04

520
AUUAAAGGCCAUUGGAGAA
UUCUCCAAUGGCCUUUAAU
521
1022
Yes
No
No
49.09
28.53

540
CAACAGAAGUUCACUCUUA
UAAGAGUGAACUUCUGUUG
541
1040
Yes
No
No
45.65
31.32

564
AAAUUGACUGCCCUUUAUA
UAUAAAGGGCAGUCAAUUU
565
1065
Yes
No
No
52.13
29.00

568
UUGACUGCCCUUUAUACAA
UUGUAUAAAGGGCAGUCAA
569
1068
Yes
No
No
47.91
31.00

618
CUAAUCAAGCUGGAUGAUA
UAUCAUCCAGCUUGAUUAG
619
1119
Yes
No
No
55.64
39.22

660
ACUUCUACCAGCUAUCUUA
UAAGAUAGCUGGUAGAAGU
661
1167
Yes
No
No
59.77
42.59

696
AGGCGAGGUUGUGUUUGAA
UUCAAACACAACCUCGCCU
697
1271
Yes
No
No
71.11
47.44

698
GGCGAGGUUGUGUUUGAUA
UAUCAAACACAACCUCGCC
699
1272
Yes
No
No
66.28
47.74

700
GCGAGGUUGUGUUUGAUAA
UUAUCAAACACAACCUCGC
701
1273
Yes
No
No
65.54
44.44

750
CUUGUCCGAGCAAACAGAA
UUCUGUUUGCUCGGACAAG
751
1373
Yes
No
No
48.42
33.40

770
AGAGCCACAUCUGUUAGUA
UACUAACAGAUGUGGCUCU
771
1404
Yes
No
No
47.87
31.94

822
UGAAUACAGCCAUGCUUUA
UAAAGCAUGGCUGUAUUCA
823
1469
Yes
Yes
Yes
44.77
23.02

830
GCUUUCCAGGCAGUUACAA
UUGUAACUGCCUGGAAAGC
831
1482
Yes
No
No
52.34
32.63

844
GCAGUUACAGAGUUUUAUA
UAUAAAACUCUGUAACUGC
845
1491
Yes
No
No
55.44
33.99

868
ACAGUUGACAUCAAAGGUA
UACCUUUGAUGUCAACUGU
869
1518
Yes
No
No
57.73
34.30

870
CAGUUGACAUCAAAGGUUA
UAACCUUUGAUGUCAACUG
871
1519
Yes
No
No
61.36
32.08

874
GUUGACAUCAAAGGUUCUA
UAGAACCUUUGAUGUCAAC
875
1521
Yes
No
No
60.17
31.68

904
CUGGCAUUGUUAACUUAGA
UCUAAGUUAACAAUGCCAG
905
1549
Yes
No
No
24.78
18.66

972
CUCCAAACCUGAGAAUUUA
UAAAUUCUCAGGUUUGGAG
973
1637
Yes
No
No
27.05
21.76

1004
AUGGAAUUUAUGACAAUUA
UAAUUGUCAUAAAUUCCAU
1005
1674
Yes
No
No
50.63
36.16

1042
ACAGAAUCAGACUGAUAUA
UAUAUCAGUCUGAUUCUGU
1043
1724
Yes
No
No
60.17
33.01

1060
UGCUGUGGGUUUUAGACCA
UGGUCUAAAACCCACAGCA
1061
1759
Yes
No
Vo
51.81
34.96

1062
GCUGUGGGUUUUAGACCAA
UUGGUCUAAAACCCACAGC
1063
1760
Yes
No
No
51.99
31.07

1064
UGGGUUUUAGACCACACUA
UAGUGUGGUCUAAAACCCA
1065
1764
Yes
Yes
Yes
120.87
320.61

1068
GGUUUUAGACCACACUAAA
UUUAGUGUGGUCUAAAACC
1069
1766
Yes
Yes
Yes
65.36
35.69

1090
UUGGGAGACGGAAGUUAAA
UUUAACUUCCGUCUCCCAA
1091
1792
Yes
No
No
57.77
30.99

1096
GACGGAAGUUAAAGAAGUA
UACUUCUUUAACUUCCGUC
1097
1798
Yes
No
No
36.69
31.20

1098
ACGGAAGUUAAAGAAGUGA
UCACUUCUUUAACUUCCGU
1099
1799
Yes
No
No
50.65
30.92

1114
CACUCCUUAAAUUAAGGGA
UCCCUUAAUUUAAGGAGUG
1115
1828
Yes
No
No
54.62
41.66

1116
ACUCCUUAAAUUAAGGGAA
UUCCCUUAAUUUAAGGAGU
1117
1829
Yes
No
No
52.74
37.90

1166
UCCAUUCAGAAUCUAGUGA
UCACUAGAUUCUGAAUGGA
1167
1882
Yes
No
No
56.52
36.88

1168
CCAUUCAGAAUCUAGUGUA
UACACUAGAUUCUGAAUGG
1169
1883
Yes
No
No
55.00
40.41

1170
AUUCAGAAUCUAGUGUGUA
UACACACUAGAUUCUGAAU
1171
1885
Yes
No
No
56.34
36.81

1182
AUCUAGUGUGUUUGGUCAA
UUGACCAAACACACUAGAU
1183
1892
Yes
No
No
49.97
32.53

1192
GUGUUUGGUCAGAUAGAAA
UUUCUAUCUGACCAAACAC
1193
1899
Yes
No
No
55.26
34.88

1212
UAGAAAAUCAUCUACGUAA
UUACGUAGAUGAUUUUCUA
1213
1912
Yes
No
No
48.24
30.58

1214
AGAAAAUCAUCUACGUAAA
UUUACGUAGAUGAUUUUCU
1215
1913
Yes
No
No
54.57
36.51

1216
AAAAUCAUCUACGUAAAUA
UAUUUACGUAGAUGAUUUU
1217
1915
Yes
No
No
53.87
38.28

1222
AUCAUCUACGUAAAUUGCA
UGCAAUUUACGUAGAUGAU
1223
1918
Yes
No
No
47.43
34.83

1244
GGACUCUGUAGCAUUUAUA
UAUAAAUGCUACAGAGUCC
1245
1950
Yes
No
No
50.88
31.70

1258
UACCCAAGAGUUCUUCUUA
UAAGAAGAACUCUUGGGUA
1259
1982
Yes
Yes
No
55.68
47.62

1292
UCACCUAAAGUCAGAAUUA
UAAUUCUGACUUUAGGUGA
1293
2018
Yes
No
No
58.53
46.28

1358
CCGUUAUUUUAGAAAUUCA
UGAAUUUCUAAAAUAACGG
1359
2089
Yes
No
No
44.55
25.12

1360
CGUUAUUUUAGAAAUUCCA
UGGAAUUUCUAAAAUAACG
1361
2090
Yes
No
No
27.08
17.25

1374
GUCCAGUGGAGCAUUACUA
UAGUAAUGCUCCACUGGAC
1375
2119
Yes
No
No
51.19
41.54

1378
AGUGGAGCAUUACUUAAAA
UUUUAAGUAAUGCUCCACU
1379
2123
Yes
No
No
56.98
42.84

1380
GUGGAGCAUUACUUAAAGA
UCUUUAAGUAAUGCUCCAC
1381
2124
Yes
No
No
51.30
42.74

1400
GAACAAGCUGCCAAAGUUA
UAACUUUGGCAGCUUGUUC
1401
2151
Yes
No
No
48.03
34.27

1488
UCCGAAUGCAUUUGCAAGA
UCUUGCAAAUGCAUUCGGA
1489
2257
Yes
No
No
67.83
43.62

1500
CUUCUGCACAAUAUGUGAA
UUCACAUAUUGUGCAGAAG
1501
2299
Yes
No
No
64.86
38.80

1610
AGCUAGUCCUUGACUGCAA
UUGCAGUCAAGGACUAGCU
1611
2473
Yes
Yes
Yes
55.51
29.25

1682
AAAGCAGUGCAUCACCUAA
UUAGGUGAUGCACUGCUUU
1683
2547
Yes
No
No
59.92
38.41

1684
GUGCAUCACCUAGCAACUA
UAGUUGCUAGGUGAUGCAC
1685
2553
Yes
No
No
56.67
38.49

1748
GCAGACCAACUGUACAAGA
UCUUGUACAGUUGGUCUGC
1749
2620
Yes
No
No
56.19
35.33

1782
CUGGGAGAACAGGAUCAAA
UUUGAUCCUGUUCUCCCAG
1783
2691
Yes
No
No
57.75
36.78

1788
GAGAACAGGAUCAAUAUGA
UCAUAUUGAUCCUGUUCUC
1789
2695
Yes
No
No
73.61
43.21

1790
AGAACAGGAUCAAUAUGUA
UACAUAUUGAUCCUGUUCU
1791
2696
Yes
No
No
64.89
39.40

1826
GGACUCAGAGAGAGUAAUA
UAUUACUCUCUCUGAGUCC
1827
2738
Yes
No

70.45
38.70

1866
GAGCUCCUACAUAAAACAA
UUGUUUUAUGUAGGAGCUC
1867
2786
Yes
No
No
55.61
36.30

1868
AGCUCCUACAUAAAACAAA
UUUGUUUUAUGUAGGAGCU
1869
2787
Yes
No
No
59.92
40.71

1870
GCUCCUACAUAAAACAAGA
UCUUGUUUUAUGUAGGAGC
1871
2788
Yes
No
No
28.05
23.06

1882
AAAACAAGUUGCAUUGAUA
UAUCAAUGCAACUUGUUUU
1883
2798
Yes
No
No
57.83
38.63

1892
AGUUGCAUUGAUUACCAUA
UAUGGUAAUCAAUGCAACU
1893
2804
Yes
No
No
49.53
31.05

1926
GCAGAAGAAGCGACAAUUA
UAAUUGUCGCUUCUUCUGC
1927
2850
Yes
No
No
46.34
30.70

1946
GAUUGUGGAUGGCAUUUUA
UAAAAUGCCAUCCACAAUC
1947
2870
Yes
Yes
No
51.01
29.16

1964
GGGUGCUGCAGACAAUAUA
UAUAUUGUCUGCAGCACCC
1965
2897
Yes
Yes
Yes
43.17
37.54

1970
CUGCAGACAAUAUAUAUAA
UUAUAUAUAUUGUCUGCAG
1971
2902
Yes
No
No
49.75
35.31

2022
CAUCACAGUCCUUGGUUAA
UUAACCAAGGACUGUGAUG
2023
2980
Yes
No
No
48.00
32.17

2026
CACAGUCCUUGGUUAUCUA
UAGAUAACCAAGGACUGUG
2027
2983
Yes
No
No
48.28
31.26

2030
CAGUCCUUGGUUAUCUUGA
UCAAGAUAACCAAGGACUG
2031
2985
Yes
No
No
50.46
36.22

2084
UUGCCUAUGCUACACUUGA
UCAAGUGUAGCAUAGGCAA
2085
3046
Yes
No
No
48.14
33.67

2088
CUAUGCUACACUUGAGUAA
UUACUCAAGUGUAGCAUAG
2089
3050
Yes
No
No
41.87
31.74

2090
UAUGCUACACUUGAGUAUA
UAUACUCAAGUGUAGCAUA
2091
3051
Yes
No
No
46.64
33.27

2094
CUACACUUGAGUAUUUCAA
UUGAAAUACUCAAGUGUAG
2095
3055
Yes
No
No
48.56
32.59

2124
UGUGAAAUCCUUAACCCUA
UAGGGUUAAGGAUUUCACA
2125
3080
Yes
No
No
52.19
35.19

2130
AUCCUUAACCCUGUUUGUA
UACAAACAGGGUUAAGGAU
2131
3086
Yes
No
No
52.13
32.29

2146
UUUGUCACCCAUUAUCCGA
UCGGAUAAUGGGUGACAAA
2147
3099
Yes
No
No
49.33
27.75

2194
GAAUUACCACAUGGGAUUA
UAAUCCCAUGUGGUAAUUC
2195
3158
Yes
Yes
No
51.85
35.54

2200
UACCACAUGGGAUUCUUGA
UCAAGAAUCCCAUGUGGUA
2201
3162
Yes
Yes
No
49.91
34.14

2204
CCACAUGGGAUUCUUGGUA
UACCAAGAAUCCCAUGUGG
2205
3164
Yes
Yes
No
49.75
33.43

2264
UCCUUUACCAAAUAACUAA
UUAGUUAUUUGGUAAAGGA
2265
3244
Yes
No
No
49.51
34.46

2290
GCAAGGAGUUAUGGAUUAA
UUAAUCCAUAACUCCUUGC
2291
3273
Yes
No
No
46.01
32.27

2308
UUAAAUGUGGCUAAACUAA
UUAGUUUAGCCACAUUUAA
2309
3288
Yes
No
No
46.98
33.49

2318
GUGGCUAAACUAGCAGAUA
UAUCUGCUAGUUUAGCCAC
2319
3294
Yes
No
No
33.99
28.52

2324
AAAGCAGCUCACAAGUCAA
UUGACUUGUGAGCUGCUUU
2325
3333
Yes
No
No
52.22
31.71

2338
GAAGGAUUAAUAAAUACGA
UCGUAUUUAUUAAUCCUUC
2339
3360
Yes
No
No
28.47
23.20

2386
AGUUAUGGACGAUGCAUAA
UUAUGCAUCGUCCAUAACU
2387
3406
Yes
No
No
63.89
34.90

2388
GUUAUGGACGAUGCAUAAA
UUUAUGCAUCGUCCAUAAC
2389
3407
Yes
No
No
53.12
34.65

2390
GGACGAUGCAUAAUGCACA
UGUGCAUUAUGCAUCGUCC
2391
3412
Yes
No
No
65.37
30.59

2392
GACGAUGCAUAAUGCACAA
UUGUGCAUUAUGCAUCGUC
2393
3413
Yes
No
No
71.34
51.55

2466
ACUGUACAAAAUAACUCUA
UAGAGUUAUUUUGUACAGU
2467
3542
Yes
No
No
73.70
46.89

2606
UAGACUUCCACUUUGUAAA
UUUACAAAGUGGAAGUCUA
2607
3704
Yes
No
No
59.07
40.22

2608
GACUUCCACUUUGUAAUUA
UAAUUACAAAGUGGAAGUC
2609
3706
Yes
No
No
62.67
36.70

2610
ACUUCCACUUUGUAAUUAA
UUAAUUACAAAGUGGAAGU
2611
3707
Yes
No
No
58.14
41.10

2632
GACAGUAAGUCCAGUAAAA
UUUUACUGGACUUACUGUC
2633
3737
Yes
No
No
56.92
35.95

2652
CCAGUAAAGCCUUAAGUGA
UCACUUAAGGCUUUACUGG
2653
3747
Yes
No
No
84.65
66.02

2678
AUAAUUCCCAAGCUUUUGA
UCAAAAGCUUGGGAAUUAU
2679
3772
Yes
No
No
59.06
38.31

2690
CUUUUGGAGGGUGAUAUAA
UUAUAUCACCCUCCAAAAG
2691
3784
Yes
No
No
58.68
35.99

2758
CACCAAGAACAUAAGAAUA
UAUUCUUAUGUUCUUGGUG
2759
3914
Yes
No
No
58.28
36.18

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 dystrophy1 (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 and is compared with controls (mock-transfected and/or control dsRNA at the same concentration as the experimental agent).

Example 4. 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 pg 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).

Example 5. Mouse Studies

Mouse models recapitulating many of the features of trinucleotide repeat expansion diseases including, HD, FA and DM1, are readily available from commercial 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 MSH3 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 R2/6 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 R2/6 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). HD Mice are genotyped using DNA derived from tail snips at weaning and the CAG repeat size is determined. Mice are randomized into groups (n=5/group) at weaning at 4 wks old and dosed with a single ICV injection of either up to a 500 μg dose of dsRNA agents (optionally encapsulated in lipid nanoparticle (LNP)) targeting MSH3 or control dsRNA agents (also optionally encapsulated in LNP). A series of dsRNA agents targeting different regions of MSH3 are tested to identify the most efficacious oligo sequence in vivo. At 12 wks 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, and the extent of CAG repeats compared with control mice. 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 MSH3 plays a role in somatic expansion of the disease allele, hemizygous YG8 FRDA animals are administered ICV with dsRNA agents targeting MSH3 or control dsRNA agents (both optionally encapsulated in LNP) targeting knockdown of MSH3 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 compared in MSH3 and control dsRNA groups as described above in Example 4.

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 MSH3 plays a role in somatic expansion of the disease allele in myotonic dystrophy, DM300-328 transgenic animals are administered ASOs targeting knockdown of MSH3 by either subcutaneous injections (sc), intraperitoneal (ip) or intravenous tail injections (iv). Mice are administered with MSH3 or control dsRNA agents (optionally encapsulated in LNP) 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 Hdh^Q111mouse 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 MSH3 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 MSH3. 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 when compared with controls indicates that the agent that reduces the level and/or activity of MSH3 is capable of decreasing the repeat which are responsible for the toxic and/or defective gene products in Huntington's disease.

Example 6. In Vitro Screening of MSH3 Knockdown

Knockdown of MSH3 in HeLa cells transfected with 10 nM dsRNA is shown in Table 12.

Two different screening protocols were utilized to screen for siRNA duplexes targeting human MSH3 to determine MSH3 knockdown, as described below.

Screening Protocol 1
Human Cell Lines

All human MSH3 targets have been screened in HeLa cells. HeLa cells were obtained from the 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% CO2 in a humidified incubator. For transfection of HeLa cells with siRNAs, cells were seeded at a density of 15,000 cells/well into 96-well tissue culture plates (#655180, GBO, Germany).

PC3 cells were obtained from ATCC (ATCC in partnership with LGC Standards, Wesel, Germany, cat. #ATCC-CRL-1435) and cultured in RPMI 1640 (#FG1215, Biochrom, Berlin, Germany), supplemented to contain 10% fetal calf serum (#1248D, Biochrom GmbH, Berlin, Germany), 25 mM Hepes (#1615, Biochrom, Berlin, Germany), 1×non-essential amino acids (#K0293; Biochrom, Berlin, Germany), 1 mM Na-Pyruvate (#10473; Biochrom, Berlin, Germany) and 100 U/ml Penicillin/100 μg/ml Streptomycin (A2213, Biochrom GmbH, Berlin, Germany) at 37° C. in an atmosphere with 5% CO2 in a humidified incubator. For transfection of PC3 cells with siRNAs, cells were seeded at a density of 15,000 cells/well into 96-well tissue culture plates (#655180, GBO, Germany).

Transfection

In all cell lines used, transfection of siRNA was carried out with Lipofectamine RNAiMax (Invitrogen/Life Technologies, Karlsruhe, Germany) according to the manufacturer's instructions for reverse transfection. The dual dose screen was performed with siRNAs in quadruplicates at 10 nM and 0.5 nM, respectively, with siRNAs targeting Aha1, Firefly-Luciferase and Factor VII as unspecific controls and a mock transfection. Dose-response experiments were done with siRNA in 10 concentrations transfected in quadruplicates, starting at 100 nM in 6-fold dilutions steps down to ˜10 fM. Mock transfected cells served as control in DRC experiments. After 24 h of incubation with siRNAs, 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-Jügesheim, Germany) following 30 minutes incubation at RT in the dark.

The Aha1-siRNA served at the same time as an unspecific control for respective target mRNA expression and as a positive control to analyze transfection efficiency with regards to Aha1 mRNA level. By hybridization with an Aha1 probeset, the other two target-unspecific controls served as controls for Aha1 mRNA level. Transfection efficiency for each 96-well plate and both doses in the dual dose screen was calculated by relating Aha1-level in wells with Aha1-siRNA (normalized to GapDH) to Aha1-level obtained with controls.

For each well, the target mRNA level was normalized to the respective GAPDH mRNA level. The activity of a given siRNA 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.

Protocol 2
1. Cell Seeding Density Evaluation

HeLa cells were optimized for growth rate over 72 h in 384 well plate format. The optimal cell seeding density was 5,000 HeLa cells per well. This allowed for efficient reverse transfection and sufficient mRNA to be measured by RTqPCR

2. Transfection Optimization

HeLa cells were reverse transfected with 10 nM and 25 nM siRNA for the following controls: NT2 (siGENOME Non-targeting Control siRNA #2, Dharmacon D-001210-02) and siTox (AllStars Hs Cell Death Control siRNA, Qiagen SI04381048) with concentrations of Lipofectamine RNAiMAX (Catalog #13778150, ThermoFisher Scientific) ranging from 0.03-0.25 μL per well. Transfection was performed in four replicates per control and per amount of Lipofectamine RNAiMAX. After 72 h the viability of the HeLa cells were measured using CTG2.0 Assay (CellTitre-Glo 2.0, Promega G924C) according to manufacturer's instructions. Briefly, a reagent volume equal to the amount of media was added per well, followed by a five-minute lysis reaction on an orbital shaker. Following a ten-minute incubation at room temperature, luminescence was measured. Lipofectamine RNAiMAX transfection reagent concentration was optimised at 0.12 μL per well (in a 384 well plate).

3. RT-qPCR Assay Optimization:

HeLa cells were reverse transfected with 10 nM and 0.5 nM siRNA for the following controls: NT2 (siGENOME Non-targeting Control siRNA #2, Dharmacon D-001210-02) and siGAPDH (siGENOME GAPDH, Dharmacon M-004253-02) using Lipofectamine RNAiMAX at 0.12 μL per well (in a 384 well plate). Twenty fours after transfection, cells were processed for RT-qPCR read-out using the Cellsto-CT 1-step TaqMan Kit (Invitrogen 4391852C and 4444436) following the manufacturer's instructions.

Briefly, cells were washed with 50 μl PBS and then lysed in 20 μl Lysis solution containing DNase I. After 5 min, lysis was stopped by addition of 2 μl STOP Solution for 2 min. Lysates were kept at −20° C. until RT-qPCR analysis or on ice for immediate RT-qPCR analysis. Cell lysates were diluted 1:1 with H2O. 3 μl of lysate was used as template in a 11 μl reaction volume.

Expression levels of GAPDH (TaqMan 4310884E) and GUSB (TaqMan 4333767F) were determined using RT-qPCR (Cells-to-CT 1-step TaqMan Kit) on a QuantStudio 6 (QS6) thermocycling instrument (Applied BioSystems). Relative quantification was determined using the ΔΔCT method, where a duplexed control GUSB was used and expression changes normalized to the reference sample (plate average of negative control siRNA transfected cells) set to 1.

4. RNAi Screen

All 1080 siRNA duplexes were resuspended in UltraPure DNase and RNase free distilled water (Invitrogen, 10977035) at 1000-fold their final assay concentration (10 μM or 0.5 μM). siRNA duplexes were dispensed in quadruplicates at 25 nL per well using the Echo 525 acoustic dispenser (LabCyte). These assay plates containing siRNA duplexes were stored at −80° C. until reverse transfection of siRNA duplexes were allowed to complex with 5 μL of Lipofectamine RNAiMAX for 20 minutes before HeLa cells were added at 5,000 cells per well (20 μl). Assay plates were kept in a cell culture incubator for 24 hours. RT-qPCR readout (using Cells-to-CT 1-step TaqMan protocol) was performed as described above.

The sense and antisense oligonucleotides of Table 12 each contain a dTdT overhang on the 3′ end. Additionally, every A and G in each sense oligonucleotide in Table 12 is a ribonucleotide. Every C and U in each sense oligonucleotide is a 2′-O-Methyl ribonucleotide.

Also, every A and G in each antisense oligonucleotide in Table 12 is a ribonucleotide. Every C and U preceding an A in the antisense oligonucleotide is a 2′-O-Methyl ribonucleotide, with one exception: U is the first nucleotide of the antisense strand, and it is a ribonucleotide.

Each nucleotide in the sense and antisense oligonucleotide in Table 12 is linked by a phosphate.

TABLE 12

SEQ

ID

NO/

SEQ
Coordinates

Mean % mRNA

SENSE

ID NO/
in

remaining

OLIGO

ANTISENSE
NM_002439.4

Std.

NO
Sense
Antisense
OLIGO NO
Begin
End
Mean
Dev.

78
ACUCUGAGCCAAAGAAAUA
UAUUUCUUUGGCUCAGAGU
79
433
450
29.16%
1.30%

82
UGAGCCAAAGAAAUGUCUA
UAGACAUUUCUUUGGCUCA
83
437
454
28.48%
3.21%

104
CUGCCCUUCCUCAAAGUAA
UUACUUUGAGGAAGGGCAG
105
511
528
66.45%
13.29%

148
AGUUCUGCCAAAAUGUACA
UGUACAUUUUGGCAGAACU
149
563
580
41.92%
0.95%

158
GUACUGAUUUUGAUGAUAA
UUAUCAUCAAAAUCAGUAC
159
577
594
34.73%
1.63%

160
ACUGAUUUUGAUGAUAUCA
UGAUAUCAUCAAAAUCAGU
161
579
596
39.18%
6.08%

190
AAGAAUGCAGUUUCUUCUA
UAGAAGAAACUGCAUUCUU
191
612
629
34.71%
1.43%

240
CAGUUUGGAUCAUCAAAUA
UAUUUGAUGAUCCAAACUG
241
684
701
34.87%
0.56%

244
GUUUGGAUCAUCAAAUACA
UGUAUUUGAUGAUCCAAAC
245
686
703
30.29%
0.25%

246
UUUGGAUCAUCAAAUACAA
UUGUAUUUGAUGAUCCAAA
247
687
704
36.80%
5.06%

250
UGGAUCAUCAAAUACAAGA
UCUUGUAUUUGAUGAUCCA
251
689
706
42.39%
1.25%

252
GGAUCAUCAAAUACAAGUA
UACUUGUAUUUGAUGAUCC
253
690
707
37.57%
4.10%

260
UACAAGUCAUGAAAAUUUA
UAAAUUUUCAUGACUUGUA
261
701
718
33.20%
3.47%

308
CCGCUAGAAUUACAAUACA
UGUAUUGUAAUUCUAGCGG
309
771
788
28.38%
2.64%

314
AGAAUUACAAUACAUAGAA
UUCUAUGUAUUGUAAUUCU
315
776
793
28.83%
0.31%

316
GAAUUACAAUACAUAGAAA
UUUCUAUGUAUUGUAAUUC
317
777
794
33.30%
2.34%

354
GUGUGGAAUGUGGAUAUAA
UUAUAUCCACAUUCCACAC
355
826
843
41.99%
10.04%

356
UGUGGAAUGUGGAUAUAAA
UUUAUAUCCACAUUCCACA
357
827
844
28.56%
4.30%

360
UGGAAUGUGGAUAUAAGUA
UACUUAUAUCCACAUUCCA
361
829
846
29.53%
4.53%

362
GGAAUGUGGAUAUAAGUAA
UUACUUAUAUCCACAUUCC
363
830
847
51.52%
3.12%

364
GAAUGUGGAUAUAAGUAUA
UAUACUUAUAUCCACAUUC
365
831
848
23.70%
0.44%

368
AUGUGGAUAUAAGUAUAGA
UCUAUACUUAUAUCCACAU
369
833
850
28.60%
3.91%

370
UGUGGAUAUAAGUAUAGAA
UUCUAUACUUAUAUCCACA
371
834
851
56.11%
7.06%

372
UGGAUAUAAGUAUAGAUUA
UAAUCUAUACUUAUAUCCA
373
836
853
66.22%
6.36%

396
GAGCUCAAUAUUUAUUGCA
UGCAAUAAAUAUUGAGCUC
379
885
902
37.54%
3.89%

414
UUUAGAUCACAACUUUAUA
UAUAAAGUUGUGAUCUAAA
415
905
922
37.46%
0.99%

416
UUAGAUCACAACUUUAUGA
UCAUAAAGUUGUGAUCUAA
417
906
923
50.98%
4.13%

418
UAGAUCACAACUUUAUGAA
UUCAUAAAGUUGUGAUCUA
419
907
924
28.29%
0.22%

474
GGUGGCAAAAGGAUAUAAA
UUUAUAUCCUUUUGCCACC
475
971
988
28.32%
2.21%

476
GUGGCAAAAGGAUAUAAGA
UCUUAUAUCCUUUUGCCAC
477
972
989
33.27%
0.44%

478
UGGCAAAAGGAUAUAAGGA
UCCUUAUAUCCUUUUGCCA
479
973
990
47.43%
20.58%

480
GGCAAAAGGAUAUAAGGUA
UACCUUAUAUCCUUUUGCC
481
974
991
25.90%
4.29%

502
GUUGUGAAGCAAACUGAAA
UUUCAGUUUGCUUCACAAC
503
996
1013
28.49%
2.40%

512
CUGAAACUGCAGCAUUAAA
UUUAAUGCUGCAGUUUCAG
513
1009
1026
40.65%
4.27%

552
ACAGAAGUUCACUCUUUUA
UAAAAGAGUGAACUUCUGU
553
1042
1059
32.99%
2.47%

558
UCUUUUCCCGGAAAUUGAA
UUCAAUUUCCGGGAAAAGA
559
1054
1071
29.47%
2.18%

560
CUUUUCCCGGAAAUUGACA
UGUCAAUUUCCGGGAAAAG
561
1055
1072
41.39%
2.43%

582
CCUUUAUACAAAAUCUACA
UGUAGAUUUUGUAUAAAGG
583
1076
1093
74.65%
11.91%

616
UGUGAAUCCCCUAAUCAAA
UUUGAUUAGGGGAUUCACA
617
1109
1126
43.41%
3.79%

618
GUGAAUCCCCUAAUCAAGA
UCUUGAUUAGGGGAUUCAC
619
1110
1127
36.19%
3.08%

634
UGGAUGAUGCUGUAAAUGA
UCAUUUACAGCAUCAUCCA
635
1129
1146
61.80%
10.95%

636
GAUGAUGCUGUAAAUGUUA
UAACAUUUACAGCAUCAUC
637
1131
1148
23.42%
1.99%

642
UAAAUGUUGAUGAGAUAAA
UUUAUCUCAUCAACAUUUA
643
1141
1158
46.72%
7.85%

646
UGUUGAUGAGAUAAUGACA
UGUCAUUAUCUCAUCAACA
647
1145
1162
29.37%
1.10%

648
GUUGAUGAGAUAAUGACUA
UAGUCAUUAUCUCAUCAAC
649
1146
1163
16.93%
2.14%

656
AGAUAAUGACUGAUACUUA
UAAGUAUCAGUCAUUAUCU
657
1153
1170
22.55%
2.42%

660
AUAAUGACUGAUACUUCUA
UAGAAGUAUCAGUCAUUAU
661
1155
1172
45.26%
4.42%

690
AUCUCUGAAAAUAAGGAAA
UUUCCUUAUUUUCAGAGAU
691
1191
1208
27.95%
1.45%

692
GAAAAUAAGGAAAAUGUUA
UAACAUUUUCCUUAUUUUC
693
1197
1214
32.77%
4.88%

718
GAGGUUGUGUUUGAUAGUA
UACUAUCAAACACAACCUC
719
1275
1292
25.27%
1.85%

720
AGGUUGUGUUUGAUAGUUA
UAACUAUCAAACACAACCU
721
1276
1293
30.55%
1.57%

722
GGUUGUGUUUGAUAGUUUA
UAAACUAUCAAACACAACC
723
1277
1294
29.47%
1.48%

796
GUGCAGGAUGACAGAAUUA
UAAUUCUGUCAUCCUGCAC
797
1422
1439
33.29%
3.25%

820
GAAAGGAUGGAUAACAUUA
UAAUGUUAUCCAUCCUUUC
821
1446
1463
28.18%
2.33%

822
AAAGGAUGGAUAACAUUUA
UAAAUGUUAUCCAUCCUUU
823
1447
1464
34.97%
4.72%

826
GGAUAACAUUUAUUUUGAA
UUCAAAAUAAAUGUUAUCC
827
1454
1471
29.04%
3.27%

848
CAGUUACAGAGUUUUAUGA
UCAUAAAACUCUGUAACUG
849
1492
1509
46.08%
2.51%

852
UUACAGAGUUUUAUGCAAA
UUUGCAUAAAACUCUGUAA
853
1495
1512
35.97%
5.19%

854
UACAGAGUUUUAUGCAAAA
UUUUGCAUAAAACUCUGUA
855
1496
1513
30.67%
1.55%

900
UUAUUUCUGGCAUUGUUAA
UUAACAAUGCCAGAAAUAA
901
1543
1560
41.90%
0.76%

914
UAGAGAAGCCUGUGAUUUA
UAAAUCACAGGCUUCUCUA
915
1564
1581
32.55%
3.19%

928
CUUUGGCUGCCAUCAUAAA
UUUAUGAUGGCAGCCAAAG
929
1585
1602
37.53%
0.23%

930
UUUGGCUGCCAUCAUAAAA
UUUUAUGAUGGCAGCCAAA
931
1586
1603
62.24%
6.30%

934
GGCUGCCAUCAUAAAAUAA
UUAUUUUAUGAUGGCAGCC
935
1589
1606
75.79%
3.96%

936
GCUGCCAUCAUAAAAUACA
UGUAUUUUAUGAUGGCAGC
937
1590
1607
39.73%
1.77%

946
UACCUCAAAGAAUUCAACA
UGUUGAAUUCUUUGAGGUA
947
1605
1622
39.67%
5.42%

948
CCUCAAAGAAUUCAACUUA
UAAGUUGAAUUCUUUGAGG
949
1607
1624
38.11%
2.55%

966
UCUCCAAACCUGAGAAUUA
UAAUUCUCAGGUUUGGAGA
967
1636
1653
29.07%
4.75%

970
AACCUGAGAAUUUUAAACA
UGUUUAAAAUUCUCAGGUU
971
1642
1659
63.55%
10.21%

972
CCUGAGAAUUUUAAACAGA
UCUGUUUAAAAUUCUCAGG
973
1644
1661
36.98%
1.63%

988
GCUAUCAAGUAAAAUGGAA
UUCCAUUUUACUUGAUAGC
989
1661
1678
35.63%
2.07%

990
CUAUCAAGUAAAAUGGAAA
UUUCCAUUUUACUUGAUAG
991
1662
1679
35.08%
2.46%

992
UAUCAAGUAAAAUGGAAUA
UAUUCCAUUUUACUUGAUA
993
1663
1680
39.55%
2.94%

994
UCAAGUAAAAUGGAAUUUA
UAAAUUCCAUUUUACUUGA
995
1665
1682
41.67%
0.76%

996
CAAGUAAAAUGGAAUUUAA
UUAAAUUCCAUUUUACUUG
997
1666
1683
46.47%
5.33%

1006
UUAUGACAAUUAAUGGAAA
UUUCCAUUAAUUGUCAUAA
1007
1681
1698
33.49%
0.60%

1020
GGAACAACAUUAAGGAAUA
UAUUCCUUAAUGUUGUUCC
1021
1695
1712
38.00%
1.90%

1032
CUGGAAAUCCUACAGAAUA
UAUUCUGUAGGAUUUCCAG
1032
1713
1730
40.41%
2.43%

1054
UCAGACUGAUAUGAAAACA
UGUUUUCAUAUCAGUCUGA
1055
1730
1747
68.98%
8.10%

1056
GACUGAUAUGAAAACCAAA
UUUGGUUUUCAUAUCAGUC
1057
1733
1750
39.79%
1.76%

1058
ACUGAUAUGAAAACCAAAA
UUUUGGUUUUCAUAUCAGU
1059
1734
1751
38.68%
2.75%

1076
UUGCUGUGGGUUUUAGACA
UGUCUAAAACCCACAGCAA
1077
1758
1775
121.44%
74.40%

1088
GUUUUAGACCACACUAAAA
UUUUAGUGUGGUCUAAAAC
1089
1767
1784
86.75%
7.70%

1096
AGACCACACUAAAACUUCA
UGAAGUUUUAGUGUGGUCU
1097
1772
1789
52.58%
5.39%

1098
CCACACUAAAACUUCAUUA
UAAUGAAGUUUUAGUGUGG
1099
1775
1792
76.51%
11.60%

1110
GGGAGACGGAAGUUAAAGA
UCUUUAACUUCCGUCUCCC
1111
1794
1811
37.03%
3.68%

1112
GGAGACGGAAGUUAAAGAA
UUCUUUAACUUCCGUCUCC
1113
1795
1812
40.15%
4.44%

1126
CCCAGCCACUCCUUAAAUA
UAUUUAAGGAGUGGCUGGG
1127
1822
1839
58.33%
3.02%

1214
UUUGGUCAGAUAGAAAAUA
UAUUUUCUAUCUGACCAAA
1215
1902
1919
45.68%
5.83%

1220
CAGAUAGAAAAUCAUCUAA
UUAGAUGAUUUUCUAUCUG
1221
1908
1925
56.12%
2.65%

1230
AAUCAUCUACGUAAAUUGA
UCAAUUUACGUAGAUGAUU
1230
1917
1934
71.94%
2.14%

1306
AGAAUUUCAAGCAAUAAUA
UAUUAUUGCUUGAAAUUCU
1307
2030
2047
36.86%
0.88%

1308
AUUUCAAGCAAUAAUACCA
UGGUAUUAUUGCUUGAAAU
1309
2033
2050
43.83%
5.54%

1310
UUUCAAGCAAUAAUACCUA
UAGGUAUUAUUGCUUGAAA
1311
2034
2051
36.53%
1.45%

1318
AUACCUGCUGUUAAUUCCA
UGGAAUUAACAGCAGGUAU
1319
2046
2063
44.51%
3.47%

1326
ACCGUUAUUUUAGAAAUUA
UAAUUUCUAAAAUAACGGU
1363
2088
2105
40.01%
3.36%

1386
UCCAGUGGAGCAUUACUUA
UAAGUAAUGCUCCACUGGA
1387
2120
2137
37.78%
4.62%

1394
GAGCAUUACUUAAAGAUAA
UUAUCUUUAAGUAAUGCUC
1395
2127
2144
42.33%
1.87%

1396
AGCAUUACUUAAAGAUACA
UGUAUCUUUAAGUAAUGCU
1397
2128
2145
38.19%
0.66%

1400
UACUUAAAGAUACUCAAUA
UAUUGAGUAUCUUUAAGUA
1401
2133
2150
88.29%
67.87%

1404
UUAAAGAUACUCAAUGAAA
UUUCAUUGAGUAUCUUUAA
1405
2136
2153
34.87%
2.77%

1424
GUUGGGGAUAAAACUGAAA
UUUCAGUUUUAUCCCCAAC
1425
2166
2183
37.58%
1.87%

1426
UUGGGGAUAAAACUGAAUA
UAUUCAGUUUUAUCCCCAA
1427
2167
2184
45.94%
2.86%

1448
UUCUGACUUCCCUUUAAUA
UAUUAAAGGGAAGUCAGAA
1449
2198
2215
42.92%
2.41%

1452
UGACUUCCCUUUAAUAAAA
UUUUAUUAAAGGGAAGUCA
1453
2201
2218
34.18%
1.53%

1454
AGAGGAAGGAUGAAAUUCA
UGAAUUUCAUCCUUCCUCU
1455
2221
2238
38.71%
4.13%

1506
AUCCUUCUGCACAAUAUGA
UCAUAUUGUGCAGAAGGAU
1507
2296
2313
78.53%
13.44%

1524
CAGGACAGGAGUUUAUGAA
UUCAUAAACUCCUGUCCUG
1525
2323
2340
41.36%
5.28%

1540
AGAACUCUGCUGUAUCUUA
UAAGAUACAGCAGAGUUCU
1541
2350
2367
39.12%
1.41%

1546
CUCUGCUGUAUCUUGUAUA
UAUACAAGAUACAGCAGAG
1547
2354
2371
42.74%
3.97%

1656
GAAUGGCUUGAUUUUCUAA
UUAGAAAAUCAAGCCAUUC
1657
2496
2513
43.75%
3.47%

1666
UUUCUAGAGAAAUUCAGUA
UACUGAAUUUCUCUAGAAA
1667
2508
2525
56.85%
11.91%

1674
AAAUUCAGUGAACAUUAUA
UAUAAUGUUCACUGAAUUU
1675
2517
2534
45.05%
5.48%

1676
AAUUCAGUGAACAUUAUCA
UGAUAAUGUUCACUGAAUU
1677
2518
2535
29.77%
2.22%

1678
AUUCAGUGAACAUUAUCAA
UUGAUAAUGUUCACUGAAU
1679
2519
2536
36.74%
3.41%

1722
UGUUGACUGCAUUUUCUCA
UGAGAAAAUGCAGUCAACA
1723
2570
2587
34.29%
3.73%

1762
AGACCAACUGUACAAGAAA
UUUCUUGUACAGUUGGUCU
1763
2622
2639
37.30%
3.16%

1766
ACUGUACAAGAAGAAAGAA
UUCUUUCUUCUUGUACAGU
1767
2628
2645
54.43%
4.31%

1768
CUGUACAAGAAGAAAGAAA
UUUCUUUCUUCUUGUACAG
1769
2629
2646
33.89%
5.86%

1836
ACUCAGAGAGAGUAAUGAA
UUCAUUACUCUCUCUGAGU
1837
2740
2757
29.94%
3.38%

1838
UCAGAGAGAGUAAUGAUAA
UUAUCAUUACUCUCUCUGA
1839
2742
2759
32.69%
1.16%

1842
AGAGAGAGUAAUGAUAAUA
UAUUAUCAUUACUCUCUCU
1843
2744
2761
35.60%
3.31%

1868
GAAAGAGCUCCUACAUAAA
UUUAUGUAGGAGCUCUUUC
1869
2782
2799
36.26%
1.38%

1886
AAACAAGUUGCAUUGAUUA
UAAUCAAUGCAACUUGUUU
1887
2799
2816
48.40%
12.84%

1888
AACAAGUUGCAUUGAUUAA
UUAAUCAAUGCAACUUGUU
1889
2800
2817
63.96%
2.64%

1964
GUGGAUGGCAUUUUCACAA
UUGUGAAAAUGCCAUCCAC
1965
2874
2891
49.72%
1.59%

1990
UGCAGACAAUAUAUAUAAA
UUUAUAUAUAUUGUCUGCA
1991
2903
2920
47.35%
2.46%

2030
GACACAGCAGAAAUAAUCA
UGAUUAUUUCUGCUGUGUC
2031
2952
2969
43.67%
8.62%

2108
AUGCUACACUUGAGUAUUA
UAAUACUCAAGUGUAGCAU
2109
3052
3069
41.76%
2.04%

2128
GAGAUGUGAAAUCCUUAAA
UUUAAGGAUUUCACAUCUC
2129
3076
3093
62.62%
4.94%

2230
AACAAGUCCCUGAUUUUGA
UCAAAAUCAGGGACUUGUU
2231
3220
3237
73.43%
10.39%

2242
UUUUGUCACCUUCCUUUAA
UUAAAGGAAGGUGACAAAA
2243
3233
3250
49.17%
4.42%

2246
UCACCUUCCUUUACCAAAA
UUUUGGUAAAGGAAGGUGA
2247
3238
3255
44.74%
5.16%

2254
CUUUACCAAAUAACUAGAA
UUCUAGUUAUUUGGUAAAG
2255
3246
3263
39.87%
3.77%

2274
AAGGAGUUAUGGAUUAAAA
UUUUAAUCCAUAACUCCUU
2275
3275
3292
41.52%
1.53%

2294
UAAAUGUGGCUAAACUAGA
UCUAGUUUAGCCACAUUUA
2295
3289
3306
39.85%
2.79%

2330
GAGCUGGAAGGAUUAAUAA
UUAUUAAUCCUUCCAGCUC
2331
3354
3371
56.05%
14.99%

2334
GGAUUAAUAAAUACGAAAA
UUUUCGUAUUUAUUAAUCC
2335
3363
3380
52.40%
5.26%

2356
ACUCAAGUAUUUUGCAAAA
UUUUGCAAAAUACUUGAGU
2357
3389
3406
35.71%
2.25%

2360
AAGUAUUUUGCAAAGUUAA
UUAACUUUGCAAAAUACUU
2361
3393
3410
41.68%
2.43%

2362
GUAUUUUGCAAAGUUAUGA
UCAUAACUUUGCAAAAUAC
2363
3395
3412
45.57%
7.28%

2448
CAACUGUACAAAAUAACUA
UAGUUAUUUUGUACAGUUG
2449
3540
3557
59.64%
10.75%

2502
ACAUGUGAGCAUAAAAUUA
UAAUUUUAUGCUCACAUGU
2503
3584
3601
40.95%
4.16%

2504
CAUGUGAGCAUAAAAUUAA
UUAAUUUUAUGCUCACAUG
2505
3585
3602
56.03%
3.39%

2516
AAUUAUGACCAUGGUAUAA
UUAUACCAUGGUCAUAAUU
2517
3598
3615
53.24%
2.72%

2518
AUUAUGACCAUGGUAUAUA
UAUAUACCAUGGUCAUAAU
2519
3599
3616
67.48%
5.75%

2578
AUAAACACUCUUGAAUAGA
UCUAUUCAAGAGUGUUUAU
2579
3689
3706
55.57%
9.42%

2580
UAAACACUCUUGAAUAGAA
UUCUAUUCAAGAGUGUUUA
2581
3690
3707
42.79%
1.54%

2592
AUAGACUUCCACUUUGUAA
UUACAAAGUGGAAGUCUAU
2593
3703
3720
65.27%
6.23%

2596
AGACUUCCACUUUGUAAUA
UAUUACAAAGUGGAAGUCU
2597
3705
3722
58.85%
9.89%

2602
CUUCCACUUUGUAAUUAGA
UCUAAUUACAAAGUGGAAG
2603
3708
3725
51.93%
7.38%

2654
CUUAAGUGGCAGAAUAUAA
UUAUAUUCUGCCACUUAAG
2655
3757
3774
61.67%
11.09%

2656
UUAAGUGGCAGAAUAUAAA
UUUAUAUUCUGCCACUUAA
2657
3758
3775
48.43%
7.02%

2686
UUUUGGAGGGUGAUAUAAA
UUUAUAUCACCCUCCAAAA
2687
3785
3802
48.16%
6.02%

2762
UCCACCAAGAACAUAAGAA
UUCUUAUGUUCUUGGUGGA
2763
3912
3929
49.77%
1.05%

2768
ACCAAGAACAUAAGAAUUA
UAAUUCUUAUGUUCUUGGU
2769
3915
3932
44.17%
3.47%

2782
UAGAAUUAUCAAGCUUUUA
UAAAAGCUUGAUAAUUCUA
2783
4290
4307
53.00%
2.15%

2844
UUGGAUGAAAUUAUUUGUA
UACAAAUAAUUUCAUCCAA
2845
4389
4406
71.32%
9.32%

2846
GAUGAAAUUAUUUGUCAUA
UAUGACAAAUAAUUUCAUC
2847
4392
4409
55.83%
6.18%

For the chemical modifications, “2m” means 2′-O-Methyl ribonucleotides, “r” means ribonucleotide, “p” means phosphate linkage, and “d” means deoxyribonucleotide.

TABLE 13

Chemical Modifications for Sense Strands

SEQ

ID

No./

Sense

No.
Sense
Sense Chemical Modifications

78
ACUCUGAGCCAAAGAAAUA
[rAp|2mCp|2mUp|2mCp|2mUp|rGp|rAp|rGp|2mCp

|2mCp|rAp|rAp|rAp|rGp|rAp

|rAp|rAp|2mUp|rAp|dTp|dT]

82
UGAGCCAAAGAAAUGUCUA
[2mUp|rGp|rAp|rGp|2mCp|2mCp|rAp|rAp|rAp|r

Gp|rAp|rAp|rAp|2mUp|rGp|2m

Up|2mCp|2mUp|rAp|dTp|dT]

104
CUGCCCUUCCUCAAAGUAA
[2mCp|2mUp|rGp|2mCp|2mCp|2mCp|2mUp|2mUp|2

mCp|2mCp|2mUp|2mCp|

rAp|rAp|rAp|rGp|2mUp|rAp|rAp|dTp|dT]

148
AGUUCUGCCAAAAUGUACA
[rAp|rGp|2mUp|2mUp|2mCp|2mUp|rGp|2mCp|2mC

p|rAp|r

Ap|rAp|rAp|2mUp|rGp|2mUp|rAp|2mCp|rAp|dT

p|dT]

158
GUACUGAUUUUGAUGAUAA
[rGp|2mUp|rAp|2mCp|2mUp|rGp|rAp|2mUp|2mUp

|2mUp|2mUp|rGp|rAp|2mU

p|rGp|rAp|2mUp|rAp|rAp|dTp|dT]

160
ACUGAUUUUGAUGAUAUCA
[rAp|2mCp|2mUp|rGp|rAp|2mUp|2mUp|2mUp|2mU

p|rGp|rAp|2mUp|rGp|rAp|2

mUp|rAp|2mUp|2mCp|rAp|dTp|dT]

190
AAGAAUGCAGUUUCUUCUA
[rAp|rAp|rGp|rAp|rAp|2mUp|rGp|2mCp|rAp|rG

p|2mUp|2mUp|2mUp|2mCp|2m

Up|2mUp|2mCp|2mUp|rAp|dTp|dT]

240
CAGUUUGGAUCAUCAAAUA
[2mCp|rAp|rGp|2mUp|2mUp|2mUp|rGp|rGp|rAp|

2mUp|2mCp|rAp|2mUp|2mC

p|rAp|rAp|rAp|2mUp|rAp|dTp|dT]

244
GUUUGGAUCAUCAAAUACA
[rGp|2mUp|2mUp|2mUp|rGp|rGp|rAp|2mUp|2mCp

|rAp|2mUp|2mCp|rAp|rAp|r

Ap|2mUp|rAp|2mCp|rAp|dTp|dT]

246
UUUGGAUCAUCAAAUACAA
[2mUp|2mUp|2mUp|rGp|rGp|rAp|2mUp|2mCp|rAp

|2mUp|2mCp|rAp|rAp|rAp|2

mUp|rAp|2mCp|rAp|rAp|dTp|dT]

250
UGGAUCAUCAAAUACAAGA
[2mUp|rGp|rGp|rAp|2mUp|2mCp|rAp|2mUp|2mCp

|rAp|rAp|rAp|2mUp|rAp|2m

Cp|rAp|rAp|rGp|rAp|dTp|dT]

252
GGAUCAUCAAAUACAAGUA
[rGp|rGp|rAp|2mUp|2mCp|rAp|2mUp|2mCp|rAp|

rAp|rAp|2mUp|rAp|2mCp|rAp|

rAp|rGp|2mUp|rAp|dTp|dT]

260
UACAAGUCAUGAAAAUUUA
[2mUp|rAp|2mCp|rAp|rAp|rGp|2mUp|2mCp|rAp|

2mUp|rGp|rAp|rAp|rAp|rAp|2

mUp|2mUp|2mUp|rAp|dTp|dT]

308
CCGCUAGAAUUACAAUACA
[2mCp|2mCp|rGp|2mCp|2mUp|rAp|rGp|rAp|rAp|

2mUp|2mUp|rAp|2mCp|rAp|r

Ap|2mUp|rAp|2mCp|rAp|dTp|dT]

314
AGAAUUACAAUACAUAGAA
[rAp|rGp|rAp|rAp|2mUp|2mUp|rAp|2mCp|rAp|r

Ap|2mUp|rAp|2mCp|rAp|2mUp|

rAp|rGp|rAp|rAp|dTp|dT]

316
GAAUUACAAUACAUAGAAA
[rGp|rAp|rAp|2mUp|2mUp|rAp|2mCp|rAp|rAp|2

mUp|rAp|2mCp|rAp|2mUp|rAp|

rGp|rAp|rAp|rAp|dTp|dT]

354
GUGUGGAAUGUGGAUAUAA
[rGp|2mUp|rGp|2mUp|rGp|rGp|rAp|rAp|2mUp|r

Gp|2mUp|rGp|rGp|rAp|2mUp|r

Ap|2mUp|rAp|rAp|dTp|dT]

356
UGUGGAAUGUGGAUAUAAA
[2mUp|rGp|2mUp|rGp|rGp|rAp|rAp|2mUp|rGp|2

mUp|rGp|rGp|rAp|2mUp|rAp|2

mUp|rAp|rAp|rAp|dTp|dT]

360
UGGAAUGUGGAUAUAAGUA
[2mUp|rGp|rGp|rAp|rAp|2mUp|rGp|2mUp|rGp|r

Gp|rAp|2mUp|rAp|2mUp|rAp|r

Ap|rGp|2mUp|rAp|dTp|dT]

362
GGAAUGUGGAUAUAAGUAA
[rGp|rGp|rAp|rAp|2mUp|rGp|2mUp|rGp|rGp|rA

p|2mUp|rAp|2mUp|rAp|rAp|rGp|

2mUp|rAp|rAp|dTp|dT]

364
GAAUGUGGAUAUAAGUAUA
[rGp|rAp|rAp|2mUp|rGp|2mUp|rGp|rGp|rAp|2m

Up|rAp|2mUp|rAp|rAp|rGp|2m

Up|rAp|2mUp|rAp|dTp|dT]

368
AUGUGGAUAUAAGUAUAGA
[rAp|2mUp|rGp|2mUp|rGp|rGp|rAp|2mUp|rAp|2

mUp|rAp|rAp|rGp|2mUp|rAp|2

mUp|rAp|rGp|rAp|dTp|dT]

370
UGUGGAUAUAAGUAUAGAA
[2mUp|rGp|2mUp|rGp|rGp|rAp|2mUp|rAp|2mUp|

rAp|rAp|rGp|2mUp|rAp|2mUp

|rAp|rGp|rAp|rAp|dTp|dT]

372
UGGAUAUAAGUAUAGAUUA
[2mUp|rGp|rGp|rAp|2mUp|rAp|2mUp|rAp|rAp|r

Gp|2mUp|rAp|2mUp|rAp|rGp|r

Ap|2mUp|2mUp|rAp|dTp|dT]

396
GAGCUCAAUAUUUAUUGCA
[rGp|rAp|rGp|2mCp|2mUp|2mCp|rAp|rAp|2mUp|

rAp|2mUp|2mUp|2mUp|rAp|2

mUp|2mUp|rGp|2mCp|rAp|dTp|dT]

414
UUUAGAUCACAACUUUAUA
[2mUp|2mUp|2mUp|rAp|rGp|rAp|2mUp|2mCp|rAp

|2mCp|rAp|rAp|2mCp|2mUp

2mUp|2mUp|rAp|2mUp|rAp|dTp|dT]

416
UUAGAUCACAACUUUAUGA
[2mUp|2mUp|rAp|rGp|rAp|2mUp|2mCp|rAp|2mCp

|rAp|rAp|2mCp|2mUp|2mUp

|2mUp|rAp|2mUp|rGp|rAp|dTp|dT]

418
UAGAUCACAACUUUAUGAA
[2mUp|rAp|rGp|rAp|2mUp|2mCp|rAp|2mCp|rAp|

rAp|2mCp|2mUp|2mUp|2mUp

rAp|2mUp|rGp|rAp|rAp|dTp|dT]

474
GGUGGCAAAAGGAUAUAAA
[rGp|rGp|2mUp|rGp|rGp|2mCp|rAp|rAp|rAp|rA

p|rGp|rGp|rAp|2mUp|rAp|2mUp|

rAp|rAp|rAp|dTp|dT]

476
GUGGCAAAAGGAUAUAAGA
[rGp|2mUp|rGp|rGp|2mCp|rAp|rAp|rAp|rAp|rG

p|rGp|rAp|2mUp|rAp|2mUp|rAp|

rAp|rGp|rAp|dTp|dT]

478
UGGCAAAAGGAUAUAAGGA
[2mUp|rGp|rGp|2mCp|rAp|rAp|rAp|rAp|rGp|rG

p|rAp|2mUp|rAp|2mUp|rAp|rAp|

rGp|rGp|rAp|dTp|dT]

480
GGCAAAAGGAUAUAAGGUA
[rGp|rGp|2mCp|rAp|rAp|rAp|rAp|rGp|rGp|rAp

|2mUp|rAp|2mUp|rAp|rAp|rGp|rG

p|2mUp|rAp|dTp|dT]

502
GUUGUGAAGCAAACUGAAA
[rGp|2mUp|2mUp|rGp|2mUp|rGp|rAp|rAp|rGp|

2mCp|rAp|rAp|rAp|2mCp|2mUp

|rGp|rAp|rAp|rAp|dTp|dT]

512
CUGAAACUGCAGCAUUAAA
[2mCp|2mUp|rGp|rAp|rAp|rAp|2mCp|2mUp|rGp|

2mCp|rAp|rGp|2mCp|rAp|2m

Up|2mUp|rAp|rAp|rAp|dTp|dT]

552
ACAGAAGUUCACUCUUUUA
[rAp|2mCp|rAp|rGp|rAp|rAp|rGp|2mUp|2mUp|2

mCp|rAp|2mCp|2mUp|2mCp|2

mUp|2mUp|2mUp|2mUp|rAp|dTp|dT]

558
UCUUUUCCCGGAAAUUGAA
[2mUp|2mCp|2mUp|2mUp|2mUp|2mUp|2mCp|2mCp|

2mCp|rGp|rGp|rAp|rAp|r

Ap|2mUp|2mUp|rGp|rAp|rAp|dTp|dT]

560
CUUUUCCCGGAAAUUGACA
[2mCp|2mUp|2mUp|2mUp|2mUp|2mCp|2mCp|2mCp|

rGp|rGp|rAp|rAp|rAp|2m

Up|2mUp|rGp|rAp|2mCp|rAp|dTp|dT]

582
CCUUUAUACAAAAUCUACA
[2mCp|2mCp|2mUp|2mUp|2mUp|rAp|2mUp|rAp|2m

Cp|rAp|rAp|rAp|rAp|2mUp

2mCp|2mUp|rAp|2mCp|rAp|dTp|dT]

616
UGUGAAUCCCCUAAUCAAA
[2mUp|rGp|2mUp|rGp|rAp|rAp|2mUp|2mCp|2mCp

|2mCp|2mCp|2mUp|rAp|rA

p|2mUp|2mCp|rAp|rAp|rAp|dTp|dT]

618
GUGAAUCCCCUAAUCAAGA
[rGp|2mUp|rGp|rAp|rAp|2mUp|2mCp|2mCp|2mCp

|2mCp|2mUp|rAp|rAp|2mU

p|2mCp|rAp|rAp|rGp|rAp|dTp|dT]

634
UGGAUGAUGCUGUAAAUGA
[2mUp|rGp|rGp|rAp|2mUp|rGp|rAp|2mUp|rGp|2

mCp|2mUp|rGp|2mUp|rAp|rAp

rAp|2mUp|rGp|rAp|dTp|dT]

636
GAUGAUGCUGUAAAUGUUA
[rGp|rAp|2mUp|rGp|rAp|2mUp|rGp|2mCp|2mUp|

rGp|2mUp|rAp|rAp|rAp|2mUp

|rGp|2mUp|2mUp|rAp|dTp|dT]

642
UAAAUGUUGAUGAGAUAAA
[2mUp|rAp|rAp|rAp|2mUp|rGp|2mUp|2mUp|rGp|

rAp|2mUp|rGp|rAp|rGp|rAp|2

mUp|rAp|rAp|rAp|dTp|dT]

646
UGUUGAUGAGAUAAUGACA
[2mUp|rGp|2mUp|2mUp|rGp|rAp|2mUp|rGp|rAp|

rGp|rAp|2mUp|rAp|rAp|2mUp

|rGp|rAp|2mCp|rAp|dTp|dT]

648
GUUGAUGAGAUAAUGACUA
[rGp|2mUp|2mUp|rGp|rAp|2mUp|rGp|rAp|rGp|r

Ap|2mUp|rAp|rAp|2mUp|rGp|r

Ap|2mCp|2mUp|rAp|dTp|dT]

656
AGAUAAUGACUGAUACUUA
[rAp|rGp|rAp|2mUp|rAp|rAp|2mUp|rGp|rAp|2m

Cp|2mUp|rGp|rAp|2mUp|rAp|2

mCp|2mUp|2mUp|rAp|dTp|dT]

660
AUAAUGACUGAUACUUCUA
[rAp|2mUp|rAp|rAp|2mUp|rGp|rAp|2mCp|2mUp|

rGp|rAp|2mUp|rAp|2mCp|2m

Up|2mUp|2mCp|2mUp|rAp|dTp|dT]

690
AUCUCUGAAAAUAAGGAAA
[rAp|2mUp|2mCp|2mUp|2mCp|2mUp|rGp|rAp|rAp

|rAp|rAp|2mUp|rAp|rAp|rGp|

rGp|rAp|rAp|rAp|dTp|dT]

692
GAAAAUAAGGAAAAUGUUA
[rGp|rAp|rAp|rAp|rAp|2mUp|rAp|rAp|rGp|rGp

|rAp|rAp|rAp|rAp|2mUp|rGp|2mU

p|2mUp|rAp|dTp|dT]

718
GAGGUUGUGUUUGAUAGUA
[rGp|rAp|rGp|rGp|2mUp|2mUp|rGp|2mUp|rGp|2

mUp|2mUp|2mUp|rGp|rAp|2m

Up|rAp|rGp|2mUp|rAp|dTp|dT]

720
AGGUUGUGUUUGAUAGUUA
[rAp|rGp|rGp|2mUp|2mUp|rGp|2mUp|rGp|2mUp|

2mUp|2mUp|rGp|rAp|2mUp|r

Ap|rGp|2mUp|2mUp|rAp|dTp|dT]

722
GGUUGUGUUUGAUAGUUUA
[rGp|rGp|2mUp|2mUp|rGp|2mUp|rGp|2mUp|2mUp

|2mUp|rGp|rAp|2mUp|rAp|r

Gp|2mUp|2mUp|2mUp|rAp|dTp|dT]

796
GUGCAGGAUGACAGAAUUA
[rGp|2mUp|rGp|2mCp|rAp|rGp|rGp|rAp|2mUp|r

Gp|rAp|2mCp|rAp|rGp|rAp|rAp|

2mUp|2mUp|rAp|dTp|dT]

820
GAAAGGAUGGAUAACAUUA
[rGp|rAp|rAp|rAp|rGp|rGp|rAp|2mUp|rGp|rGp

|rAp|2mUp|rAp|rAp|2mCp|rAp|2

mUp|2mUp|rAp|dTp|dT]

822
AAAGGAUGGAUAACAUUUA
[rAp|rAp|rAp|rGp|rGp|rAp|2mUp|rGp|rGp|rAp

|2mUp|rAp|rAp|2mCp|rAp|2mUp|

2mUp|2mUp|rAp|dTp|dT]

826
GGAUAACAUUUAUUUUGAA
[rGp|rGp|rAp|2mUp|rAp|rAp|2mCp|rAp|2mUp|2

mUp|2mUp|rAp|2mUp|2mUp|2

mUp|2mUp|rGp|rAp|rAp|dTp|dT]

848
CAGUUACAGAGUUUUAUGA
[2mCp|rAp|rGp|2mUp|2mUp|rAp|2mCp|rAp|rGp|

rAp|rGp|2mUp|2mUp|2mUp|2

mUp|rAp|2mUp|rGp|rAp|dTp|dT]

852
UUACAGAGUUUUAUGCAAA
[2mUp|2mUp|rAp|2mCp|rAp|rGp|rAp|rGp|2mUp|

2mUp|2mUp|2mUp|rAp|2mU

p|rGp|2mCp|rAp|rAp|rAp|dTp|dT]

854
UACAGAGUUUUAUGCAAAA
[2mUp|rAp|2mCp|rAp|rGp|rAp|rGp|2mUp|2mUp|

2mUp|2mUp|rAp|2mUp|rGp|2

mCp|rAp|rAp|rAp|rAp|dTp|dT]

900
UUAUUUCUGGCAUUGUUAA
[2mUp|2mUp|rAp|2mUp|2mUp|2mUp|2mCp|2mUp|r

Gp|rGp|2mCp|rAp|2mUp|

2mUp|rGp|2mUp|2mUp|rAp|rAp|dTp|dT]

914
UAGAGAAGCCUGUGAUUUA
[2mUp|rAp|rGp|rAp|rGp|rAp|rAp|rGp|2mCp|2m

Cp|2mUp|rGp|2mUp|rGp|rAp|2

mUp|2mUp|2mUp|rAp|dTp|dT]

928
CUUUGGCUGCCAUCAUAAA
[2mCp|2mUp|2mUp|2mUp|rGp|rGp|2mCp|2mUp|rG

p|2mCp|2mCp|rAp|2mUp|

2mCp|rAp|2mUp|rAp|rAp|rAp|dTp|dT]

930
UUUGGCUGCCAUCAUAAAA
[2mUp|2mUp|2mUp|rGp|rGp|2mCp|2mUp|rGp|2mC

p|2mCp|rAp|2mUp|2mCp|

rAp|2mUp|rAp|rAp|rAp|rAp|dTp|dT]

934
GGCUGCCAUCAUAAAAUAA
[rGp|rGp|2mCp|2mUp|rGp|2mCp|2mCp|rAp|2mUp

|2mCp|rAp|2mUp|rAp|rAp|r

Ap|rAp|2mUp|rAp|rAp|dTp|dT]

936
GCUGCCAUCAUAAAAUACA
[rGp|2mCp|2mUp|rGp|2mCp|2mCp|rAp|2mUp|2mC

p|rAp|2mUp|rAp|rAp|rAp|r

Ap|2mUp|rAp|2mCp|rAp|dTp|dT]

946
UACCUCAAAGAAUUCAACA
[2mUp|rAp|2mCp|2mCp|2mUp|2mCp|rAp|rAp|rAp

|rGp|rAp|rAp|2mUp|2mUp|2

mCp|rAp|rAp|2mCp|rAp|dTp|dT]

948
CCUCAAAGAAUUCAACUUA
[2mCp|2mCp|2mUp|2mCp|rAp|rAp|rAp|rGp|rAp|

rAp|2mUp|2mUp|2mCp|rAp|r

Ap|2mCp|2mUp|2mUp|rAp|dTp|dT]

966
UCUCCAAACCUGAGAAUUA
[2mUp|2mCp|2mUp|2mCp|2mCp|rAp|rAp|rAp|2mC

p|2mCp|2mUp|rGp|rAp|rG

p|rAp|rAp|2mUp|2mUp|rAp|dTp|dT]

970
AACCUGAGAAUUUUAAACA
[rAp|rAp|2mCp|2mCp|2mUp|rGp|rAp|rGp|rAp|r

Ap|2mUp|2mUp|2mUp|2mUp|r

Ap|rAp|rAp|2mCp|rAp|dTp|dT]

972
CCUGAGAAUUUUAAACAGA
[2mCp|2mCp|2mUp|rGp|rAp|rGp|rAp|rAp|2mUp|

2mUp|2mUp|2mUp|rAp|rAp|r

Ap|2mCp|rAp|rGp|rAp|dTp|dT]

988
GCUAUCAAGUAAAAUGGAA
[rGp|2mCp|2mUp|rAp|2mUp|2mCp|rAp|rAp|rGp|

2mUp|rAp|rAp|rAp|rAp|2mUp|

rGp|rGp|rAp|rAp|dTp|dT]

990
CUAUCAAGUAAAAUGGAAA
[2mCp|2mUp|rAp|2mUp|2mCp|rAp|rAp|rGp|2mUp

|rAp|rAp|rAp|rAp|2mUp|rGp|

rGp|rAp|rAp|rAp|dTp|dT]

992
UAUCAAGUAAAAUGGAAUA
[2mUp|rAp|2mUp|2mCp|rAp|rAp|rGp|2mUp|rAp|

rAp|rAp|rAp|2mUp|rGp|rGp|rA

p|rAp|2mUp|rAp|dTp|dT]

994
UCAAGUAAAAUGGAAUUUA
[2mUp|2mCp|rAp|rAp|rGp|2mUp|rAp|rAp|rAp|r

Ap|2mUp|rGp|rGp|rAp|rAp|2mU

p|2mUp|2mUp|rAp|dTp|dT]

996
CAAGUAAAAUGGAAUUUAA
[2mCp|rAp|rAp|rGp|2mUp|rAp|rAp|rAp|rAp|2m

Up|rGp|rGp|rAp|rAp|2mUp|2mU

p|2mUp|rAp|rAp|dTp|dT]

1006
UUAUGACAAUUAAUGGAAA
[2mUp|2mUp|rAp|2mUp|rGp|rAp|2mCp|rAp|rAp|

2mUp|2mUp|rAp|rAp|2mUp|r

Gp|rGp|rAp|rAp|rAp|dTp|dT]

1020
GGAACAACAUUAAGGAAUA
[rGp|rGp|rAp|rAp|2mCp|rAp|rAp|2mCp|rAp|2m

Up|2mUp|rAp|rAp|rGp|rGp|rAp|

rAp|2mUp|rAp|dTp|dT]

1032
CUGGAAAUCCUACAGAAUA
[2mCp|2mUp|rGp|rGp|rAp|rAp|rAp|2mUp|2mCp|

2mCp|2mUp|rAp|2mCp|rAp|r

Gp|rAp|rAp|2mUp|rAp|dTp|dT]

1054
UCAGACUGAUAUGAAAACA
[2mUp|2mCp|rAp|rGp|rAp|2mCp|2mUp|rGp|rAp|

2mUp|rAp|2mUp|rGp|rAp|rAp

|rAp|rAp|2mCp|rAp|dTp|dT]

1056
GACUGAUAUGAAAACCAAA
[rGp|rAp|2mCp|2mUp|rGp|rAp|2mUp|rAp|2mUp|

rGp|rAp|rAp|rAp|rAp|2mCp|2

mCp|rAp|rAp|rAp|dTp|dT]

1058
ACUGAUAUGAAAACCAAAA
[rAp|2mCp|2mUp|rGp|rAp|2mUp|rAp|2mUp|rGp|

rAp|rAp|rAp|rAp|2mCp|2mCp|

rAp|rAp|rAp|rAp|dTp|dT]

1076
UUGCUGUGGGUUUUAGACA
[2mUp|2mUp|rGp|2mCp|2mUp|rGp|2mUp|rGp|rGp

|rGp|2mUp|2mUp|2mUp|2

mUp|rAp|rGp|rAp|2mCp|rAp|dTp|dT]

1088
GUUUUAGACCACACUAAAA
[rGp|2mUp|2mUp|2mUp|2mUp|rAp|rGp|rAp|2mCp

|2mCp|rAp|2mCp|rAp|2mC

p|2mUp|rAp|rAp|rAp|rAp|dTp|dT]

1096
AGACCACACUAAAACUUCA
[rAp|rGp|rAp|2mCp|2mCp|rAp|2mCp|rAp|2mCp|

2mUp|rAp|rAp|rAp|rAp|2mCp|

2mUp|2mUp|2mCp|rAp|dTp|dT]

1098
CCACACUAAAACUUCAUUA
[2mCp|2mCp|rAp|2mCp|rAp|2mCp|2mUp|rAp|rAp

|rAp|rAp|2mCp|2mUp|2mUp

|2mCp|rAp|2mUp|2mUp|rAp|dTp|dT]

1110
GGGAGACGGAAGUUAAAGA
[rGp|rGp|rGp|rAp|rGp|rAp|2mCp|rGp|rGp|rAp

|rAp|rGp|2mUp|2mUp|rAp|rAp|rA

p|rGp|rAp|dTp|dT]

1112
GGAGACGGAAGUUAAAGAA
[rGp|rGp|rAp|rGp|rAp|2mCp|rGp|rGp|rAp|rAp

|rGp|2mUp|2mUp|rAp|rAp|rAp|rG

p|rAp|rAp|dTp|dT]

1126
CCCAGCCACUCCUUAAAUA
[2mCp|2mCp|2mCp|rAp|rGp|2mCp|2mCp|rAp|2mC

p|2mUp|2mCp|2mCp|2mU

p|2mUp|rAp|rAp|rAp|2mUp|rAp|dTp|dT]

1214
UUUGGUCAGAUAGAAAAUA
[2mUp|2mUp|2mUp|rGp|rGp|2mUp|2mCp|rAp|rGp

|rAp|2mUp|rAp|rGp|Ap|Ap

|rAp|rAp|2mUp|rAp|dTp|dT]

1220
CAGAUAGAAAAUCAUCUAA
[2mCp|rAp|rGp|rAp|2mUp|rAp|rGp|rAp|rAp|rA

p|rAp|2mUp|2mCp|rAp|2mUp|2

mCp|2mUp|rAp|rAp|dTp|dT]

1230
AAUCAUCUACGUAAAUUGA
[rAp|rAp|2mUp|2mCp|rAp|2mUp|2mCp|2mUp|rAp

|2mCp|rGp|2mUp|rAp|rAp|r

Ap|2mUp|2mUp|rGp|rAp|dTp|dT]

1306
AGAAUUUCAAGCAAUAAUA
[rAp|rGp|rAp|rAp|2mUp|2mUp|2mUp|2mCp|rAp|

rAp|rGp|2mCp|rAp|rAp|2mUp|

rAp|rAp|2mUp|rAp|dTp|dT]

1308
AUUUCAAGCAAUAAUACCA
[rAp|2mUp|2mUp|2mUp|2mCp|rAp|rAp|rGp|2mCp

|rAp|rAp|2mUp|rAp|rAp|2m

Up|rAp|2mCp|2mCp|rAp|dTp|dT]

1310
UUUCAAGCAAUAAUACCUA
[2mUp|2mUp|2mUp|2mCp|rAp|rAp|rGp|2mCp|rAp

|rAp|2mUp|rAp|rAp|2mUp|r

Ap|2mCp|2mCp|2mUp|rAp|dTp|dT]

1318
AUACCUGCUGUUAAUUCCA
[rAp|2mUp|rAp|2mCp|2mCp|2mUp|rGp|2mCp|2mU

p|rGp|2mUp|2mUp|rAp|rA

p|2mUp|2mUp|2mCp|2mCp|rAp|dTp|dT]

1326
ACCGUUAUUUUAGAAAUUA
[rAp|2mCp|2mCp|rGp|2mUp|2mUp|rAp|2mUp|2mU

p|2mUp|2mUp|rAp|rGp|rA

p|rAp|rAp|2mUp|2mUp|rAp|dTp|dT]

1386
UCCAGUGGAGCAUUACUUA
[2mUp|2mCp|2mCp|rAp|rGp|2mUp|rGp|rGp|rAp|

rGp|2mCp|rAp|2mUp|2mUp|r

Ap|2mCp|2mUp|2mUp|rAp|dTp|dT]

1394
GAGCAUUACUUAAAGAUAA
[rGp|rAp|rGp|2mCp|rAp|2mUp|2mUp|rAp|2mCp|

2mUp|2mUp|rAp|rAp|rAp|rGp

|rAp|2mUp|rAp|rAp|dTp|dT]

1396
AGCAUUACUUAAAGAUACA
[rAp|rGp|2mCp|rAp|2mUp|2mUp|rAp|2mCp|2mUp

|2mUp|rAp|rAp|rAp|rGp|rAp|

2mUp|rAp|2mCp|rAp|dTp|dT]

1400
UACUUAAAGAUACUCAAUA
[2mUp|rAp|2mCp|2mUp|2mUp|rAp|rAp|rAp|rGp|

rAp|2mUp|rAp|2mCp|2mUp|2

mCp|rAp|rAp|2mUp|rAp|dTp|dT]

1404
UUAAAGAUACUCAAUGAAA
[2mUp|2mUp|rAp|rAp|rAp|rGp|rAp|2mUp|rAp|2

mCp|2mUp|2mCp|rAp|rAp|2m

Up|rGp|rAp|rAp|rAp|dTp|dT]

1424
GUUGGGGAUAAAACUGAAA
[rGp|2mUp|2mUp|rGp|rGp|rGp|rGp|rAp|2mUp|r

Ap|rAp|rAp|rAp|2mCp|2mUp|r

Gp|rAp|rAp|rAp|dTp|dT]

1426
UUGGGGAUAAAACUGAAUA
[2mUp|2mUp|rGp|rGp|rGp|rGp|rAp|2mUp|rAp|r

Ap|rAp|rAp|2mCp|2mUp|rGp|r

Ap|rAp|2mUp|rAp|dTp|dT]

1448
UUCUGACUUCCCUUUAAUA
[2mUp|2mUp|2mCp|2mUp|rGp|rAp|2mCp|2mUp|2m

Up|2mCp|2mCp|2mCp|2

mUp|2mUp|2mUp|rAp|rAp|2mUp|rAp|dTp|dT]

1452
UGACUUCCCUUUAAUAAAA
[2mUp|rGp|rAp|2mCp|2mUp|2mUp|2mCp|2mCp|2m

Cp|2mUp|2mUp|2mUp|rA

2mUp|rAp|2mUp|rAp|dTp|dT]

1454
AGAGGAAGGAUGAAAUUCA
[rAp|rGp|rAp|rGp|rGp|rAp|rAp|rGp|rGp|rAp|

2mUp|rGp|rAp|rAp|rAp|2mUp|2mU

p|2mCp|rAp|dTp|dT]

1506
AUCCUUCUGCACAAUAUGA
[rAp|2mUp|2mCp|2mCp|2mUp|2mUp|2mCp|2mUp|r

Gp|2mCp|rAp|2mCp|rAp|r

Ap|2mUp|rAp|2mUp|rGp|rAp|dTp|dT]

1524
CAGGACAGGAGUUUAUGAA
[2mCp|rAp|rGp|rGp|rAp|2mCp|rAp|rGp|rGp|rA

p|rGp|2mUp|2mUp|2mUp|rAp|2

mUp|rGp|rAp|rAp|dTp|dT]

1540
AGAACUCUGCUGUAUCUUA
[rAp|rGp|rAp|rAp|2mCp|2mUp|2mCp|2mUp|rGp|

2mCp|2mUp|rGp|2mUp|rAp|2

mUp|2mCp|2mUp|2mUp|rAp|dTp|dT]

1546
CUCUGCUGUAUCUUGUAUA
[2mCp|2mUp|2mCp|2mUp|rGp|2mCp|2mUp|rGp|2m

Up|rAp|2mUp|2mCp|2mU

p|2mUp|rGp|2mUp|rAp|2mUp|rAp|dTp|dT]

1656
GAAUGGCUUGAUUUUCUAA
[rGp|rAp|rAp|2mUp|rGp|rGp|2mCp|2mUp|2mUp|

rGp|rAp|2mUp|2mUp|2mUp|2

mUp|2mCp|2mUp|rAp|rAp|dTp|dT]

1666
UUUCUAGAGAAAUUCAGUA
[2mUp|2mUp|2mUp|2mCp|2mUp|rAp|rGp|rAp|rGp

|rAp|rAp|rAp|2mUp|2mUp|2

mCp|rAp|rGp|2mUp|rAp|dTp|dT]

1674
AAAUUCAGUGAACAUUAUA
[rAp|rAp|rAp|2mUp|2mUp|2mCp|rAp|rGp|2mUp|

rGp|rAp|rAp|2mCp|rAp|2mUp|

2mUp|rAp|2mUp|rAp|dTp|dT]

1676
AAUUCAGUGAACAUUAUCA
[rAp|rAp|2mUp|2mUp|2mCp|rAp|rGp|2mUp|rGp|

rAp|rAp|2mCp|rAp|2mUp|2m

Up|rAp|2mUp|2mCp|rAp|dTp|dT]

1678
AUUCAGUGAACAUUAUCAA
[rAp|2mUp|2mUp|2mCp|rAp|rGp|2mUp|rGp|rAp|

rAp|2mCp|rAp|2mUp|2mUp|r

Ap|2mUp|2mCp|rAp|rAp|dTp|dT]

1722
UGUUGACUGCAUUUUCUCA
[2mUp|rGp|2mUp|2mUp|rGp|rAp|2mCp|2mUp|rGp

|2mCp|rAp|2mUp|2mUp|2

mUp|2mUp|2mCp|2mUp|2mCp|rAp|dTp|dT]

1762
AGACCAACUGUACAAGAAA
[rAp|rGp|rAp|2mCp|2mCp|rAp|rAp|2mCp|2mUp|

rGp|2mUp|rAp|2mCp|rAp|rAp|

rGp|rAp|rAp|rAp|dTp|dT]

1766
ACUGUACAAGAAGAAAGAA
[rAp|2mCp|2mUp|rGp|2mUp|rAp|2mCp|rAp|rAp

|rGp|rAp|rAp|rGp|rAp|rAp|rAp|r

Gp|rAp|rAp|dTp|dT]

1768
CUGUACAAGAAGAAAGAAA
[2mCp|2mUp|rGp|2mUp|rAp|2mCp|rAp|rAp|rGp|

rAp|rAp|rGp|rAp|rAp|rAp|rGp|

rAp|rAp|rAp|dTp|dT]

1836
ACUCAGAGAGAGUAAUGAA
[rAp|2mCp|2mUp|2mCp|rAp|rGp|rAp|rGp|rAp|r

Gp|rAp|rGp|2mUp|rAp|rAp|2m

Up|rGp|rAp|rAp|dTp|dT]

1838
UCAGAGAGAGUAAUGAUAA
[2mUp|2mCp|rAp|rGp|rAp|rGp|rAp|rGp|rAp|rG

p|2mUp|rAp|rAp|2mUp|rGp|rAp|

2mUp|rAp|rAp|dTp|dT]

1842
AGAGAGAGUAAUGAUAAUA
[rAp|rGp|rAp|rGp|rAp|rGp|rAp|rGp|2mUp|rAp

|rAp|2mUp|rGp|rAp|2mUp|rAp|rA

p|2mUp|rAp|dTp|dT]

1868
GAAAGAGCUCCUACAUAAA
[rGp|rAp|rAp|rAp|rGp|rAp|rGp|2mCp|2mUp|2m

Cp|2mCp|2mUp|rAp|2mCp|rAp

|2mUp|rAp|rAp|rAp|dTp|dT]

1886
AAACAAGUUGCAUUGAUUA
[rAp|rAp|rAp|2mCp|rAp|rAp|rGp|2mUp|2mUp|r

Gp|2mCp|rAp|2mUp|2mUp|rGp

|rAp|2mUp|2mUp|rAp|dTp|dT]

1888
AACAAGUUGCAUUGAUUAA
[rAp|rAp|2mCp|rAp|rAp|rGp|2mUp|2mUp|rGp|2

mCp|rAp|2mUp|2mUp|rGp|rAp

|2mUp|2mUp|rAp|rAp|dTp|dT]

1964
GUGGAUGGCAUUUUCACAA
[rGp|2mUp|rGp|rGp|rAp|2mUp|rGp|rGp|2mCp|r

Ap|2mUp|2mUp|2mUp|2mUp|

2mCp|rAp|2mCp|rAp|rAp|dTp|dT]

1990
UGCAGACAAUAUAUAUAAA
[2mUp|rGp|2mCp|rAp|rGp|rAp|2mCp|rAp|rAp|2

mUp|rAp|2mUp|rAp|2mUp|rAp|

2mUp|rAp|rAp|rAp|dTp|dT]

2030
GACACAGCAGAAAUAAUCA
[rGp|rAp|2mCp|rAp|2mCp|rAp|rGp|2mCp|rAp|r

Gp|rAp|rAp|rAp|2mUp|rAp|rAp|

2mUp|2mCp|rAp|dTp|dT]

2108
AUGCUACACUUGAGUAUUA
[rAp|2mUp|rGp|2mCp|2mUp|rAp|2mCp|rAp|2mCp

|2mUp|2mUp|rGp|rAp|rGp|2

mUp|rAp|2mUp|2mUp|rAp|dTp|dT]

2128
GAGAUGUGAAAUCCUUAAA
[rGp|rAp|rGp|rAp|2mUp|rGp|2mUp|rGp|rAp|rA

p|rAp|2mUp|2mCp|2mCp|2mUp

2mUp|rAp|rAp|rAp|dTp|dT]

2230
AACAAGUCCCUGAUUUUGA
[rAp|rAp|2mCp|rAp|rAp|rGp|2mUp|2mCp|2mCp|

2mCp|2mUp|rGp|rAp|2mUp|2

mUp|2mUp|2mUp|rGp|rAp|dTp|dT]

2242
UUUUGUCACCUUCCUUUAA
[2mUp|2mUp|2mUp|2mUp|rGp|2mUp|2mCp|rAp|2m

Cp|2mCp|2mUp|2mUp|2

mCp|2mCp|2mUp|2mUp|2mUp|rAp|rAp|dTp|dT]

2246
UCACCUUCCUUUACCAAAA
[2mUp|2mCp|rAp|2mCp|2mCp|2mUp|2mUp|2mCp|2

mCp|2mUp|2mUp|2mUp|

rAp|2mCp|2mCp|rAp|rAp|rAp|rAp|dTp|dT]

2254
CUUUACCAAAUAACUAGAA
[2mCp|2mUp|2mUp|2mUp|rAp|2mCp|2mCp|rAp|rA

p|rAp|2mUp|rAp|rAp|2mCp

|2mUp|rAp|rGp|rAp|rAp|dTp|dT]

2274
AAGGAGUUAUGGAUUAAAA
[rAp|rAp|rGp|rGp|rAp|rGp|2mUp|2mUp|rAp|2m

Up|rGp|rGp|rAp|2mUp|2mUp|r

Ap|rAp|rAp|rAp|dTp|dT]

2294
UAAAUGUGGCUAAACUAGA
[2mUp|rAp|rAp|rAp|2mUp|rGp|2mUp|rGp|rGp|2

mCp|2mUp|rAp|rAp|rAp|2mCp

|2mUp|rAp|rGp|rAp|dTp|dT]

2330
GAGCUGGAAGGAUUAAUAA
[rGp|rAp|rGp|2mCp|2mUp|rGp|rGp|rAp|rAp|rG

p|rGp|rAp|2mUp|2mUp|rAp|rAp|

2mUp|rAp|rAp|dTp|dT]

2334
GGAUUAAUAAAUACGAAAA
[rGp|rGp|rAp|2mUp|2mUp|rAp|rAp|2mUp|rAp|r

Ap|rAp|2mUp|rAp|2mCp|rGp|rA

p|rAp|rAp|rAp|dTp|dT]

2356
ACUCAAGUAUUUUGCAAAA
[rAp|2mCp|2mUp|2mCp|rAp|rAp|rGp|2mUp|rAp|

2mUp|2mUp|2mUp|2mUp|rG

p|2mCp|rAp|rAp|rAp|rAp|dTp|dT]

2360
AAGUAUUUUGCAAAGUUAA
[rAp|rAp|rGp|2mUp|rAp|2mUp|2mUp|2mUp|2mUp

|rGp|2mCp|rAp|rAp|rAp|rGp

|2mUp|2mUp|rAp|rAp|dTp|dT]

2362
GUAUUUUGCAAAGUUAUGA
[rGp|2mUp|rAp|2mUp|2mUp|2mUp|2mUp|rGp|2mC

p|rAp|rAp|rAp|rGp|2mUp|2

mUp|rAp|2mUp|rGp|rAp|dTp|dT]

2448
CAACUGUACAAAAUAACUA
[2mCp|rAp|rAp|2mCp|2mUp|rGp|2mUp|rAp|2mCp

|rAp|rAp|rAp|rAp|2mUp|rAp|

rAp|2mCp|2mUp|rAp|dTp|dT]

2502
ACAUGUGAGCAUAAAAUUA
[rAp|2mCp|rAp|2mUp|rGp|2mUp|rGp|rAp|rGp|2

mCp|rAp|2mUp|rAp|rAp|rAp|rA

p|2mUp|2mUp|rAp|dTp|dT]

2504
CAUGUGAGCAUAAAAUUAA
[2mCp|rAp|2mUp|rGp|2mUp|rGp|rAp|rGp|2mCp|

rAp|2mUp|rAp|rAp|rAp|rAp|2

mUp|2mUp|rAp|rAp|dTp|dT]

2516
AAUUAUGACCAUGGUAUAA
[rAp|rAp|2mUp|2mUp|rAp|2mUp|rGp|rAp|2mCp|

2mCp|rAp|2mUp|rGp|rGp|2m

Up|rAp|2mUp|rAp|rAp|dTp|dT]

2518
AUUAUGACCAUGGUAUAUA
[rAp|2mUp|2mUp|rAp|2mUp|rGp|rAp|2mCp|2mCp

|rAp|2mUp|rGp|rGp|2mUp|r

Ap|2mUp|rAp|2mUp|rAp|dTp|dT]

2578
AUAAACACUCUUGAAUAGA
[rAp|2mUp|rAp|rAp|rAp|2mCp|rAp|2mCp|2mUp|

2mCp|2mUp|2mUp|rGp|rAp|r

Ap|2mUp|rAp|rGp|rAp|dTp|dT]

2580
UAAACACUCUUGAAUAGAA
[2mUp|rAp|rAp|rAp|2mCp|rAp|2mCp|2mUp|2mCp

|2mUp|2mUp|rGp|rAp|rAp|2

mUp|rAp|rGp|rAp|rAp|dTp|dT]

2592
AUAGACUUCCACUUUGUAA
[rAp|2mUp|rAp|rGp|rAp|2mCp|2mUp|2mUp|2mCp

|2mCp|rAp|2mCp|2mUp|2m

Up|2mUp|rGp|2mUp|rAp|rAp|dTp|dT]

2596
AGACUUCCACUUUGUAAUA
[rAp|rGp|rAp|2mCp|2mUp|2mUp|2mCp|2mCp|rAp

|2mCp|2mUp|2mUp|2mUp|r

Gp|2mUp|rAp|rAp|2mUp|rAp|dTp|dT]

2602
CUUCCACUUUGUAAUUAGA
[2mCp|2mUp|2mUp|2mCp|2mCp|rAp|2mCp|2mUp|2

mUp|2mUp|rGp|2mUp|rA

p|rAp|2mUp|2mUp|rAp|rGp|Ap|dTp|dT]

2654
CUUAAGUGGCAGAAUAUAA
[2mCp|2mUp|2mUp|rAp|rAp|rGp|2mUp|rGp|rGp|

2mCp|rAp|rGp|rAp|rAp|2mUp

|rAp|2mUp|rAp|rAp|dTp|dT]

2656
UUAAGUGGCAGAAUAUAAA
[2mUp|2mUp|rAp|rAp|rGp|2mUp|rGp|rGp|2mCp|

rAp|rGp|rAp|rAp|2mUp|rAp|2

mUp|rAp|rAp|rAp|dTp|dT]

2686
UUUUGGAGGGUGAUAUAAA
[2mUp|2mUp|2mUp|2mUp|rGp|rGp|rAp|rGp|rGp|

rGp|2mUp|rGp|rAp|2mUp|rA

p|2mUp|rAp|rAp|rAp|dTp|dT]

2762
UCCACCAAGAACAUAAGAA
[2mUp|2mCp|2mCp|rAp|2mCp|2mCp|rAp|rAp|rGp

|rAp|rAp|2mCp|rAp|2mUp|r

Ap|rAp|rGp|rAp|rAp|dTp|dT]

2768
ACCAAGAACAUAAGAAUUA
[rAp|2mCp|2mCp|rAp|rAp|rGp|rAp|rAp|2mCp|

rAp|2mUp|rAp|rAp|rGp|rAp|rAp|

2mUp|2mUp|rAp|dTp|dT]

2782
UAGAAUUAUCAAGCUUUUA
[2mUp|rAp|rGp|rAp|rAp|2mUp|2mUp|rAp|2mUp|

2mCp|rAp|rAp|rGp|2mCp|2m

Up|2mUp|2mUp|2mUp|rAp|dTp|dT]

2844
UUGGAUGAAAUUAUUUGUA
[2mUp|2mUp|rGp|rGp|rAp|2mUp|rGp|rAp|rAp|r

Ap|2mUp|2mUp|rAp|2mUp|2m

Up|2mUp|rGp|2mUp|rAp|dTp|dT]

2846
GAUGAAAUUAUUUGUCAUA
[rGp|rAp|2mUp|rGp|rAp|rAp|rAp|2mUp|2mUp|r

Ap|2mUp|2mUp|2mUp|rGp|2m

Up|2mCp|rAp|2mUp|rAp|dTp|dT]

For the chemical modifications, “2m” means 2′-O-Methyl ribonucleotides, “r” means ribonucleotide, “p” means phosphate linkage, and “d” means deoxyribonucleotide.

TABLE 14

Chemical Modifications for Antisense Strands

SEQ ID

No./

Antisense

No.
Antisense
Antisense Chemical Modifications

79
UAUUUCUUUGGCUCAGAGU
[rUp|rAp|rUp|rUp|rUp|rCp|rUp|rUp|rUp|

rGp|rGp|rCp|rUp|2mCp|rAp|rGp|rAp|rG

p|rUp|dTp|dT]

83
UAGACAUUUCUUUGGCUCA
[rUp|rAp|rGp|rAp|2mCp|rAp|rUp|rUp|rUp

|rCp|rUp|rUp|rUp|rGp|rGp|rCp|rUp|2

mCp|rAp|dTp|dT]

105
UUACUUUGAGGAAGGGCAG
[rUp|2mUp|rAp|rCp|rUp|rUp|rUp|rGp|rAp

|rGp|rGp|rAp|rAp|rGp|rGp|rGp|2mCp|

rAp|rGp|dTp|dT]

149
UGUACAUUUUGGCAGAACU
[rUp|rGp|2mUp|rAp|2mCp|rAp|rUp|rUp|rU

p|rUp|rGp|rGp|2mCp|rAp|rGp|rAp|rA

p|rCp|rUp|dTp|dT]

159
UUAUCAUCAAAAUCAGUAC
[rUp|2mUp|rAp|rUp|2mCp|rAp|rUp|2mCp|r

Ap|rAp|rAp|rAp|rUp|2mCp|rAp|rGp|

2mUp|rAp|rCp|dTp|dT]

161
UGAUAUCAUCAAAAUCAGU
[rUp|rGp|rAp|2mUp|rAp|rUp|2mCp|rAp|rU

p|2mCp|rAp|rAp|rAp|rAp|rUp|2mCp|r

Ap|rGp|rUp|dTp|dT]

191
UAGAAGAAACUGCAUUCUU
[rUp|rAp|rGp|rAp|rAp|rGp|rAp|rAp|rAp|

rCp|rUp|rGp|2mCp|rAp|rUp|rUp|rCp|rU

p|rUp|dTp|dT]

241
UAUUUGAUGAUCCAAACUG
[rUp|rAp|rUp|rUp|rUp|rGp|rAp|rUp|rGp|

rAp|rUp|rCp|2mCp|rAp|rAp|rAp|rCp|rU

p|rGp|dTp|dT]

245
UGUAUUUGAUGAUCCAAAC
[rUp|rGp|2mUp|rAp|rUp|rUp|rUp|rGp|rAp

|rUp|rGp|rAp|rUp|rCp|2mCp|rAp|rAp|r

Ap|rCp|dTp|dT]

247
UUGUAUUUGAUGAUCCAAA
[rUp|rUp|rGp|2mUp|rAp|rUp|rUp|rUp|rGp

|rAp|rUp|rGp|rAp|rUp|rCp|2mCp|rAp|r

Ap|rAp|dTp|dT]

251
UCUUGUAUUUGAUGAUCCA
[rUp|rCp|rUp|rUp|rGp|2mUp|rAp|rUp|rUp

|rUp|rGp|rAp|rUp|rGp|rAp|rUp|rCp|2

mCp|rAp|dTp|dT]

253
UACUUGUAUUUGAUGAUCC
[rUp|rAp|rCp|rUp|rUp|rGp|2mUp|rAp|rUp

|rUp|rUp|rGp|rAp|rUp|rGp|rAp|rUp|rC

p|rCp|dTp|dT]

261
UAAAUUUUCAUGACUUGUA
[rUp|rAp|rAp|rAp|rUp|rUp|rUp|rUp|2mCp

|rAp|rUp|rGp|rAp|rCp|rUp|rUp|rGp|2m

Up|rAp|dTp|dT]

309
UGUAUUGUAAUUCUAGCGG
[rUp|rGp|2mUp|rAp|rUp|rUp|rGp|2mUp|rAp

|rAp|rUp|rUp|rCp|2mUp|rAp|rGp|rC

p|rGp|rGp|dTp|dT]

315
UUCUAUGUAUUGUAAUUCU
[rUp|rUp|rCp|2mUp|rAp|rUp|rGp|2mUp|rA

p|rUp|rUp|rGp|2mUp|rAp|rAp|rUp|rU

p|rCp|rUp|dTp|dT]

317
UUUCUAUGUAUUGUAAUUC
[rUp|rUp|rUp|rCp|2mUp|rAp|rUp|rGp|2mU

p|rAp|rUp|rUp|rGp|2mUp|rAp|rAp|rU

p|rUp|rCp|dTp|dT]

355
UUAUAUCCACAUUCCACAC
[rUp|2mUp|rAp|2mUp|rAp|rUp|rCp|2mCp|r

Ap|2mCp|rAp|rUp|rUp|rCp|2mCp|rA

p|2mCp|rAp|rCp|dTp|dT]

357
UUUAUAUCCACAUUCCACA
[rUp|rUp|2mUp|rAp|2mUp|rAp|rUp|rCp|2m

Cp|rAp|2mCp|rAp|rUp|rUp|rCp|2mC

p|rAp|2mCp|rAp|dTp|dT]

361
UACUUAUAUCCACAUUCCA
[rUp|rAp|rCp|rUp|2mUp|rAp|2mUp|rAp|rU

p|rCp|2mCp|rAp|2mCp|rAp|rUp|rUp|r

Cp|2mCp|rAp|dTp|dT]

363
UUACUUAUAUCCACAUUCC
[rUp|2mUp|rAp|rCp|rUp|2mUp|rAp|2mUp|r

Ap|rUp|rCp|2mCp|rAp|2mCp|rAp|rU

p|rUp|rCp|rCp|dTp|dT]

365
UAUACUUAUAUCCACAUUC
[rUp|rAp|2mUp|rAp|rCp|rUp|2mUp|rAp|2m

Up|rAp|rUp|rCp|2mCp|rAp|2mCp|rA

p|rUp|rUp|rCp|dTp|dT]

369
UCUAUACUUAUAUCCACAU
[rUp|rCp|2mUp|rAp|2mUp|rAp|rCp|rUp|2m

Up|rAp|2mUp|rAp|rUp|rCp|2mCp|rA

p|2mCp|rAp|rUp|dTp|dT]

371
UUCUAUACUUAUAUCCACA
[rUp|rUp|rCp|2mUp|rAp|2mUp|rAp|rCp|rU

p|2mUp|rAp|2mUp|rAp|rUp|rCp|2mC

p|rAp|2mCp|rAp|dTp|dT]

373
UAAUCUAUACUUAUAUCCA
[rUp|rAp|rAp|rUp|rCp|2mUp|rAp|2mUp|rA

p|rCp|rUp|2mUp|rAp|2mUp|rAp|rUp|r

Cp|2mCp|rAp|dTp|dT]

379
UGCAAUAAAUAUUGAGCUC
[rUp|rGp|2mCp|rAp|rAp|2mUp|rAp|rAp|rA

p|2mUp|rAp|rUp|rUp|rGp|rAp|rGp|rC

p|rUp|rCp|dTp|dT]

415
UAUAAAGUUGUGAUCUAAA
[rUp|rAp|2mUp|rAp|rAp|rAp|rGp|rUp|rUp

|rGp|rUp|rGp|rAp|rUp|rCp|2mUp|rAp|r

Ap|rAp|dTp|dT]

417
UCAUAAAGUUGUGAUCUAA
[rUp|2mCp|rAp|2mUp|rAp|rAp|rAp|rGp|rU

p|rUp|rGp|rUp|rGp|rAp|rUp|rCp|2mU

p|rAp|rAp|dTp|dT]

419
UUCAUAAAGUUGUGAUCUA
[rUp|rUp|2mCp|rAp|2mUp|rAp|rAp|rAp|rG

p|rUp|rUp|rGp|rUp|rGp|rAp|rUp|rCp|

2mUp|rAp|dTp|dT]

475
UUUAUAUCCUUUUGCCACC
[rUp|rUp|2mUp|rAp|2mUp|rAp|rUp|rCp|rC

p|rUp|rUp|rUp|rUp|rGp|rCp|2mCp|rA

p|rCp|rCp|dTp|dT]

477
UCUUAUAUCCUUUUGCCAC
[rUp|rCp|rUp|2mUp|rAp|2mUp|rAp|rUp|r

Cp|rCp|rUp|rUp|rUp|rUp|rGp|rCp|2mC

p|rAp|rCp|dTp|dT]

479
UCCUUAUAUCCUUUUGCCA
[rUp|rCp|rCp|rUp|2mUp|rAp|2mUp|rAp|rU

p|rCp|rCp|rUp|rUp|rUp|rUp|rGp|rCp|

2mCp|rAp|dTp|dT]

481
UACCUUAUAUCCUUUUGCC
[rUp|rAp|rCp|rCp|rUp|2mUp|rAp|2mUp|rA

p|rUp|rCp|rCp|rUp|rUp|rUp|rUp|rGp|r

Cp|rCp|dTp|dT]

503
UUUCAGUUUGCUUCACAAC
[rUp|rUp|rUp|2mCp|rAp|rGp|rUp|rUp|rUp

|rGp|rCp|rUp|rUp|2mCp|rAp|2mCp|rA

p|rAp|rCp|dTp|dT]

513
UUUAAUGCUGCAGUUUCAG
[rUp|rUp|2mUp|rAp|rAp|rUp|rGp|rCp|rUp

|rGp|2mCp|rAp|rGp|rUp|rUp|rUp|2mC

p|rAp|rGp|dTp|dT]

553
UAAAAGAGUGAACUUCUGU
[rUp|rAp|rAp|rAp|rAp|rGp|rAp|rGp|rUp|

rGp|rAp|rAp|rCp|rUp|rUp|rCp|rUp|rGp|

rUp|dTp|dT]

559
UUCAAUUUCCGGGAAAAGA
[rUp|rUp|2mCp|rAp|rAp|rUp|rUp|rUp|rCp

|rCp|rGp|rGp|rGp|rAp|rAp|rAp|rAp|rG

p|rAp|dTp|dT]

561
UGUCAAUUUCCGGGAAAAG
[rUp|rGp|rUp|2mCp|rAp|rAp|rUp|rUp|rUp

|rCp|rCp|rGp|rGp|rGp|rAp|rAp|rAp|rA

p|rGp|dTp|dT]

583
UGUAGAUUUUGUAUAAAGG
[rUp|rGp|2mUp|rAp|rGp|rAp|rUp|rUp|rUp

|rUp|rGp|2mUp|rAp|2mUp|rAp|rAp|rA

p|rGp|rGp|dTp|dT]

617
UUUGAUUAGGGGAUUCACA
[rUp|rUp|rUp|rGp|rAp|rUp|2mUp|rAp|rGp

|rGp|rGp|rGp|rAp|rUp|rUp|2mCp|rAp|

2mCp|rAp|dTp|dT]

619
UCUUGAUUAGGGGAUUCAC
[rUp|rCp|rUp|rUp|rGp|rAp|rUp|2mUp|rAp

|rGp|rGp|rGp|rGp|rAp|rUp|rUp|2mCp|

rAp|rCp|dTp|dT]

635
UCAUUUACAGCAUCAUCCA
[rUp|2mCp|rAp|rUp|rUp|2mUp|rAp|2mCp|r

Ap|rGp|2mCp|rAp|rUp|2mCp|rAp|rU

p|rCp|2mCp|rAp|dTp|dT]

637
UAACAUUUACAGCAUCAUC
[rUp|rAp|rAp|2mCp|rAp|rUp|rUp|2mUp|rA

p|2mCp|rAp|rGp|2mCp|rAp|rUp|2mC

p|rAp|rUp|rCp|dTp|dT]

643
UUUAUCUCAUCAACAUUUA
[rUp|rUp|2mUp|rAp|rUp|rCp|rUp|2mCp|rA

p|rUp|2mCp|rAp|rAp|2mCp|rAp|rUp|r

Up|2mUp|rAp|dTp|dT]

647
UGUCAUUAUCUCAUCAACA
[rUp|rGp|rUp|2mCp|rAp|rUp|2mUp|rAp|rU

p|rCp|rUp|2mCp|rAp|rUp|2mCp|rAp|

rAp|2mCp|rAp|dTp|dT]

649
UAGUCAUUAUCUCAUCAAC
[rUp|rAp|rGp|rUp|2mCp|rAp|rUp|2mUp|rA

p|rUp|rCp|rUp|2mCp|rAp|rUp|2mCp|

rAp|rAp|rCp|dTp|dT]

657
UAAGUAUCAGUCAUUAUCU
[rUp|rAp|rAp|rGp|2mUp|rAp|rUp|2mCp|rA

p|rGp|rUp|2mCp|rAp|rUp|2mUp|rAp|

rUp|rCp|rUp|dTp|dT]

661
UAGAAGUAUCAGUCAUUAU
[rUp|rAp|rGp|rAp|rAp|rGp|2mUp|rAp|rUp

|2mCp|rAp|rGp|rUp|2mCp|rAp|rUp|2

mUp|rAp|rUp|dTp|dT]

691
UUUCCUUAUUUUCAGAGAU
[rUp|rUp|rUp|rCp|rCp|rUp|2mUp|rAp|rUp

|rUp|rUp|rUp|2mCp|rAp|rGp|rAp|rGp|r

Ap|rUp|dTp|dT]

693
UAACAUUUUCCUUAUUUUC
[rUp|rAp|rAp|2mCp|rAp|rUp|rUp|rUp|rUp

|rCp|rCp|rUp|2mUp|rAp|rUp|rUp|rUp|r

Up|rCp|dTp|dT]

719
UACUAUCAAACACAACCUC
[rUp|rAp|rCp|2mUp|rAp|rUp|2mCp|rAp|rA

p|rAp|2mCp|rAp|2mCp|rAp|rAp|rCp|r

Cp|rUp|rCp|dTp|dT]

721
UAACUAUCAAACACAACCU
[rUp|rAp|rAp|rCp|2mUp|rAp|rUp|2mCp|rA

p|rAp|rAp|2mCp|rAp|2mCp|rAp|rAp|r

Cp|rCp|rUp|dTp|dT]

723
UAAACUAUCAAACACAACC
[rUp|rAp|rAp|rAp|rCp|2mUp|rAp|rUp|2mC

p|rAp|rAp|rAp|2mCp|rAp|2mCp|rAp|r

Ap|rCp|rCp|dTp|dT]

797
UAAUUCUGUCAUCCUGCAC
[rUp|rAp|rAp|rUp|rUp|rCp|rUp|rGp|rUp|

2mCp|rAp|rUp|rCp|rCp|rUp|rGp|2mCp|r

Ap|rCp|dTp|dT]

821
UAAUGUUAUCCAUCCUUUC
[rUp|rAp|rAp|rUp|rGp|rUp|2mUp|rAp|rUp

|rCp|2mCp|rAp|rUp|rCp|rCp|rUp|rUp|r

Up|rCp|dTp|dT]

823
UAAAUGUUAUCCAUCCUUU
[rUp|rAp|rAp|rAp|rUp|rGp|rUp|2mUp|rAp

|rUp|rCp|2mCp|rAp|rUp|rCp|rCp|rUp|r

Up|rUp|dTp|dT]

827
UUCAAAAUAAAUGUUAUCC
[rUp|rUp|2mCp|rAp|rAp|rAp|rAp|2mUp|rA

p|rAp|rAp|rUp|rGp|rUp|2mUp|rAp|rU

p|rCp|rCp|dTp|dT]

849
UCAUAAAACUCUGUAACUG
[rUp|2mCp|rAp|2mUp|rAp|rAp|rAp|rAp|rC

p|rUp|rCp|rUp|rGp|2mUp|rAp|rAp|rC

p|rUp|rGp|dTp|dT]

853
UUUGCAUAAAACUCUGUAA
[rUp|rUp|rUp|rGp|2mCp|rAp|2mUp|rAp|rA

p|rAp|rAp|rCp|rUp|rCp|rUp|rGp|2mu

p|rAp|rAp|dTp|dT]

855
UUUUGCAUAAAACUCUGUA
[rUp|rUp|rUp|rUp|rGp|2mCp|rAp|2mUp|rA

p|rAp|rAp|rAp|rCp|rUp|rCp|rUp|rGp|

2mUp|rAp|dTp|dT]

901
UUAACAAUGCCAGAAAUAA
[rUp|2mUp|rAp|rAp|2mCp|rAp|rAp|rUp|rG

p|rCp|2mCp|rAp|rGp|rAp|rAp|rAp|2m

Up|rAp|rAp|dTp|dT]

915
UAAAUCACAGGCUUCUCUA
[rUp|rAp|rAp|rAp|rUp|2mCp|rAp|2mCp|rA

p|rGp|rGp|rCp|rUp|rUp|rCp|rUp|rCp|

2mUp|rAp|dTp|dT]

929
UUUAUGAUGGCAGCCAAAG
[rUp|rUp|2mUp|rAp|rUp|rGp|rAp|rUp|rGp

|rGp|2mCp|rAp|rGp|rCp|2mCp|rAp|rA

p|rAp|rGp|dTp|dT]

931
UUUUAUGAUGGCAGCCAAA
[rUp|rUp|rUp|2mUp|rAp|rUp|rGp|rAp|rUp

|rGp|rGp|2mCp|rAp|rGp|rCp|2mCp|rA

p|rAp|rAp|dTp|dT]

935
UUAUUUUAUGAUGGCAGCC
[rUp|2mUp|rAp|rUp|rUp|rUp|2mUp|rAp|rU

p|rGp|rAp|rUp|rGp|rGp|2mCp|rAp|rG

p|rCp|rCp|dTp|dT]

937
UGUAUUUUAUGAUGGCAGC
[rUp|rGp|2mUp|rAp|rUp|rUp|rUp|2mUp|rA

p|rUp|rGp|rAp|rUp|rGp|rGp|2mCp|rA

p|rGp|rCp|dTp|dT]

947
UGUUGAAUUCUUUGAGGUA
[rUp|rGp|rUp|rUp|rGp|rAp|rAp|rUp|rUp|

rCp|rUp|rUp|rUp|rGp|rAp|rGp|rGp|2mU

p|rAp|dTp|dT]

949
UAAGUUGAAUUCUUUGAGG
[rUp|rAp|rAp|rGp|rUp|rUp|rGp|rAp|rAp|

rUp|rUp|rCp|rUp|rUp|rUp|rGp|rAp|rGp|

rGp|dTp|dT]

967
UAAUUCUCAGGUUUGGAGA
[rUp|rAp|rAp|rUp|rUp|rCp|rUp|2mCp|rAp

|rGp|rGp|rUp|rUp|rUp|rGp|rGp|rAp|rG

p|rAp|dTp|dT]

971
UGUUUAAAAUUCUCAGGUU
[rUp|rGp|rUp|rUp|2mUp|rAp|rAp|rAp|rAp

|rUp|rUp|rCp|rUp|2mCp|rAp|rGp|rGp|r

Up|rUp|dTp|dT]

973
UCUGUUUAAAAUUCUCAGG
[rUp|rCp|rUp|rGp|rUp|rUp|2mUp|rAp|rA

p|rAp|rAp|rUp|rUp|rCp|rUp|2mCp|rAp|r

Gp|rGp|dTp|dT]

989
UUCCAUUUUACUUGAUAGC
[rUp|rUp|rCp|2mCp|rAp|rUp|rUp|rUp|2mU

p|rAp|rCp|rUp|rUp|rGp|rAp|2mUp|rA

p|rGp|rCp|dTp|dT]

991
UUUCCAUUUUACUUGAUAG
[rUp|rUp|rUp|rCp|2mCp|rAp|rUp|rUp|rUp

|2mUp|rAp|rCp|rUp|rUp|rGp|rAp|2mU

p|rAp|rGp|dTp|dT]

993
UAUUCCAUUUUACUUGAUA
[rUp|rAp|rUp|rUp|rCp|2mCp|rAp|rUp|rUp

|rUp|2mUp|rAp|rCp|rUp|rUp|rGp|rAp|

2mUp|rAp|dTp|dT]

995
UAAAUUCCAUUUUACUUGA
[rUp|rAp|rAp|rAp|rUp|rUp|rCp|2mCp|rAp

|rUp|rUp|rUp|2mUp|rAp|rCp|rUp|rUp|r

Gp|rAp|dTp|dT]

997
UUAAAUUCCAUUUUACUUG
[rUp|2mUp|rAp|rAp|rAp|rUp|rUp|rCp|2mC

p|rAp|rUp|rUp|rUp|2mUp|rAp|rCp|rU

p|rUp|rGp|dTp|dT]

1007
UUUCCAUUAAUUGUCAUAA
[rUp|rUp|rUp|rCp|2mCp|rAp|rUp|2mUp|rA

p|rAp|rUp|rUp|rGp|rUp|2mCp|rAp|2

mUp|rAp|rAp|dTp|dT]

1021
UAUUCCUUAAUGUUGUUCC
[rUp|rAp|rUp|rUp|rCp|rCp|rUp|2mUp|rAp

|rAp|rUp|rGp|rUp|rUp|rGp|rUp|rUp|rC

p|rCp|dTp|dT]

1032
UAUUCUGUAGGAUUUCCAG
[rUp|rAp|rUp|rUp|rCp|rUp|rGp|2mUp|rA

p|rGp|rGp|rAp|rUp|rUp|rUp|rCp|2mCp|

rAp|rGp|dTp|dT]

1055
UGUUUUCAUAUCAGUCUGA
[rUp|rGp|rUp|rUp|rUp|rUp|2mCp|rAp|2mU

p|rAp|rUp|2mCp|rAp|rGp|rUp|rCp|rU

p|rGp|rAp|dTp|dT]

1057
UUUGGUUUUCAUAUCAGUC
[rUp|rUp|rUp|rGp|rGp|rUp|rUp|rUp|rUp

|2mCp|rAp|2mUp|rAp|rUp|2mCp|rAp|rG

p|rUp|rCp|dTp|dT]

1059
UUUUGGUUUUCAUAUCAGU
[rUp|rUp|rUp|rUp|rGp|rGp|rUp|rUp|rUp|

rUp|2mCp|rAp|2mUp|rAp|rUp|2mCp|rA

p|rGp|rUp|dTp|dT]

1077
UGUCUAAAACCCACAGCAA
[rUp|rGp|rUp|rCp|2mUp|rAp|rAp|rAp|rAp

|rCp|rCp|2mCp|rAp|2mCp|rAp|rGp|2

mCp|rAp|rAp|dTp|dT]

1089
UUUUAGUGUGGUCUAAAAC
[rUp|rUp|rUp|2mUp|rAp|rGp|rUp|rGp|rUp

|rGp|rGp|rUp|rCp|2mUp|rAp|rAp|rAp|

rAp|rCp|dTp|dT]

1097
UGAAGUUUUAGUGUGGUCU
[rUp|rGp|rAp|rAp|rGp|rUp|rUp|rUp|2mUp

|rAp|rGp|rUp|rGp|rUp|rGp|rGp|rUp|rC

p|rUp|dTp|dT]

1099
UAAUGAAGUUUUAGUGUGG
[rUp|rAp|rAp|rUp|rGp|rAp|rAp|rGp|rUp|

rUp|rUp|2mUp|rAp|rGp|rUp|rGp|rUp|rG

p|rGp|dTp|dT]

1111
UCUUUAACUUCCGUCUCCC
[rUp|rCp|rUp|rUp|2mUp|rAp|rAp|rCp|rUp

|rUp|rCp|rCp|rGp|rUp|rCp|rUp|rCp|rC

p|rCp|dTp|dT]

1113
UUCUUUAACUUCCGUCUCC
[rUp|rUp|rCp|rUp|rUp|2mUp|rAp|rAp|rCp

|rUp|rUp|rCp|rCp|rGp|rUp|rCp|rUp|rC

p|rCp|dTp|dT]

1127
UAUUUAAGGAGUGGCUGGG
[rUp|rAp|rUp|rUp|2mUp|rAp|rAp|rGp|rGp

|rAp|rGp|rUp|rGp|rGp|rCp|rUp|rGp|rG

p|rGp|dTp|dT]

1215
UAUUUUCUAUCUGACCAAA
[rUp|rAp|rUp|rUp|rUp|rUp|rCp|2mUp|rAp

|rUp|rCp|rUp|rGp|rAp|rCp|2mCp|rAp|r

Ap|rAp|dTp|dT]

1221
UUAGAUGAUUUUCUAUCUG
[rUp|2mUp|rAp|rGp|rAp|rUp|rGp|rAp|rUp

|rUp|rUp|rUp|rCp|2mUp|rAp|rUp|rCp|r

Up|rGp|dTp|dT]

1230
UCAAUUUACGUAGAUGAUU
[rUp|2mCp|rAp|rAp|rUp|rUp|2mUp|rAp|rC

p|rGp|2mUp|rAp|rGp|rAp|rUp|rGp|rA

prUp|rUp|dTp|dT]

1307
UAUUAUUGCUUGAAAUUCU
[rUp|rAp|rUp|2mUp|rAp|rUp|rUp|rGp|rCp

|rUp|rUp|rGp|rAp|rAp|rAp|rUp|rUp|rC

p|rUp|dTp|dT]

1309
UGGUAUUAUUGCUUGAAAU
[rUp|rGp|rGp|2mUp|rAp|rUp|2mUp|rAp|rU

p|rUp|rGp|rCp|rUp|rUp|rGp|rAp|rAp|

rAp|rUp|dTp|dT]

1311
UAGGUAUUAUUGCUUGAAA
[rUp|rAp|rGp|rGp|2mUp|rAp|rUp|2mUp|rA

p|rUp|rUp|rGp|rCp|rUp|rUp|rGp|rAp|

rAp|rAp|dTp|dT]

1319
UGGAAUUAACAGCAGGUAU
[rUp|rGp|rGp|rAp|rAp|rUp|2mUp|rAp|rAp

|2mCp|rAp|rGp|2mCp|rAp|rGp|rGp|2

mUp|rAp|rUp|dTp|dT]

1363
UAAUUUCUAAAAUAACGGU
[rUp|rAp|rAp|rUp|rUp|rUp|rCp|2mUp|rAp

|rAp|rAp|rAp|2mUp|rAp|rAp|rCp|rGp|r

Gp|rUp|dTp|dT]

1387
UAAGUAAUGCUCCACUGGA
[rUp|rAp|rAp|rGp|2mUp|rAp|rAp|rUp|rGp

|rCp|rUp|rCp|2mCp|rAp|rCp|rUp|rGp|r

Gp|rAp|dTp|dT]

1395
UUAUCUUUAAGUAAUGCUC
[rUp|2mUp|rAp|rUp|rCp|rUp|rUp|2mUp|rA

p|rAp|rGp|2mUp|rAp|rAp|rUp|rGp|rC

p|rUp|rCp|dTp|dT]

1397
UGUAUCUUUAAGUAAUGCU
[rUp|rGp|2mUp|rAp|rUp|rCp|rUp|rUp|2mU

p|rAp|rAp|rGp|2mUp|rAp|rAp|rUp|rG

p|rCp|rUp|dTp|dT]

1401
UAUUGAGUAUCUUUAAGUA
[rUp|rAp|rUp|rUp|rGp|rAp|rGp|2mUp|rAp

|rUp|rCp|rUp|rUp|2mUp|rAp|rAp|rGp|

2mUp|rAp|dTp|dT]

1405
UUUCAUUGAGUAUCUUUAA
[rUp|rUp|rUp|2mCp|rAp|rUp|rUp|rGp|rAp

|rGp|2mUp|rAp|rUp|rCp|rUp|rUp|2mU

p|rAp|rAp|dTp|dT]

1425
UUUCAGUUUUAUCCCCAAC
[rUp|rUp|rUp|2mCp|rAp|rGp|rUp|rUp|rUp

|2mUp|rAp|rUp|rCp|rCp|rCp|2mCp|rA

p|rAp|rCp|dTp|dT]

1427
UAUUCAGUUUUAUCCCCAA
[rUp|rAp|rUp|rUp|2mCp|rAp|rGp|rUp|rUp

|rUp|2mUp|rAp|rUp|rCp|rCp|rCp|2mC

p|rAp|rAp|dTp|dT]

1449
UAUUAAAGGGAAGUCAGAA
[rUp|rAp|rUp|2mUp|rAp|rAp|rAp|rGp|rGp

|rGp|rAp|rAp|rGp|rUp|2mCp|rAp|rGp|r

Ap|rAp|dTp|dT]

1453
UUUUAUUAAAGGGAAGUCA
[rUp|rUp|rUp|2mUp|rAp|rUp|2mUp|rAp|rA

p|rAp|rGp|rGp|rGp|rAp|rAp|rGp|rUp|

2mCp|rAp|dTp|dT]

1455
UGAAUUUCAUCCUUCCUCU
[rUp|rGp|rAp|rAp|rUp|rUp|rUp|2mCp|rAp

|rUp|rCp|rCp|rUp|rUp|rCp|rCp|rUp|rC

p|rUp|dTp|dT]

1507
UCAUAUUGUGCAGAAGGAU
[rUp|2mCp|rAp|2mUp|rAp|rUp|rUp|rGp|rU

p|rGp|2mCp|rAp|rGp|rAp|rAp|rGp|rG

p|rAp|rUp|dTp|dT]

1525
UUCAUAAACUCCUGUCCUG
[rUp|rUp|2mCp|rAp|2mUp|rAp|rAp|rAp|rC

p|rUp|rCp|rCp|rUp|rGp|rUp|rCp|rCp|r

Up|rGp|dTp|dT]

1541
UAAGAUACAGCAGAGUUCU
[rUp|rAp|rAp|rGp|rAp|2mUp|rAp|2mCp|rA

p|rGp|2mCp|rAp|rGp|rAp|rGp|rUp|rU

p|rCp|rUp|dTp|dT]

1547
UAUACAAGAUACAGCAGAG
[rUp|rAp|2mUp|rAp|2mCp|rAp|rAp|rGp|rA

p|2mUp|rAp|2mCp|rAp|rGp|2mCp|rA

p|rGp|rAp|rGp|dTp|dT]

1657
UUAGAAAAUCAAGCCAUUC
[rUp|2mUp|rAp|rGp|rAp|rAp|rAp|rAp|rUp

|2mCp|rAp|rAp|rGp|rCp|2mCp|rAp|rU

p|rUp|rCp|dTp|dT]

1667
UACUGAAUUUCUCUAGAAA
[rUp|rAp|rCp|rUp|rGp|rAp|rAp|rUp|rUp|

rUp|rCp|rUp|rCp|2mUp|rAp|rGp|rAp|rA

p|rAp|dTp|dT]

1675
UAUAAUGUUCACUGAAUUU
[rUp|rAp|2mUp|rAp|rAp|rUp|rGp|rUp|rUp

|2mCp|rAp|rCp|rUp|rGp|rAp|rAp|rUp|r

Up|rUp|dTp|dT]

1677
UGAUAAUGUUCACUGAAUU
[rUp|rGp|rAp|2mUp|rAp|rAp|rUp|rGp|rUp

|rUp|2mCp|rAp|rCp|rUp|rGp|rAp|rAp|r

Up|rUp|dTp|dT]

1679
UUGAUAAUGUUCACUGAAU
[rUp|rUp|rGp|rAp|2mUp|rAp|rAp|rUp|rGp

|rUp|rUp|2mCp|rAp|rCp|rUp|rGp|rAp|r

Ap|rUp|dTp|dT]

1723
UGAGAAAAUGCAGUCAACA
[rUp|rGp|rAp|rGp|rAp|rAp|rAp|rAp|rUp|

rGp|2mCp|rAp|rGp|rUp|2mCp|rAp|rAp|

2mCp|rAp|dTp|dT]

1763
UUUCUUGUACAGUUGGUCU
[rUp|rUp|rUp|rCp|rUp|rUp|rGp|2mUp|rAp

|2mCp|rAp|rGp|rUp|rUp|rGp|rGp|rUp|

rCp|rUp|dTp|dT]

1767
UUCUUUCUUCUUGUACAGU
[rUp|rUp|rCp|rUp|rUp|rUp|rCp|rUp|rUp|

rCp|rUp|rUp|rGp|2mUp|rAp|2mCp|rAp|r

Gp|rUp|dTp|dT]

1769
UUUCUUUCUUCUUGUACAG
[rUp|rUp|rUp|rCp|rUp|rUp|rUp|rCp|rUp|

rUp|rCp|rUp|rUp|rGp|2mUp|rAp|2mCp|

rAp|rGp|dTp|dT]

1837
UUCAUUACUCUCUCUGAGU
[rUp|rUp|2mCp|rAp|rUp|2mUp|rAp|rCp|rU

p|rCp|rUp|rCp|rUp|rCp|rUp|rGp|rAp|r

Gp|rUp|dTp|dT]

1839
UUAUCAUUACUCUCUCUGA
[rUp|2mUp|rAp|rUp|2mCp|rAp|rUp|2mUp|r

Ap|rCp|rUp|rCp|rUp|rCp|rUp|rCp|ru

p|rGp|rAp|dTp|dT]

1843
UAUUAUCAUUACUCUCUCU
[rUp|rAp|rUp|2mUp|rAp|rUp|2mCp|rAp|rU

p|2mUp|rAp|rCp|rUp|rCp|rUp|rCp|rU

p|rCp|rUp|dTp|dT]

1869
UUUAUGUAGGAGCUCUUUC
[rUp|rUp|2mUp|rAp|rUp|rGp|2mUp|rAp|rG

p|rGp|rAp|rGp|rCp|rUp|rCp|rUp|rUp|

rUp|rCp|dTp|dT]

1887
UAAUCAAUGCAACUUGUUU
[rUp|rAp|rAp|rUp|2mCp|rAp|rAp|rUp|rGp

|2mCp|rAp|rAp|rCp|rUp|rUp|rGp|rUp|r

Up|rUp|dTp|dT]

1889
UUAAUCAAUGCAACUUGUU
[rUp|2mUp|rAp|rAp|rUp|2mCp|rAp|rAp|rU

p|rGp|2mCp|rAp|rAp|rCp|rUp|rUp|rG

p|rUp|rUp|dTp|dT]

1965
UUGUGAAAAUGCCAUCCAC
[rUp|rUp|rGp|rUp|rGp|rAp|rAp|rAp|rAp|

rUp|rGp|rCp|2mCp|rAp|rUp|rCp|2mCp|r

Ap|rCp|dTp|dT]

1991
UUUAUAUAUAUUGUCUGCA
[rUp|rUp|2mUp|rAp|2mUp|rAp|2mUp|rAp|2

mUp|rAp|rUp|rUp|rGp|rUp|rCp|rUp|

rGp|2mCp|rAp|dTp|dT]

2031
UGAUUAUUUCUGCUGUGUC
[rUp|rGp|rAp|rUp|2mUp|rAp|rUp|rUp|rUp

|rCp|rUp|rGp|rCp|rUp|rGp|rUp|rGp|rU

p|rCp|dTp|dT]

2109
UAAUACUCAAGUGUAGCAU
[rUp|rAp|rAp|2mUp|rAp|rCp|rUp|2mCp|rA

p|rAp|rGp|rUp|rGp|2mUp|rAp|rGp|2

mCp|rAp|rUp|dTp|dT]

2129
UUUAAGGAUUUCACAUCUC
[rUp|rUp|2mUp|rAp|rAp|rGp|rGp|rAp|rUp

|rUp|rUp|2mCp|rAp|2mCp|rAp|rUp|rC

p|rUp|rCp|dTp|dT]

2231
UCAAAAUCAGGGACUUGUU
[rUp|2mCp|rAp|rAp|rAp|rAp|rUp|2mCp|rA

p|rGp|rGp|rGp|rAp|rCp|rUp|rUp|rGp|r

Up|rUp|dTp|dT]

2243
UUAAAGGAAGGUGACAAAA
[rUp|2mUp|rAp|rAp|rAp|rGp|rGp|rAp|rAp

|rGp|rGp|rUp|rGp|rAp|2mCp|rAp|rAp|r

Ap|rAp|dTp|dT]

2247
UUUUGGUAAAGGAAGGUGA
[rUp|rUp|rUp|rUp|rGp|rGp|2mUp|rAp|rA

p|rAp|rGp|rGp|rAp|rAp|rGp|rGp|rUp|rG

p|rAp|dTp|dT]

2255
UUCUAGUUAUUUGGUAAAG
[rUp|rUp|rCp|2mUp|rAp|rGp|rUp|2mUp|rA

p|rUp|rUp|rUp|rGp|rGp|2mUp|rAp|rA

p|rAp|rGp|dTp|dT]

2275
UUUUAAUCCAUAACUCCUU
[rUp|rUp|rUp|2mUp|rAp|rAp|rUp|rCp|2m

Cp|rAp|2mUp|rAp|rAp|rCp|rUp|rCp|rC

p|rUp|rUp|dTp|dT]

2295
UCUAGUUUAGCCACAUUUA
[rUp|rCp|2mUp|rAp|rGp|rUp|rUp|2mUp|rA

p|rGp|rCp|2mCp|rAp|2mCp|rAp|rUp|

rUp|2mUp|rAp|dTp|dT]

2331
UUAUUAAUCCUUCCAGCUC
[rUp|2mUp|rAp|rUp|2mUp|rAp|rAp|rUp|rC

p|rCp|rUp|rUp|rCp|2mCp|rAp|rGp|rC

p|rUp|rCp|dTp|dT]

2335
UUUUCGUAUUUAUUAAUCC
[rUp|rUp|rUp|rUp|rCp|rGp|2mUp|rAp|rUp

|rUp|2mUp|rAp|rUp|2mUp|rAp|rAp|rU

p|rCp|rCp|dTp|dT]

2357
UUUUGCAAAAUACUUGAGU
[rUp|rUp|rUp|rUp|rGp|2mCp|rAp|rAp|rAp

|rAp|2mUp|rAp|rCp|rUp|rUp|rGp|rAp|r

Gp|rUp|dTp|dT]

2361
UUAACUUUGCAAAAUACUU
[rUp|2mUp|rAp|rAp|rCp|rUp|rUp|rUp|rGp

|2mCp|rAp|rAp|rAp|rAp|2mUp|rAp|rC

p|rUp|rUp|dTp|dT]

2363
UCAUAACUUUGCAAAAUAC
[rUp|2mCp|rAp|2mUp|rAp|rAp|rCp|rUp|rU

p|rUp|rGp|2mCp|rAp|rAp|rAp|rAp|2m

Up|rAp|rCp|dTp|dT]

2449
UAGUUAUUUUGUACAGUUG
[rUp|rAp|rGp|rUp|2mUp|rAp|rUp|rUp|rUp

|rUp|rGp|2mUp|rAp|2mCp|rAp|rGp|rU

p|rUp|rGp|dTp|dT]

2503
UAAUUUUAUGCUCACAUGU
[rUp|rAp|rAp|rUp|rUp|rUp|2mUp|rAp|rUp

|rGp|rCp|rUp|2mCp|rAp|2mCp|rAp|rU

p|rGp|rUp|dTp|dT]

2505
UUAAUUUUAUGCUCACAUG
[rUp|2mUp|rAp|rAp|rUp|rUp|rUp|2mUp|rA

p|rUp|rGp|rCp|rUp|2mCp|rAp|2mCp|

rAp|rUp|rGp|dTp|dT]

2517
UUAUACCAUGGUCAUAAUU
[rUp|2mUp|rAp|2mUp|rAp|rCp|2mCp|rAp|r

Up|rGp|rGp|rUp|2mCp|rAp|2mUp|r

Ap|rAp|rUp|rUp|dTp|dT]

2519
UAUAUACCAUGGUCAUAAU
[rUp|rAp|2mUp|rAp|2mUp|rAp|rCp|2mCp|r

Ap|rUp|rGp|rGp|rUp|2mCp|rAp|2m

Up|rAp|rAp|rUp|dTp|dT]

2579
UCUAUUCAAGAGUGUUUAU
[rUp|rCp|2mUp|rAp|rUp|rUp|2mCp|rAp|rA

p|rGp|rAp|rGp|rUp|rGp|rUp|rUp|2mU

p|rAp|rUp|dTp|dT]

2581
UUCUAUUCAAGAGUGUUUA
[rUp|rUp|rCp|2mUp|rAp|rUp|rUp|2mCp|rA

p|rAp|rGp|rAp|rGp|rUp|rGp|rUp|rUp

2mUp|rAp|dTp|dT]

2593
UUACAAAGUGGAAGUCUAU
[rUp|2mUp|rAp|2mCp|rAp|rAp|rAp|rGp|rU

p|rGp|rGp|rAp|rAp|rGp|rUp|rCp|2mU

p|rAp|rUp|dTp|dT]

2597
UAUUACAAAGUGGAAGUCU
[rUp|rAp|rUp|2mUp|rAp|2mCp|rAp|rAp|rA

p|rGp|rUp|rGp|rGp|rAp|rAp|rGp|rUp|r

Cp|rUp|dTp|dT]

2603
UCUAAUUACAAAGUGGAAG
[rUp|rCp|2mUp|rAp|rAp|rUp|2mUp|rAp|2m

Cp|rAp|rAp|rAp|rGp|rUp|rGp|rGp|rA

p|rAp|rGp|dTp|dT]

2655
UUAUAUUCUGCCACUUAAG
[rUp|2mUp|rAp|2mUp|rAp|rUp|rUp|rCp|rU

p|rGp|rCp|2mCp|rAp|rCp|rUp|2mUp|

rAp|rAp|rGp|dTp|dT]

2657
UUUAUAUUCUGCCACUUAA
[rUp|rUp|2mUp|rAp|2mUp|rAp|rUp|rUp|rC

p|rUp|rGp|rCp|2mCp|rAp|rCp|rUp|2

mUp|rAp|rAp|dTp|dT]

2687
UUUAUAUCACCCUCCAAAA
[rUp|rUp|2mUp|rAp|2mUp|rAp|rUp|2mCp|r

Ap|rCp|rCp|rCp|rUp|rCp|2mCp|rAp|

rAp|rAp|rAp|dTp|dT]

2763
UUCUUAUGUUCUUGGUGGA
[rUp|rUp|rCp|rUp|2mUp|rAp|rUp|rGp|rUp

|rUp|rCp|rUp|rUp|rGp|rGp|rUp|rGp|rG

p|rAp|dTp|dT]

2769
UAAUUCUUAUGUUCUUGGU
[rUp|rAp|rAp|rUp|rUp|rCp|rUp|2mUp|rAp

|rUp|rGp|rUp|rUp|rCp|rUp|rUp|rGp|rG

p|rUp|dTp|dT]

2783
UAAAAGCUUGAUAAUUCUA
[rUp|rAp|rAp|rAp|rAp|rGp|rCp|rUp|rUp|

rGp|rAp|2mUp|rAp|rAp|rUp|rUp|rCp|2m

Up|rAp|dTp|dT]

2845
UACAAAUAAUUUCAUCCAA
[rUp|rAp|2mCp|rAp|rAp|rAp|2mUp|rAp|rA

p|rUp|rUp|rUp|2mCp|rAp|rUp|rCp|2m

Cp|rAp|rAp|dTp|dT]

2847
UAUGACAAAUAAUUUCAUC
[rUp|rAp|rUp|rGp|rAp|2mCp|rAp|rAp|rAp

|2mUp|rAp|rAp|rUp|rUp|rUp|2mCprA

p|rUp|rCp|dTp|dT]

Example 7. MSH3 siRNA Screening Data and Knockdown Data for Selected Duplexes

Various screenings were implemented to determine potential lead siRNA duplex candidates.

Selected siRNA duplexes targeting human MSH3 were screened in Hela cells at 10 nM and 0.5 nM doses to determine potential lead siRNA candidates. The screening results are provided below in Table 15. The sense and antisense oligonucleotides in Table 15 each include a dTdT overhang on the 3′ end.

Additionally, every A and G in each sense oligonucleotide in Table 15 is a ribonucleotide. Every C and U in each sense oligonucleotide is a 2′-O-Methyl ribonucleotide.

Also, every A and Gin each antisense oligonucleotide in Table 15 is a ribonucleotide. Every C and U preceding an A in the antisense oligonucleotide is a 2′-O-Methyl ribonucleotide, with one exception: U is the first nucleotide of the antisense strand, and it is a ribonucleotide.

Each nucleotide in the sense and antisense oligonucleotide in Table 15 is linked by a phosphate.

TABLE 15

SEQ ID

SEQ ID NO./

NO./Sense

Antisense

Sense
No.
antisense
No.

GCCAAAAUGUACUGAUUUA
156
UAAAUCAGUACAUUUUGGC
157

GUGGAAUGUGGAUAUAAGA
358
UCUUAUAUCCACAUUCCAC
359

GAUAUAAGUAUAGAUUCUA
374
UAGAAUCUAUACUUAUAUC
375

GCCAUUUAGAUCACAACUA
412
UAGUUGUGAUCUAAAUGGC
413

CGAGGUUGUGUUUGAUAGA
716
UCUAUCAAACACAACCUCG
717

UGAAUACAGCCAUGCUUUA
832
UAAAGCAUGGCUGUAUUCA
833

CUGGCAUUGUUAACUUAGA
906
UCUAAGUUAACAAUGCCAG
907

CUCCAAACCUGAGAAUUUA
968
UAAAUUCUCAGGUUUGGAG
969

UGGGUUUUAGACCACACUA
1084
UAGUGUGGUCUAAAACCCA
1085

GGUUUUAGACCACACUAAA
1086
UUUAGUGUGGUCUAAAACC
1087

GACGGAAGUUAAAGAAGUA
1114
UACUUCUUUAACUUCCGUC
1115

CUCCAUUCAGAAUCUAGUA
1186
UACUAGAUUCUGAAUGGAG
1187

CCGUUAUUUUAGAAAUUCA
1364
UGAAUUUCUAAAAUAACGG
1365

CGUUAUUUUAGAAAUUCCA
1366
UGGAAUUUCUAAAAUAACG
1367

GGAGCAUUACUUAAAGAUA
1392
UAUCUUUAAGUAAUGCUCC
1393

AGCUAGUCCUUGACUGCAA
1642
UUGCAGUCAAGGACUAGCU
1643

GCUCCUACAUAAAACAAGA
1874
UCUUGUUUUAUGUAGGAGC
1875

GGGUGCUGCAGACAAUAUA
1982
UAUAUUGUCUGCAGCACCC
1983

GACAAUAUAUAUAAAGGAA
1992
UUCCUUUAUAUAUAUUGUC
1993

GUGGCUAAACUAGCAGAUA
2302
UAUCUGCUAGUUUAGCCAC
2303

GAAGGAUUAAUAAAUACGA
2332
UCGUAUUUAUUAAUCCUUC
2333

GGACGAUGCAUAAUGCACA
2386
UGUGCAUUAUGCAUCGUCC
2387

CUGUCUUCCUAACUUUUCA
2550
UGAAAAGUUAGGAAGACAG
2551

CCAGUAAAGCCUUAAGUGA
2642
UCACUUAAGGCUUUACUGG
2643

Based on the results of Table 15, the following siRNA duplex candidates were selected for further testing and characterization.

- dsRNA duplex of SENSE OLIGO NO: 156/ANTISENSE OLIGO: 157
- dsRNA duplex of SENSE OLIGO NO: 906/ANTISENSE OLIGO NO: 907
- dsRNA duplex of SENSE OLIGO NO: 968/ANTISENSE OLIGO NO: 1969
- dsRNA duplex of SENSE OLIGO NO: 1392/ANTISENSE OLIGO NO: 1393
- dsRNA duplex of SENSE OLIGO NO: 1366/ANTISENSE OLIGO NO: 1367
- dsRNA duplex of SENSE OLIGO NO: 1874/ANTISENSE OLIGO NO: 1875

The dose response curves for the selected candidates are shown in FIGS. 1-6. The IC₂₀, IC₅₀, and IC₅₀values for the candidates are shown below in Table 16. The sense and antisense oligonucleotides of Table 16 each contain a dTdT overhang on the 3′ end. Additionally, every A and Gin each sense oligonucleotide in Table 16 is a ribonucleotide. Every C and U in each sense oligonucleotide is a 2′-O-Methyl ribonucleotide.

Also, every A and G in each antisense oligonucleotide in Table 16 is a ribonucleotide. Every C and U preceding an A in the antisense oligonucleotide is a 2′-O-Methyl ribonucleotide, with one exception: U is the first nucleotide of the antisense strand, and it is a ribonucleotide.

Each nucleotide in the sense and antisense oligonucleotide in Table 16 is linked by a phosphate.

TABLE 16

SEQ ID No.

Sense

No./

Antisense
IC₂₀ @
IC₅₀ @
IC₈₀ @

Sense
Antisense
No.
[nM]
[nM]
[nM]

CUGGCAUUGUUAACUUAGA
UCUAAGUUAACAAUGCCAG
968/969
0.0016
0.0081
#N/A

GGAGCAUUACUUAAAGAUA
UAUCUUUAAGUAAUGCUCC
1392/1393
0.0037
0.0213
1.1670

CUCCAAACCUGAGAAUUUA
UAAAUUCUCAGGUUUGGAG
1084/1085
0.0008
0.0172
#N/A

CGUUAUUUUAGAAAUUCCA
UGGAAUUUCUAAAAUAACG
1366/1367
0.0049
0.0343
#N/A

GCCAAAAUGUACUGAUUUA
UAAAUCAGUACAUUUUGGC
156/157
0.0040
0.0196
#N/A

GCUCCUACAUAAAACAAGA
UCUUGUUUUAUGUAGGAGC
1874/1875
0.0173
0.1013
#N/A

RNAi Dose Response Screen

Twelve siRNA duplexes targeting MSH3 were tested using a Hela cell-based assay. The siRNA duplex strands and the EC₅₀results are provided below in Table 17. The sense and antisense oligonucleotides of Table 18 each contain a dTdT overhang on the 3′ end. Additionally, every A and Gin each sense oligonucleotide in Table 17 is a ribonucleotide. Every C and U in each sense oligonucleotide is a 2′-O-Methyl ribonucleotide.

Also, every A and G in each antisense oligonucleotide in Table 17 is a ribonucleotide. Every C and U preceding an A in the antisense oligonucleotide is a 2′-O-Methyl ribonucleotide, with one exception: U is the first nucleotide of the antisense strand, and it is a ribonucleotide.

Each nucleotide in the sense and antisense oligonucleotide in Table 17 is linked by a phosphate.

The non linear regression curves depicting the mean, the standard deviation, and the RQ values for each of the tested siRNAs at ten concentrations are plotted together in FIG. 7.

TABLE 17

SEQ ID

NO.

Sense

No./

Anti-

sense
EC50

Sense
Antisense
No.
(pM)

AUACAAGUCAUGAAAAUUA
UAAUUUUCAUGACUUGUAU
258/259
41.09

CGCUAGAAUUACAAUACAA
UUGUAUUGUAAUUCUAGCG
310/311
129.2

UAGAAUUACAAUACAUAGA
UCUAUGUAUUGUAAUUCUA
312/313
120

AGCCCGAGAGCUCAAUAUA
UAUAUUGAGCUCUCGGGCU
388/389
109.9

GCCCGAGAGCUCAAUAUUA
UAAUAUUGAGCUCUCGGGC
390/391
179.9

CGAGAGCUCAAUAUUUAUA
UAUAAAUAUUGAGCUCUCG
392/393
18.49

GAGAGCUCAAUAUUUAUUA
UAAUAAAUAUUGAGCUCUC
394/395
191.7

UUUAUUGCCAUUUAGAUCA
UGAUCUAAAUGGCAAUAAA
402/403
184.4

AGAUCACAACUUUAUGACA
UGUCAUAAAGUUGUGAUCU
420/421
374.3

CACAGACUGUUUGUUCAUA
UAUGAACAAACAGUCUGUG
460/461
176.4

UCACCUAAAGUCAGAAUUA
UAAUUCUGACUUUAGGUGA
1302/1303
74.96

CAGAGAGAGUAAUGAUAAA
UUUAUCAUUACUCUCUCUG
1840/1841
30.16

The 108 siRNA duplexes selected for dose response validation had new source plates created to allow each dose to be diluted 1000-fold in the final assay plate. The final siRNA concentrations in the assay plates were as follows:

- 100 nM
- 16.67 nM
- 2.77 nM
- 462 pM
- 77.1 pM
- 12.9 pM
- 2.14 pM
- 357 fM
- 59.5 fM
- UltraPure H2O Control

After this initial dispense the RNAi screen protocol was followed. The screening results for the top four candidates are provided in FIGS. 8A-8D.

Example 8: Confirmatory Screen of Top Sequences in Primary Human Hepatocytes (PHH)

Primary Human Hepatocytes (PHH) were used to select potent siRNAs targeting the human MSH3 transcript for further in vivo testing. The siRNAs used in this study were selected from in vitro activity screens. Twenty MSH3 siRNA from the primary screens were screened by transient transfection at 0.1 nM and 2 nM in PHH. The eight siRNAs that showed significant KD (>75% at 2 nM) activity were further evaluated by dose response curves (DRC) in PHH. A mouse/cyno/human cross-reactive siRNA (SENSE OLIGO NO. 832/ANTISENSE OLIGO NO. 833) with known MSH3 knockdown activity in mouse was included as a reference.

Testing Top Eight siRNAs for DRCs by Transient Transfection in PHH

PHH cat. #Hu8350 were obtained from (Thermo Scientific; Cambridge, MA, USA). Cells were plated at 35K per well in Hepatocyte plating media cat. #CM3000 and maintained in Hepatocyte maintenance media cat. #CM4000 (Thermo Scientific; Cambridge, MA, USA) in a 96 well collagen coated plate cat. #12-565-909 (Fisher Scientific; Cambridge, MA, USA) and incubated at 37° C. with 5% CO2 in a humidified incubator for each experiment.

Transfection

Five to six hours post plating, the cells were washed with maintenance media and incubated at 37° C. Twenty-four hours post-thaw, siRNAs were diluted starting at 100 nM with a 4-fold dilution to 0.000154 nM, complexed with 0.3 μl of RNAi Max cat. #13-778-150 (Fisher Scientific; Cambridge, MA, USA) and added to cells. Twenty-four hours post transfection, RNA was extracted using Quick-RNA 96 kit cat. #R1053 (Zymo Research; Irvine, CA, USA) according to manufacturer's instructions and samples were eluted in 22 μl nuclease-free water. Total RNA was quantified using Quant-iT RiboGreen RNA assay kit cat. #R11490 (Invitrogen; Carlsbad, CA, USA) and 80 ngs was used to generate cDNA with SuperScript IV VILO Master Mix cat. #11-756-500 (Thermo Scientific; Cambridge, MA, USA) according to manufacturer's instructions. The RNA was treated with DNAse I to remove any genomic DNA during RNA extraction and any remainder of it was accounted for with the addition of a cDNA reaction lacking reverse transcriptase (−RT control). 10% of the cDNA reaction was used for qPCR using PrimeTime Gene Expression Master Mix cat. #1055772 (Integrated DNA Technologies (IDT); Coralville, IA, USA) along with the hydrolysis probes for the gene of interest and housekeeper GUSB or TBP (IDT; Coralville, IA, USA) on a LightCycler 480 II (Roche; Basel, Switzerland).

Data Analysis

The GUSB and TBP genes were used as housekeeping controls. The signal threshold for target MSH3 was set based on background signal from −RT control samples and crossing point (Cp) determined for each probe and sample. ΔCp was calculated as Cp[target]−Cp[housekeeping]. The average ΔCp for Mock treated samples was established for each target gene. The ΔΔCp was calculated as the ΔCp−Average [Mock] ΔCp, and relative expression as 2{circumflex over ( )}−(ΔΔCp). Relative fold-change in target expression calculated by 2−ΔΔCp, was averaged between the two housekeepers and analyzed in GraphPad Prism. For generating DRCs, the concentration of each treatment was converted to the Log values, IC50 for each target was calculated by analyzing the relative expression using the equation for “Non-linear regression curve fit” with Prism. The equation was fit to Log (Inhibitor) Vs response-variable slope using (Four parameters).

DRCs and IC₅₀Graphs

The siRNAs with highest activity in the dual-dose screen were used to generate the dose response curves. The graphs in FIGS. 9A-9I shows IC₅₀analysis for the target knock down measured by qPCR. The X-axis represents the concentration of siRNA transfected and the Y-axis represents the relative MSH3 target remaining. The IC₅₀for each target was calculated using the equation for “Non-linear regression curve fit” with Prism. The equation was fit to Log (Inhibitor) Vs response-variable slope using (Four parameters).

FIGS. 9A-9I show that there was good transfection efficiency between plates by the control MSH3 siRNA knock down in different plates. The DRCs for siRNA knock down were generated in four different plates. A commercially available SMART pool MSH3 siRNA was used to compare the transfection efficiency between different plates at 2 nM. The X-axis represents the concentration of siRNA transfection on different plates and Y-axis represents the percentage of target remaining. The fold change in MSH3 expression from the four plates is shown in FIG. 10. The siRNAs in each plate are provided in Table 18. The IC₅₀with R²values and max KD for the top eight siRNAs are shown in Table 19.

The sense and antisense oligonucleotides of Tables 18 and 19 each contain a dTdT overhang on the 3′ end. Additionally, every A and G in each sense oligonucleotide in Tables 18 and 19 is a ribonucleotide. Every C and U in each sense oligonucleotide is a 2′-O-Methyl ribonucleotide.

Also, every A and Gin each antisense oligonucleotide in Tables 18 and 19 is a ribonucleotide. Every C and U preceding an A in the antisense oligonucleotide is a 2′-O-Methyl ribonucleotide, with one exception: U is the first nucleotide of the antisense strand, and it is a ribonucleotide.

Each nucleotide in the sense and antisense oligonucleotide in Tables 18 and 19 is linked by a phosphate.

TABLE 18

Seq id/

Seq id/

SENSE

ANTISENSE

OLIGO

OLIGO

Plates
Sense
NO
Antisense
NO

Plate 1
CGUUAUUUUA
1366
UGGAAUUUCU
1367

GAAAUUCCA

AAAAUAACG

Plate 1
GCUCCUACAU
1874
UCUUGUUUUA
1875

AAAACAAGA

UGUAGGAGC

Plate 2
CAACAGAAGU
550
UAAGAGUGAA
551

UCACUCUUA

CUUCUGUUG

Plate 2
UCACCUAAAG
1302
UAAUUCUGAC
1303

UCAGAAUUA

UUUAGGUGA

Plate 3
AGCCCGAGAG
388
UAUAUUGAGC
389

CUCAAUAUA

UCUCGGGCU

Plate 3
UUUAUUGCCA
402
UGAUCUAAAU
403

UUUAGAUCA

GGCAAUAAA

Plate 4
GUUGAUGAGA
7450
UAGUCAUUAU
745

UAAUGACUA

CUCAUCAAC

Plate 4
AGAUAAUGAC
656
UAAGUAUCAG
657

UGAUACUUA

UCAUUAUCU

TABLE 19

SEQ ID

NO.

Sense

No./

Anti-

Max

sense
IC50

KD
Rank-

Sense
Antisense
No.
[nM]
R²
(%)
ing

GUUGAUGAGA
UAGUCAUUAU
648/
0.003
0.95
88
1

UAAUGACUA
CUCAUCAAC
649

AGAUAAUGAC
UAAGUAUCAG
656/
0.007
0.84
80
2

UGAUACUUA
UCAUUAUCU
657

UGAAUACAGC
UAAAGCAUGG
832/
0.006498
0.92
80
3

CAUGCUUUA
CUGUAUUCA
833

AGAUCACAAC
UGUCAUAAAG
420/
0.007
0.78
72
4

UUUAUGACA
UUGUGAUCU
421

CGUUAUUUUA
UGGAAUUUCU
1366/
0.009
0.92
80
5

GAAAUUCCA
AAAAUAACG
1367

UCACCUAAAG
UAAUUCUGAC
1302/
0.009
0.93
83
6

UCAGAAUUA
UUUAGGUGA
1303

CAACAGAAGU
UAAGAGUGAA
550/
0.013
0.85
75
7

UCACUCUUA
CUUCUGUUG
551

ACUUCUACCA
UAAGAUAGCU
672/
0.015
0.86
69
8

GCUAUCUUA
GGUAGAAGU
673

GCUCCUACAU
UCUUGUUUUA
1874/
0.087
0.92
79
9

AAAACAAGA
UGUAGGAGC
1875

This study identified eight siRNAs targeting MSH3 with half maximal inhibitory concentration (IC₅₀) by transfection in PHH in the low pM range. The results of the eight candidates selected from the siRNA screening at 0.5 nM and 10 nM doses and the resulting IC₅₀are provided below in Table 20. The siRNA of Sense No. 656/Antisense No. 657 was used as a control.

TABLE 20

dsRNA

SENSE OLIGO No./
% target remaining

ANTISENSE OLIGO No.
0.5 nM
10 nM
IC₅₀(nM)

SENSE OLIGO No. 832/
44.7735672
23.0173288
N/A

ANTISENSE OLIGO No.

833

SENSE OLIGO No. 1366/
27.0833733
17.2481438
34.349054

ANTISENSE OLIGO No.

1367

SENSE OLIGO No. 1874/
28.0472485
23.0624968
101.305208

ANTISENSE OLIGO No.

1875

SENSE OLIGO No. 550/
34.4598214
24.5834182
18.49

ANTISENSE OLIGO No.

551

SENSE OLIGO No. 1302/
36.1222642
25.7249608
74.96

ANTISENSE OLIGO No.

1303

SENSE OLIGO No. 420/
37.1536281
25.4957144
109.9

ANTISENSE OLIGO No.

421

SENSE OLIGO No. 672/
33.8159421
24.8627536
184.4

ANTISENSE OLIGO No.

673

SENSE OLIGO No. 648/
N/A
16.9266119
N/A

ANTISENSE OLIGO No.

649

SENSE OLIGO No. 656/
N/A
22.5483683
N/A

ANTISENSE OLIGO No.

657

This example demonstrates that the siRNAs targeting MSH3 identified in the screening studies showed confirmed MSH3 knockdown in vitro.

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 following, in general, the principles 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.

In addition to the various aspects described herein, the present disclosure includes the following aspects numbered E1 through E108. This list of aspects is presented as an exemplary list and the application is not limited to these particular aspects.

- E1. 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 MSH3 gene, and wherein the dsRNA comprises a duplex structure of between 15 and 30 linked nucleosides in length.
- E2. A dsRNA for reducing expression of MSH3 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 MSH3 gene, and wherein the dsRNA comprises a duplex structure of between 15 and 30 linked nucleosides in length.
- E3. The dsRNA of E1 or E2 comprising a duplex structure of between 19 and 23 linked nucleosides in length.
- E4. The dsRNA of any one of E1-E3, 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.
- E5. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at one or more of positions 566-589, 678-701, 875-989, 1019-1088, 1370-1393, 1466-1569, 1721-1849, 1879-2038, 2086-2171, 2783-2806, 2847-2922, 3043-3119, 3241-3314, 3330-3353, or 3701-3792 of the MSH3 gene.
- E6. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at one or more of positions 876-989, 1019-1088, 1370-1393, 1889-1938, or 3241-3314 of the MSH3 gene.
- E7. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at one or more of positions 876-989, 1019-1088, 1370-1393, 1466-1569, 1756-1849, 1879-2038, 2086-2171, 2783-2806, 2847-2922, 3043-3119, 3241-3314, 3330-3353, or 3701-3792 of the MSH3 gene.
- E8. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at one or more of positions 678-701, 875-989, 1019-1088, 1370-1393, 1466-1569, 1721-1849, 1879-2038, 2086-2171, 2783-2806, 2847-2922, 3043-3119, 3241-3314, 3330-3353, and 3701-3792 of the MSH3 gene.
- E9. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at positions 879-921 of the MSH3 gene.
- E10. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at one or more of positions 875-989, 1019-1088, 1370-1393, 1466-1569, 1721-1849, 1879-1970, 2783-2806, 2847-2922, 3043-3119, 3241-3314, 3330-3353, or 3703-3792 of the MSH3 gene.
- E11. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at one or more of positions 566-589, 678-701, 875-989, 1019-1088, 1370-1393, 1466-1569, 1721-1849, 1879-2038, 2086-2171, 2783-2806, 2847-2922, 3043-3119, 3241-3314, 3330-3353, or 3701-3792 of the MSH3 gene.
- E12. The dsRNA of any one of E1-E4, wherein the antisense strand comprises an antisense nucleobase sequence selected from Table 3, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), and the sense strand comprises a sense nucleobase sequence complementary to the antisense nucleobase sequence.
- E13. The dsRNA of any one of E1-E4, wherein the antisense nucleobase sequence consists of an antisense strand in Table 3, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), and the sense nucleobase sequence consists of a sequence complementary to the antisense nucleobase sequence.
- E14. The dsRNA of any one of E1-E4, wherein the sense strand comprises a sense nucleobase sequence selected from Table 3, and the antisense strand comprises an antisense nucleobase sequence complementary to the sense nucleobase sequence.
- E15. The dsRNA of any one of E1-E4, wherein the sense nucleobase sequence consists of a sense strand in Table 3, and the antisense nucleobase sequence consists of a sequence complementary to the sense nucleobase sequence.
- E16. The dsRNA of any one of E1-E4, wherein the sense strand comprises a sense nucleobase sequence selected from Tables 4-10, and the antisense strand comprises an antisense nucleobase sequence complementary to the sense nucleobase sequence.
- E17. The dsRNA of any one of E1-E4, wherein the sense nucleobase sequence consists of a sense strand in any one of Tables 4-10, and the antisense nucleobase sequence consists of a sequence complementary to the sense nucleobase sequence.
- E18. The dsRNA of any one of E1-E4, wherein the antisense strand comprises an antisense nucleobase sequence selected from Table 11, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), and the sense strand comprises a sense nucleobase sequence complementary to the antisense nucleobase sequence.
- E19. The dsRNA of any one of E1-E4, wherein the antisense nucleobase sequence consists of an antisense strand in Table 11, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), and the sense nucleobase sequence consists of a sequence complementary to the antisense nucleobase sequence
- E20. The dsRNA of any one of E1-E4, wherein the sense strand comprises a sense nucleobase sequence selected from Table 11, and the antisense strand comprises an antisense nucleobase sequence complementary to the sense nucleobase sequence.
- E21. The dsRNA of any one of E1-E4, wherein the sense nucleobase sequence consists of a sense strand in Table 11, and the antisense nucleobase sequence consists of a sequence complementary to the sense nucleobase sequence.
- E22. The dsRNA of any one of E1-E21, wherein the dsRNA comprises at least one alternative nucleobase, at least one alternative internucleoside linkage, and/or at least one alternative sugar moiety.
- E23. The dsRNA of E22, wherein at least one alternative internucleoside linkage is a phosphorothioate internucleoside linkage.
- E24. The dsRNA of E22, wherein at least one alternative internucleoside linkage is a 2′-alkoxy internucleoside linkage.
- E25. The dsRNA of E22, wherein at least one alternative internucleoside linkage is an alkyl phosphate internucleoside linkage.
- E26. The dsRNA of E22, wherein at least one alternative nucleobase is 5′-methylcytosine, pseudouridine, or 5-methoxyuridine.
- E27. The dsRNA of E22, wherein at least one alternative sugar moiety is 2′-OMe or a bicyclic nucleic acid.
- E28. The dsRNA of E22, wherein the dsRNA comprises at least one 2′-OMe sugar moiety and at least one phosphorothioate internucleoside linkage.
- E29. The dsRNA of any one of E1-E28, 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.
- E30. The dsRNA of any one of E1-E29, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 140, 156, 234, 240, 246, 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.
- E31. The dsRNA of any one of E1-E29, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 2264, 2290, 2308, or 2318.
- E32. The dsRNA of any one of E1-E29, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.
- E33. The dsRNA of any one of E1-E29, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 234, 240, 246, 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.
- E34. The dsRNA of any one of E1-E29, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1866, 1868, 1870, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.
- E35. The dsRNA of any one of E1-E29, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 140, 156, 234, 240, 246, 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.
- E36. The dsRNA of any one of E1-E29, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 141, 157, 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E37. The dsRNA of any one of E1-E29, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 2265, 2291, 2309, or 2319, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E38. The dsRNA of any one of E1-E29, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E39. The dsRNA of any one of E1-E29, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E40. The dsRNA of any one of E1-E29, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E41. The dsRNA of any one of E1-E29, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 141, 157, 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E42. The dsRNA of any one of E1-E29, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 140, 156, 234, 240, 246, 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.
- E43. The dsRNA of any one of E1-E29, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 2264, 2290, 2308, or 2318.
- E44. The dsRNA of any one of E1-E29, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.
- E45. The dsRNA of any one of E1-E29, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 234, 240, 246, 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.
- E46. The dsRNA of any one of E1-E29, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.
- E47. The dsRNA of any one of E1-E29, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 140, 156, 234, 240, 246, 380, 382, 386, 388, 396, 406, 418, 464, 478, 520, 540, 564, 568, 750, 822, 830, 844, 868, 870, 874, 904, 1042, 1060, 1062, 1064, 1068, 1090, 1096, 1098, 1114, 1116, 1166, 1168, 1170, 1182, 1192, 1212, 1214, 1216, 1222, 1244, 1258, 1292, 1358, 1360, 1374, 1378, 1380, 1400, 1866, 1868, 1870, 1882, 1892, 1926, 1946, 1964, 1970, 2084, 2088, 2090, 2094, 2124, 2130, 2146, 2264, 2290, 2308, 2318, 2324, 2606, 2608, 2610, 2632, 2652, 2678, or 2690.
- E48. The dsRNA of any one of E1-E29, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 141, 157, 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691.
- E49. The dsRNA of any one of E1-E29, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 2265, 2291, 2309, or 2319, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E50. The dsRNA of any one of E1-E29, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E51. The dsRNA of any one of E1-E29, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E52. The dsRNA of any one of E1-E29, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E53. The dsRNA of any one of E1-E29, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 141, 157, 235, 241, 247, 381, 383, 387, 389, 397, 407, 419, 465, 479, 521, 541, 565, 569, 751, 823, 831, 845, 869, 871, 875, 905, 1043, 1061, 1063, 1065, 1069, 1091, 1097, 1099, 1115, 1117, 1167, 1169, 1171, 1183, 1193, 1213, 1215, 1217, 1223, 1245, 1259, 1293, 1359, 1361, 1375, 1379, 1381, 1401, 1867, 1869, 1871, 1883, 1893, 1927, 1947, 1965, 1971, 2085, 2089, 2091, 2095, 2125, 2131, 2147, 2265, 2291, 2309, 2319, 2325, 2607, 2609, 2611, 2633, 2653, 2679, or 2691, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E54. The dsRNA of any one of E1-E53, 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.
- E55. The dsRNA of any one of E1-E53, 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.
- E56. The dsRNA of any one of E1-E53, 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.
- E57. The dsRNA of any one of E1-E53, wherein the dsRNA exhibits at least 70% mRNA inhibition at a 10 nM dsRNA concentration when determined using a cell assay when compared with a control cell.
- E58. The dsRNA of any one of E1-E53, 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.
- E59. The dsRNA of any one of E1-E53, 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.
- E60. The dsRNA of any one of E1-E59, wherein the antisense strand is complementary to at least 17 contiguous nucleotides of an MSH3 gene.
- E61. The dsRNA of any one of E1-E59, wherein the antisense strand is complementary to at least 19 contiguous nucleotides of an MSH3 gene.
- E62. The dsRNA of any one of E1-E59, wherein the antisense strand is complementary to 19 contiguous nucleotides of an MSH3 gene.
- E63. The dsRNA of any one of E1-E59, wherein the sense strand is complementary to at least 17 contiguous nucleotides of an MSH3 gene.
- E64. The dsRNA of any one of E1-E59, wherein the sense strand is complementary to at least 19 contiguous nucleotides of an MSH3 gene.
- E65. The dsRNA of any one of E1-E59, wherein the sense strand is complementary to 19 contiguous nucleotides of an MSH3 gene.
- E66. The dsRNA of any one of E1-E65, 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.
- E67. A pharmaceutical composition comprising one or more dsRNAs of any one of E1-E66 and a pharmaceutically acceptable carrier.
- E68. A composition comprising one or more dsRNAs of any one of E1-E66 and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome.
- E69. A vector encoding at least one strand of the dsRNA of any one of E1-E66.
- E70. A cell comprising the vector of E69.
- E71. A method of reducing transcription of MSH3 in a cell, the method comprising contacting the cell with the dsRNA of any one of E1-E66, the pharmaceutical composition of E67, the composition of E68, the vector of E69, or the cell of E70 for a time sufficient to obtain degradation of an mRNA transcript of MSH3, thereby reducing expression of MSH3 in the cell.
- E72. 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 E1-E66, the pharmaceutical composition of E67, the composition of E68, the vector of E69, or the cell of E70.
- E73. A method of reducing the level and/or activity of MSH3 in a cell of a subject identified as having a nucleotide repeat expansion disorder, the method comprising contacting the cell with the dsRNA of any one of E1-E66, the pharmaceutical composition of E67, the composition of E68, the vector of E69, or the cell of E70.
- E74. A method for reducing expression of MSH3 in a cell comprising contacting the cell with the dsRNA of any one of E1-E66, the pharmaceutical composition of E67, the composition of E68, the vector of E69, or the cell of E70 and maintaining the cell for a time sufficient to obtain degradation of an mRNA transcript of MSH3, thereby reducing expression of MSH3 in the cell.
- E75. A method of decreasing nucleotide repeat expansion in a cell, the method comprising contacting the cell with the dsRNA of any one of E1-E66, the pharmaceutical composition of E67, the composition of E68, the vector of E69, or the cell of E70.
- E76. The method of E74 or E75, wherein the cell is in a subject.
- E77. The method of any one of E72, E73, and E76, wherein the subject is a human.
- E78. The method of any one of E71 and 73-E76, wherein the cell is a cell of the central nervous system or a muscle cell.
- E79. The method of any one of E72, E73, and E76-E78, wherein the subject is identified as having a nucleotide repeat expansion disorder.
- E80. The method of any one of E72, E73, and E75-E79 wherein the nucleotide repeat expansion disorder is a polyglutamine disease.
- E81. The method of E80, 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.
- E82. The method of any one of E72, E73, and E75-E79, wherein the nucleotide repeat expansion disorder is a non-polyglutamine disease.
- E83. The method of E82, 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.
- E84. A dsRNA of any one of E1-E66, the pharmaceutical composition of E67, the composition of E68, the vector of E69, or the cell of E70 for use in prevention or treatment of a nucleotide repeat expansion disorder.
- E85. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E84, wherein the nucleotide 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.
- E86. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E84 or E85, wherein the nucleotide repeat expansion disorder is Huntington's disease.
- E87. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E84 or E85, wherein the nucleotide repeat expansion disorder is Friedreich's ataxia.
- E88. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E84 or E85, wherein the nucleotide repeat expansion disorder is myotonic dystrophy type 1.
- E89. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of any of E84-E88, wherein the dsRNA, pharmaceutical composition, composition, vector, or cell is administered intrathecally.
- E90. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of any of E84-E88, wherein the dsRNA, pharmaceutical composition, composition, vector, or cell is administered intraventricularly.
- E91. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of any of E84-E88, wherein the dsRNA, pharmaceutical composition, composition, vector, or cell is administered intramuscularly.
- E92. A method of treating, preventing, or delaying progression of a disorder in a subject in need thereof wherein the subject is suffering from nucleotide repeat expansion disorder, comprising administering to said subject the dsRNA of any one of E1-E66, the pharmaceutical composition of E67, the composition of E68, the vector of E69, or the cell of E70.
- E93. The method of E92, further comprising administering at least one additional therapeutic agent.
- E94. The method of E93, wherein at least one additional therapeutic agent is an antisense oligonucleotide that hybridizes to an mRNA encoding the Huntingtin gene.
- E95. A method of preventing or delaying progression of a nucleotide repeat expansion disorder in a subject, the method comprising administering to the subject the dsRNA of any one of E1-E66, the pharmaceutical composition of E67, the composition of E68, the vector of E69, or the cell of E70 in an amount effective to delay progression of a nucleotide repeat expansion disorder of the subject.
- E96. The method of E95, wherein the nucleotide 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.
- E97. The method of E95 or E96, wherein the nucleotide repeat expansion disorder is Huntington's disease.
- E98. The method of E95 or E96, wherein the nucleotide repeat expansion disorder is Friedrich's ataxia.
- E99. The method of E95 or E96, wherein the nucleotide repeat expansion disorder is myotonic dystrophy type 1.
- E100. The method of any of E95 or E96, further comprising administering at least one additional therapeutic agent.
- E101. The method of E100, wherein at least one additional therapeutic agent is an antisense oligonucleotide that hybridizes to an mRNA encoding the Huntingtin gene.
- E102. The method of any of E94-E101, wherein progression of the nucleotide 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.
- E103. A dsRNA of any one of E1-E66, the pharmaceutical composition of E67, the composition of E68, the vector of E69, or the cell of E70, for use in preventing or delaying progression of a nucleotide repeat expansion disorder in a subject.
- E104. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E103, wherein the nucleotide 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.
- E105. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E103 or E104, wherein the nucleotide repeat expansion disorder is Huntington's disease.
- E106. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E103 or E104, wherein the nucleotide repeat expansion disorder is Friedrich's ataxia.
- E107. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E103 or E104, wherein the nucleotide repeat expansion disorder is myotonic dystrophy type 1.
- E108. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of any one of E103-E107, wherein progression of the nucleotide 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.
- E109. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of any one of claims E103-E107, wherein progression of the nucleotide 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 at least 20 years or more, when compared with a predicted progression.
- E110. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at one or more of positions 430-453, 508-531, 560-599, 609-632, 681-721, 768-797, 823-856, 882-927, 968-1029, 1039-1096, 1106-1175, 1188-1217, 1272-1297, 1419-1474, 1489-1516, 1540-1627, 1633-1815, 1819-1842, 1899-1937, 2027-2066, 2085-2108, 2117-2156, 2163-2187, 2195-2241, 2293-2343, 2347-2374, 2493-2539, 2567-2590, 2619-2649, 2737-2764, 2779-2820, 2871-2894, 2900-2923, 2949-2972, 3049-3096, 3217-3266, 3272-3309, 3351-3383, 3386-3415, 3537-3560, 3581-3619, 3686-3728, 3754-3778, 3782-3805, 3909-3935, 4287-4310, or 4386-4412 of the MSH3 gene.
- E111. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at one or more of positions 508-531, 827-856, 903-926, 1073-1096, 1126-1149, 1583-1609, 1639-1662, 1727-1750, 1755-1795, 1819-1842, 1905-1937, 2130-2153, 2293-2316, 2505-2528, 2625-2648, 2797-2820, 3073-3096, 3217-3240, 3351-3383, 3686-3728, 3754-3777, 4287-4310, or 4386-4412 of the MSH3 gene.
- E112. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at one or more of positions 508-531, 833-856, 1073-1096, 1126-1149, 1583-1609, 1639-1662, 1727-1750, 1755-1795, 1914-1937, 2130-2153, 2293-2316, 2797-2820, 3073-3096, 3217-3240, 3596-3619, 3700-3723, 3754-3777, or 4386-4409 of the MSH3 gene.
- E113. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at one or more of positions 1073-1096, 1586-1609, 1755-1795, 1914-1937, 2130-2153, 2293-2316, 3217-3240, or 4386-4409 of the MSH3 gene
- E114. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at positions 908-925 of the MSH3 gene.
- E115. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at positions 1167-1184 of the MSH3 gene.
- E116. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at positions 1143-1166 of the MSH3 gene.
- E117. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at positions 1150-1173 of the MSH3 gene
- E118. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at positions 2090-2107 of the MSH3 gene
- E119. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at positions 1040-1057 of the MSH3 gene
- E120. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at positions 2018-2035 of the MSH3 gene
- E121. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at positions 1469-1486 of the MSH3 gene
- E122. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at positions 1128-1151 of the MSH3 gene.
- E123. The dsRNA of any one of E1-E4, wherein the region the sense or antisense strand is complementary to is at least 15 contiguous nucleotides of an MSH3 gene corresponding to a sequence of reference mRNA NM_002439.4 at positions 828-851 of the MSH3 gene.
- E124. The dsRNA of any one of E1-E4, wherein the antisense strand comprises an antisense nucleobase sequence selected from Table 12, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), and the sense strand comprises a sense nucleobase sequence complementary to the antisense nucleobase sequence.
- E125. The dsRNA of any one of E1-E4, wherein the antisense nucleobase sequence consists of an antisense strand in Table 12, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), and the sense nucleobase sequence consists of a sequence complementary to the antisense nucleobase sequence.
- E126. The dsRNA of any one of E1-E4, wherein the sense strand comprises a sense nucleobase sequence selected from Table 12, and the antisense strand comprises an antisense nucleobase sequence complementary to the sense nucleobase sequence.
- E127. The dsRNA of any one of E1-E4, wherein the sense nucleobase sequence consists of a sense strand in Table 12, and the antisense nucleobase sequence consists of a sequence complementary to the sense nucleobase sequence.
- E128. The dsRNA of any one of E109-E127, wherein the dsRNA comprises at least one alternative nucleobase, at least one alternative internucleoside linkage, at least one alternative sugar moiety, or a combination thereof, optionally wherein the sense strand is selected from Table 13 and the antisense strand is selected from Table 14.
- E129. The dsRNA of E127, wherein at least one alternative internucleoside linkage is a phosphorothioate internucleoside linkage.
- E130. The dsRNA of E127, wherein at least one alternative internucleoside linkage is a 2′-alkoxy internucleoside linkage.
- E131. The dsRNA of E127, wherein at least one alternative internucleoside linkage is an alkyl phosphate internucleoside linkage.
- E132. The dsRNA of E127, wherein at least one alternative nucleobase is 5′-methylcytosine, pseudouridine, or 5-methoxyuridine.
- E133. The dsRNA of E127, wherein at least one alternative sugar moiety is 2′-OMe or a bicyclic nucleic acid.
- E134. The dsRNA of E127, wherein the dsRNA comprises at least one 2′-OMe sugar moiety and at least one phosphorothioate internucleoside linkage.
- E135. The dsRNA of any one of E110-E134, 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.
- E136. The dsRNA of any one of E110-135, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 78, 82, 104, 148, 158, 160, 190, 240, 244, 246, 250, 252, 260, 308, 314, 316, 354,356, 360, 362, 364, 368, 370, 372, 396, 414, 416, 418, 474, 476,478, 480, 502, 512, 552 558, 560, 582, 616, 618, 634, 636, 642, 646, 648, 656, 660, 690, 692, 718, 720, 722, 796, 820, 822, 826, 848, 852, 854, 900, 914, 928, 930, 934,936, 946, 948, 966, 970,972, 988, 990, 992, 994, 996, 1006, 1020, 1032, 1054, 1056, 1058, 1076, 1088, 1096, 1098, 1110, 1112, 1126, 1214, 1220, 1230, 1306, 1308, 1310, 1318, 1326, 1386, 1394, 1396, 1400, 1404, 1424, 1426, 1448, 1452, 1454, 1506, 1524, 1540, 1546, 1656, 1666, 1674, 1676, 1678, 1722, 1762, 1766, 1768, 1836, 1838, 1842, 1868, 1886, 1888, 1964, 1990, 2030, 2108, 2128, 2230, 2242, 2246, 2254, 2274, 2294, 2330, 2334, 2356, 2360, 2362, 2448, 2502, 2504, 2516, 2518, 2578, 2580, 2592, 2596, 2602, 2654, 2656, 2686, 2762, 2768, 2782, 2844, or 2846.
- E137. The dsRNA of any one of E110-E135, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 362, 370, 372, 416, 582, 634, 930, 934, 970, 1054, 1076, 1088, 1096, 1098, 1126, 1220, 1230, 1400, 1506, 1666, 1766, 1888, 2128, 2230, 2330, 2334, 2448, 2504, 2516, 2518, 2578, 2592, 2596, 2602, 2654, 2782, 2844, or 2846.
- E138. The dsRNA of any one of E110-E135, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 104, 372, 582, 634, 930, 934, 970, 1054, 1076, 1088, 1098, 1230, 1400, 1506, 1888, 2128, E2230, 2518, 2592, 2654, or 2844.
- E139. The dsRNA of any one of E110-E135, wherein the sense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 582, 934, 1076, 1088, 1098, 1230, 1400, 1506, 2230, or 2844.
- E140. The dsRNA of any one of E110-E135, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 78, 82, 104, 148, 158, 160, 190, 240, 244, 246, 250, 252, 260, 308, 314, 316, 354, 356, 360, 362, 364, 368, 370, 372, 396, 414, 416, 418, 474, 476, 478, 480, 502, 512, 552, 558, 560, 582, 616, 618, 634, 636, 642, 646, 648, 656, 660, 690, 692, 718, 720, 722, 796, 820, 822, 826, 848, 852, 854, 900, 914, 928, 930, 934, 936, 946, 948, 966, 970, 972, 988, 990, 992, 994, 996, 1006, 1020, 1032, 1054, 1056, 1058, 1076, 1088, 1096, 1098, 1110, 1112, 1126, 1214, 1220, 1230, 1306, 1308, 1310, 1318, 1326, 1386, 1394, 1396, 1400, 1404, 1424, 1426, 1448, 1452, 1454, 1506, 1524, 1540, 1546, 1656, 1666, 1674, 1676, 1678, 1722, 1762, 1766, 1768, 1836, 1838, 1842, 1868, 1886, 1888, 1964, 1990, 2030, 2108, 2128, 2230, 2242, 2246, 2254, 2274, 2294, 2330, 2334, 2356, 2360, 2362, 2448, 2502, 2504, 2516, 2518, 2578, 2580, 2592, 2596, 2602, 2654, 2656, 2686, 2762, 2768, 2782, 2844, or 2846.
- E141. The dsRNA of any one of E110-E135, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 104, 362, 370, 372, 416, 582, 634, 930, 934, 970, 1054, 1076, 1088, 1096, 1098, 1126, 1220, 1230, 1400, 1506, 1666, 1766, 1888, 2128, 2230, 2330, 2334, 2448, 2504, 2516 2518, 2578, 2592, 2596, 2602, 2654, 2782, 2844, or 2846.
- E142. The dsRNA of any one of E110-E135, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 104, 372, 582, 634, 930, 934, 970, 1054, 1076, 1088, 1098, 1230, 1400, 1506, 1888, 2128, 2230, 2518, 2592, 2654, or 2844.
- E143. The dsRNA of any one of E110-E135, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 582, 934, 1076, 1088, 1098, 1230, 1400, 1506, 2230, or 2844
- E144. The dsRNA of any one of E110-E135, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 78, 82, 104, 148, 158, 160, 190, 240, 244, 246, 250, 252, 260, 308, 314, 316, 354, 356, 360, 362, 364, 368, 370, 372, 396, 414, 416, 418, 474, 476,478, 480, 502, 512, 552 558, 560, 582, 616, 618, 634, 636, 642, 646, 648, 656, 660, 690, 692, 718, 720, 722, 796, 820, 822, 826, 848, 852, 854, 900, 914, 928, 930, 934,936, 946, 948, 966, 970,972, 988, 990, 992, 994, 996, 1006, 1020, 1032, 1054, 1056, 1058, 1076, 1088, 1096, 1098, 1110, 1112, 1126, 1214, 1220, 1230, 1306, 1308, 1310, 1318, 1326, 1386, 1394, 1396, 1400, 1404, 1424, 1426, 1448, 1452, 1454, 1506, 1524, 1540, 1546, 1656, 1666, 1674, 1676, 1678, 1722, 1762, 1766, 1768, 1836, 1838, 1842, 1868, 1886, 1888, 1964, 1990, 2030, 2108, 2128, 2230, 2242, 2246, 2254, 2274, 2294, 2330, 2334, 2356, 2360, 2362, 2448, 2502, 2504, 2516, 2518, 2578, 2580, 2592, 2596, 2602, 2654, 2656, 2686, 2762, 2768, 2782, 2844, or 2846 and an overhang of 1-4 nucleotides.
- E145. The dsRNA of any one of E110-E135, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 104, 362, 370, 372, 416, 582, 634, 930, 934, 970, 1054, 1076, 1088, 1096, 1098, 1126, 1220, 1230, 1400, 1506, 1666, 1766, 1888, 2128, 2230, 2330, 2334, 2448, 2504, 2516, 2518, 2578, 2592, 2596, 2602, 2654, 2782, 2844, or 2846 and an overhang of 1-4 nucleotides.
- E146. The dsRNA of any one of E110-E135, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 104, 372, 582, 634,930, 934, 970, 1054, 1076, 1088, 1098, 1230, 1400, 1506, 1888, 2128, 2230, 2518, 2592, 2654, or 2844 and an overhang of 1-4 nucleotides.
- E147. The dsRNA of any one of E110-E135, wherein the sense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 582, 934, 1076, 1088, 1098, 1230, 1400, 1506, 2230, or 2844 and an overhang of 1-4 nucleotides.
- E148. The dsRNA of any one of E110-E135, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 79, 83, 105, 149, 159, 161, 191, 241, 245, 247, 251, 253, 261, 309, 315, 317, 355, 357, 361, 363, 365, 369, 371, 373, 379, 415, 417, 419, 475, 477, 479, 481, 503, 513, 553, 559, 561, 583, 617, 619, 635, 637, 643, 647, 649, 657, 661, 691, 693, 719, 721, 723, 797, 821, 823, 827, 849, 853, 855, 901, 915, 929, 931, 935, 937, 947, 949, 967, 971, 973, 989, 991, 993, 995, 997, 1007, 1021, 1032, 1055, 1057, 1059, 1077, 1089, 1097, 1099, 1111, 1113, 1127, 1215, 1221, 1230, 1307, 1309, 1311, 1319, 1363, 1387, 1395, 1397, 1401, 1405, 1425, 1427, 1449, 1453, 1455, 1507, 1525, 1541, 1547, 1657, 1667, 1675, 1677, 1679, 1723, 1763, 1767, 1769, 1837, 1839, 1843, 1869, 1887, 1889, 1965, 1991, 2031, 2109, 2129, 2231, 2243, 2247, 2255, 2275, 2295, 2331, 2335, 2357, 2361, 2363, 2449, 2503, 2505, 2517, 2519, 2579, 2581, 2593, 2597, 2603, 2655, 2657, 2687, 2763, 2769, 2783, 2845, or 2847, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E149. The dsRNA of any one of E110-E135, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 104, 362, 370, 372, 416, 582, 634, 930, 934, 970, 1054, 1076, 1088, 1096, 1098, 1126, 1220, 1230, 1400, 1506, 1666, 1766, 1888, 2128, 2230, 2330, 2334, 2448, 2504, 2516, 2518, 2578, 2592, 2596, 2602, 2654, 278, 2844, or 2846, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E150. The dsRNA of any one of E110-E135, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 104, 372, 582, 634, 930, 934, 970, 1054, 1076, 1088, 1098, 1230, 1400, 1506, 1888, 2128, 2230, 2518, 2592, 2654, or 2844, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E151. The dsRNA of any one of E110-E135, wherein the antisense strand comprises a nucleobase sequence of any one of SEQ ID NOs: 582, 934, 1076, 1088, 1098, 1230, 1400, 1506, 2230, or 2844, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G)
- E152. The dsRNA of any one of E110-E135, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 79, 83, 105, 149, 159, 161, 191, 241, 245, 247, 251, 253, 261, 309, 315, 317, 355, 357, 361, 363, 365, 369, 371, 373, 379, 415, 417, 419, 475, 477, 479, 481, 503, 513, 553, 559, 561, 583, 617, 619, 635, 637, 643, 647, 649, 657, 661, 691, 693, 719, 721, 723, 797, 821, 823, 827, 849, 853, 855, 901, 915, 929, 931, 935, 937, 947, 949, 967, 971, 973, 989, 991, 993, 995, 997, 1007, 1021, 1032, 1055, 1057, 1059, 1077, 1089, 1097, 1099, 1111, 1113, 1127, 1215, 1221, 1230, 1307, 1309, 1311, 1319, 1363, 1387, 1395, 1397, 1401, 1405, 1425, 1427, 1449, 1453, 1455, 1507, 1525, 1541, 1547, 1657, 1667, 1675, 1677, 1679, 1723, 1763, 1767, 1769, 1837, 1839, 1843, 1869, 1887, 1889, 1965, 1991, 2031, 2109, 2129, 2231, 2243, 2247, 2255, 2275, 2295, 2331, 2335, 2357, 2361, 2363, 2449, 2503, 2505, 2517, 2519, 2579, 2581, 2593, 2597, 2603, 2655, 2657, 2687, 2763, 2769, 2783, 2845, or 2847, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E153. The dsRNA of any one of E110-E135, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 104, 362, 370, 372, 416, 582, 634, 930, 934, 970, 1054, 1076, 1088, 1096, 1098, 1126, 1220, 1230, 1400, 1506, 1666, 1766, 1888, 2128, 2230, 2330, 2334, 2448, 2504, 2516, 2518, 2578, 2592, 2596, 2602, 2654, 2782, 2844, or 2846, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E154. The dsRNA of any one of E110-E135, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 104, 372, 582, 634, 930, 934, 970, 1054, 1076, 1088, 1098, 1230, 1400, 1506, 1888, 2128, 2230, 2518, 2592, 2654, or 2844, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E155. The dsRNA of any one of E110-E135, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 582, 934, 1076, 1088, 1098, 1230, 1400, 1506, 2230, or 2844, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G)
- E156. The dsRNA of any one of E110-E135, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 79, 83, 105, 149, 159, 161, 191, 241, 245, 247, 251, 253, 261, 309, 315, 317, 355, 357, 361, 363, 365, 369, 371, 373, 379, 415, 417, 419, 475, 477, 479, 481, 503, 513, 553, 559, 561, 583, 617, 619, 635, 637, 643, 647, 649, 657, 661, 691, 693, 719, 721, 723, 797, 821, 823, 827, 849, 853, 855, 901, 915, 929, 931, 935, 937, 947, 949, 967, 971, 973, 989, 991, 993, 995, 997, 1007, 1021, 1032, 1055, 1057, 1059, 1077, 1089, 1097, 1099, 1111, 1113, 1127, 1215, 1221, 1230, 1307, 1309, 1311, 1319, 1363, 1387, 1395, 1397, 1401, 1405, 1425, 1427, 1449, 1453, 1455, 1507, 1525, 1541, 1547, 1657, 1667, 1675, 1677, 1679, 1723, 1763, 1767, 1769, 1837, 1839, 1843, 1869, 1887, 1889, 1965, 1991, 2031, 2109, 2129, 2231, 2243, 2247, 2255, 2275, 2295, 2331, 2335, 2357, 2361, 2363, 2449, 2503, 2505, 2517, 2519, 2579, 2581, 2593, 2597, 2603, 2655, 2657, 2687, 2763, 2769, 2783, 2845, or 2847, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), and an overhang of 1-4 nucleotides.
- E157. The dsRNA of any one of E110-E135, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 104, 362, 370, 372, 416, 582, 634, 930, 934, 970, 1054, 1076, 1088, 1096, 1098, 1126, 1220, 1230, 1400, 1506, 1666, 1766, 1888, 2128, 2230, 2330, 2334, 2448, 2504, 2516, 2518, 2578, 2592, 2596, 2602, 2654, 2782, 2844, or 2846, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), and an overhang of 1-4 nucleotides.
- E158. The dsRNA of any one of E110-E135, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 104, 372, 582, 634, 930, 934, 970, 1054, 1076, 1088, 1098, 1230, 1400, 1506, 1888, 2128, E2230, 2518, 2592, 2654, or 2844, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), and an overhang of 1-4 nucleotides.
- E159. The dsRNA of any one of E110-E135, wherein the antisense strand consists of a nucleobase sequence of any one of SEQ ID NOs: 582, 934, 1076, 1088, 1098, 1230, 1400, 1506, 2230, or 2844, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G), and an overhang of 1-4 nucleotides.
- E160. The dsRNA of E136, wherein the sense strand comprises a nucleobase sequence of SEQ ID NO: 656.
- E161. The dsRNA of E136, wherein the sense strand comprises a nucleobase sequence of SEQ ID NO: 636.
- E162. The dsRNA of E136, wherein the sense strand comprises a nucleobase sequence of SEQ ID NO: 364.
- E163. The dsRNA of E136, wherein the sense strand comprises a nucleobase sequence of SEQ ID NO: 648.
- E164. The dsRNA of E136, wherein the sense strand comprises a nucleobase sequence of SEQ ID NO: E1366.
- E165. The dsRNA of E136, wherein the sense strand comprises a nucleobase sequence of SEQ ID NO: 550.
- E166. The dsRNA of E136, wherein the sense strand comprises a nucleobase sequence of SEQ ID NO: 1874.
- E167. The dsRNA of E136, wherein the sense strand comprises a nucleobase sequence of SEQ ID NO: E1302.
- E168. The dsRNA of E136, wherein the sense strand comprises a nucleobase sequence of SEQ ID NO: 420.
- E169. The dsRNA of E136, wherein the sense strand comprises a nucleobase sequence of SEQ ID NO: 672.
- E170. The dsRNA of E136, wherein the sense strand comprises a nucleobase sequence of SEQ ID NO: 832.
- E171. The dsRNA of E148, wherein the antisense strand comprises a nucleobase sequence of SEQ ID NO: 649, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E172. The dsRNA of E148, wherein the antisense strand comprises a nucleobase sequence of SEQ ID NO: 657, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E173. The dsRNA of E148, wherein the antisense strand comprises a nucleobase sequence of SEQ ID NO: 637, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E174. The dsRNA of E148, wherein the antisense strand comprises a nucleobase sequence of SEQ ID NO: 365, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E175. The dsRNA of E148, wherein the antisense strand comprises a nucleobase sequence of SEQ ID NO: E1367, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E176. The dsRNA of E148, wherein the antisense strand comprises a nucleobase sequence of SEQ ID NO: 551, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E177. The dsRNA of E148, wherein the antisense strand comprises a nucleobase sequence of SEQ ID NO: 1875, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E178. The dsRNA of E148, wherein the antisense strand comprises a nucleobase sequence of SEQ ID NO: 1303, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E179. The dsRNA of E148, wherein the antisense strand comprises a nucleobase sequence of SEQ ID NO: 421, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E180. The dsRNA of E148, wherein the antisense strand comprises a nucleobase sequence of SEQ ID NO: 673, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E181. The dsRNA of E148, wherein the antisense strand comprises a nucleobase sequence of SEQ ID NO: 833, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E182. The dsRNA of any one of E110-E135, wherein the sense strand is any one of Sense Oligo Nos: 78, 82, 104, 148, 158, 160, 190, 240, 244, 246, 250, 252, 260, 308, 314, 316, 354, 356, 360, 362, 364, 368, 370, 372, 396, 414, 416, 418, 474, 476,478, 480, 502, 512, 552 558, 560, 582, 616, 618, 634, 636, 642, 646, 648, 656, 660, 690, 692, 718, 720, 722, 796, 820, 822, 826, 848, 852, 854, 900, 914, 928, 930, 934,936, 946, 948, 966, 970,972, 988, 990, 992, 994, 996, 1006, 1020, 1032, 1054, 1056, 1058, 1076, 1088, 1096, 1098, 1110, 1112, 1126, 1214, 1220, 1230, 1306, 1308, 1310, 1318, 1326, 1386, 1394, 1396, 1400, 1404, 1424, 1426, 1448, 1452, 1454, 1506, 1524, 1540, 1546, 1656, 1666, 1674, 1676, 1678, 1722, 1762, 1766, 1768, 1836, 1838, 1842, 1868, 1886, 1888, 1964, 1990, 2030, 2108, 2128, 2230, 2242, 2246, 2254, 2274, 2294, 2330, 2334, 2356, 2360, 2362, 2448, 2502, 2504, 2516, 2518, 2578, 2580, 2592, 2596, 2602, 2654, 2656, 2686, 2762, 2768, 2782, 2844, or 2846.
- E183. The dsRNA of any one of E110-E135, wherein the sense strand is any one of Sense Oligo Nos: 362, 370, 372, 416, 582, 634, 930, 934, 970, 1054, 1076, 1088, 1096, 1098, 1126, 1220, 1230, 1400, 1506, 1666, 1766, 1888, 2128, 2230, 2330, 2334, 2448, 2504, 2516 2518, 2578, 2592, 2596, 2602, 2654, 2782, 2844, or 2846.
- E184. The dsRNA of any one of E110-E135, wherein the sense strand is any one of Sense Oligo Nos: 104, 372, 582, 634, 930, 934, 970, 1054, 1076, 1088, 1098, 1230, 1400, 1506, 1888, 2128, 2230, 2518, 2592, 2654, or 2844.
- E185. The dsRNA of any one of E110-E135, wherein the sense strand is any one of Sense Oligo Nos: 582, 934, 1076, 1088, 1098, 1230, 1400, 1506, 2230, or 2844.
- E186. The dsRNA of any one of E110-E135, wherein the antisense strand is any one of Antisense Oligo Nos: 79, 83, 105, 149, 159, 161, 191, 241, 245, 247, 251, 253, 261, 309, 315, 317, 355, 357, 361, 363, 365, 369, 371, 373, 379, 415, 417, 419, 475, 477, 479, 481, 503, 513, 553, 559, 561, 583, 617, 619, 635, 637, 643, 647, 649, 657, 661, 691, 693, 719, 721, 723, 797, 821, 823, 827, 849, 853, 855, 901, 915, 929, 931, 935, 937, 947, 949, 967, 971, 973, 989, 991, 993, 995, 997, 1007, 1021, 1032, 1055, 1057, 1059, 1077, 1089, 1097, 1099, 1111, 1113, 1127, 1215, 1221, 1230, 1307, 1309, 1311, 1319, 1363, 1387, 1395, 1397, 1401, 1405, 1425, 1427, 1449, 1453, 1455, 1507, 1525, 1541, 1547, 1657, 1667, 1675, 1677, 1679, 1723, 1763, 1767, 1769, 1837, 1839, 1843, 1869, 1887, 1889, 1965, 1991, 2031, 2109, 2129, 2231, 2243, 2247, 2255, 2275, 2295, 2331, 2335, 2357, 2361, 2363, 2449, 2503, 2505, 2517, 2519, 2579, 2581, 2593, 2597, 2603, 2655, 2657, 2687, 2763, 2769, 2783, 2845, or 2847, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E187. The dsRNA of any one of E110-E135, wherein the antisense strand is any one of Antisense Oligo Nos: 104, 362, 370, 372, 416, 582, 634, 930, 934, 970, 1054, 1076, 1088, 1096, 1098, 1126, 1220, 1230, 1400, 1506, 1666, 1766, 1888, 2128, 2230, 2330, 2334, 2448, 2504, 2516, 2518, 2578, 2592, 2596, 2602, 2654, 2782, 2844, or 2846, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E188. The dsRNA of any one of E110-E135, wherein the antisense strand is any one of Antisense Oligo Nos: 104, 372, 582, 634, 930, 934, 970, 1054, 1076, 1088, 1098, 1230, 1400, 1506, 1888, 2128, 2230, 2518, 2592, 2654, or 2844, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E189. The dsRNA of any one of E110-E135, wherein the antisense strand is any one of Antisense Oligo Nos: 582, 934, 1076, 1088, 1098, 1230, 1400, 1506, 2230, or 2844, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E190. The dsRNA of E136, wherein the sense strand is Sense Oligo No: 656.
- E191. The dsRNA of E136, wherein the sense strand is Sense Oligo No: 636.
- E192. The dsRNA of E136, wherein the sense strand Sense Oligo No: 364.
- E193. The dsRNA of E136, wherein the sense strand is Sense Oligo No: 648.
- E194. The dsRNA of E136, wherein the sense strand is Sense Oligo No: 1366.
- E195. The dsRNA of E136, wherein the sense strand is Sense Oligo No: 550.
- E196. The dsRNA of E136, wherein the sense strand Sense Oligo No: 1874.
- E197. The dsRNA of E136, wherein the sense strand Sense Oligo No: E1302.
- E198. The dsRNA of E136, wherein the sense strand is Sense Oligo No: 420.
- E199. The dsRNA of E135, wherein the sense strand Sense Oligo No: 672.
- E200. The dsRNA of E136, wherein the sense strand is Sense Oligo No: 832.
- E201. The dsRNA of E148, wherein the antisense strand is Antisense Oligo No: 649, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E202. The dsRNA of E148, wherein the antisense strand is Antisense Oligo No: 657, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E203. The dsRNA of E148, wherein the antisense strand is Antisense Oligo No: 637, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E204. The dsRNA of E148, wherein the antisense strand is Antisense Oligo No: 365, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E205. The dsRNA of E148, wherein the antisense strand is Antisense Oligo No: E1367, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E206. The dsRNA of E148, wherein the antisense strand is Antisense Oligo No: 551, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E207. The dsRNA of E148, wherein the antisense strand is Antisense Oligo No: 1875, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E208. The dsRNA of E148, wherein the antisense strand is Antisense Oligo No: 1303, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E209. The dsRNA of E148, wherein the antisense strand is Antisense Oligo No: 421, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E210. The dsRNA of E148, wherein the antisense strand is Antisense Oligo No: 673, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E211. The dsRNA of E148, wherein the antisense strand is Antisense Oligo No 833, wherein the 5′ nucleotide represented by U can be any nucleotide (e.g., U, A, C, G).
- E212. The dsRNA of any one of E110-E211, wherein the dsRNA exhibits at least 50% mRNA inhibition at a 10 nM dsRNA concentration when determined using a cell assay compared with a control cell.
- E213. The dsRNA of any one of E110-E211, wherein the dsRNA exhibits at least 60% mRNA inhibition at a 10 nM dsRNA concentration when determined using a cell assay compared with a control cell.
- E214. The dsRNA of any one of E110-E211, wherein the dsRNA exhibits at least 70% mRNA inhibition at a 10 nM dsRNA concentration when determined using a cell assay when compared with a control cell.
- E215. The dsRNA of any one of E110-E211, wherein the sense strand is complementary to at least 17 contiguous nucleotides of an MSH3 gene.
- E216. The dsRNA of any one of E110-E211, wherein the sense strand is complementary to at least 19 contiguous nucleotides of an MSH3 gene.
- E217. The dsRNA of any one of E110-E211, wherein the antisense strand is complementary to 17 contiguous nucleotides of an MSH3 gene.
- E218. The dsRNA of any one of E110-E211, wherein the antisense strand is complementary to at least 19 contiguous nucleotides of an MSH3 gene.
- E219. The dsRNA of any one of E110-E211, 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.
- E220. A pharmaceutical composition comprising one or more dsRNAs of any one of E110-E219 and a pharmaceutically acceptable carrier.
- E221. A composition comprising one or more dsRNAs of any one of E110-E219 and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome.
- E222. A vector encoding at least one strand of the dsRNA of any one of E110-E219.
- E223. A cell comprising the vector of E222.
- E224. A method of reducing transcription of MSH3 in a cell, the method comprising contacting the cell with the dsRNA of any one of E110-E219, the pharmaceutical composition of E220, the composition of E221, the vector of E222, or the cell of E223 for a time sufficient to obtain degradation of an mRNA transcript of MSH3, thereby reducing expression of MSH3 in the cell.
- E225. A method of treating, preventing, or delaying progression of a nucleotide repeat expansion disorder in a subject in need thereof, the method comprising administering to the subject the dsRNA of any one of E110-E219, the pharmaceutical composition of E220, the composition of E221, the vector of E222, or the cell of E223.
- E226. A method of reducing the level and/or activity of MSH3 in a cell of a subject identified as having a nucleotide repeat expansion disorder, the method comprising contacting the cell with the dsRNA of any one of E of any one of E110-E219, the pharmaceutical composition of E220, the composition of E221, the vector of E222, or the cell of E223.
- E227. A method for reducing expression of MSH3 in a cell comprising contacting the cell with the dsRNA of any one of E of any one of E110-E219, the pharmaceutical composition of E220, the composition of E221, the vector of E222, or the cell of E223 and maintaining the cell for a time sufficient to obtain degradation of an mRNA transcript of MSH3, thereby reducing expression of MSH3 in the cell.

228. A method of decreasing nucleotide repeat expansion in a cell, the method comprising contacting the cell with the dsRNA of any one of E of any one of E110-E219, the pharmaceutical composition of E220, the composition of E221, the vector of E222, or the cell of E223.

- E229. The method of any one of E224-E228, wherein the cell is in a subject.
- E230. The method of any one of E224-E229, wherein the subject is a human.
- E231. The method of any one of E224-E230, wherein the cell is a cell of the central nervous system or a muscle cell
- E232. The method of any one of E225-E226 or E229-E231, wherein the subject is identified as having a nucleotide repeat expansion disorder.
- E233. The method of any one of E232, wherein the subject is identified as having a trinucleotide repeat expansion disorder.
- E234. The method of E233, wherein the trinucleotide repeat expansion disorder is a polyglutamine disease.
- E235. The method of E234, 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.
- E236. The method of E233, wherein the trinucleotide repeat expansion disorder is a non-polyglutamine disease.
- E237. The method of E236, 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.
- E238. A dsRNA of any one of E of any one of E110-E219, the pharmaceutical composition of E220, the composition of E221, the vector of E222, or the cell of E223, for use in prevention or treatment of a nucleotide repeat expansion disorder.
- E239. The dsRNA of E238, wherein the nucleotide repeat expansion disorder is a trinucleotide repeat expansion disorder.
- E240. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E238 or E239, wherein the nucleotide 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.
- E241. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E238 or E239, wherein the trinucleotide repeat expansion disorder is Huntington's disease.
- E242. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E238 or E239, wherein the trinucleotide repeat expansion disorder is Friedreich's ataxia.
- E243. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E238 or E239, wherein the trinucleotide repeat expansion disorder is myotonic dystrophy type 1.
- E244. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of any of E238 or E239, wherein the dsRNA, pharmaceutical composition, composition, vector, or cell is administered intrathecally.
- E245. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of any of E238 or E239, wherein the dsRNA, pharmaceutical composition, composition, vector, or cell is administered intraventricularly.
- E246. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of any of E238 or E239, wherein the dsRNA, pharmaceutical composition, composition, vector, or cell is administered intramuscularly.
- E247. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of any of E238 or E239, wherein the dsRNA, pharmaceutical composition, composition, vector, or cell is administered intracerebroventricularly.
- E248. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of any of E238 or E239, wherein the dsRNA, pharmaceutical composition, composition, vector, or cell is administered intraocularly.
- E249. 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 any one of E110-E219, the pharmaceutical composition of E220, the composition of E221, the vector of E222, or the cell of E223.
- E250. The method of E249, further comprising administering at least one additional therapeutic agent.
- E251. The method of E250, wherein at least one additional therapeutic agent is an antisense oligonucleotide that hybridizes to an mRNA encoding the Huntingtin gene.
- E252. 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 s E110-E219, the pharmaceutical composition of E220, the composition of E221, the vector of 222, or the cell of E223 in an amount effective to delay progression of a nucleotide repeat expansion disorder of the subject.
- E253. The method of E252, wherein the nucleotide repeat expansion disorder is a trinucleotide repeat expansion disorder.
- E254. The method of E253, 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.
- E255. The method of E253 or E254, wherein the trinucleotide repeat expansion disorder is Huntington's disease.
- E256. The method of E253 or E254, wherein the trinucleotide repeat expansion disorder is Friedrich's ataxia.
- E257. The method of E253 or E254, wherein the trinucleotide repeat expansion disorder is myotonic dystrophy type 1.
- E258. The method of E253 or E254, wherein the trinucleotide repeat expansion disorder is a Spinocerebellar ataxia (SCA).
- E259. The method of E258, wherein the SCA is Spinocerebellar ataxia type 1 (SCA1).
- E260. The method of E258, wherein the SCA is Spinocerebellar ataxia type 10 (SCA10).
- E261. The method of E258, wherein the SCA is Spinocerebellar ataxia type 12 (SCA12).
- E262. The method of E258, wherein the SCA is Spinocerebellar ataxia type 17 (SCA17).
- E263. The method of E258, wherein the SCA is Spinocerebellar ataxia type 2 (SCA2).
- E264. The method of E258, wherein the SCA is Spinocerebellar ataxia type 3 (SCA3)/Machado-Joseph Disease.
- E265. The method of E258, wherein the SCA is Spinocerebellar ataxia type 45 (SCA45).
- E266. The method of E258, wherein the SCA is Spinocerebellar ataxia type 6 (SCA6).
- E267. The method of E258, wherein the SCA is Spinocerebellar ataxia type 7 (SCA7).
- E268. The method of E258, wherein the SCA is Spinocerebellar ataxia type 8 (SCA8).
- E269. The method of any of E249-E268, further comprising administering at least one additional therapeutic agent.
- E270. The method of 269, wherein at least one additional therapeutic agent is an antisense oligonucleotide that hybridizes to an mRNA encoding the Huntingtin gene.
- E271. The method of any of E249-E270, 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.
- E272. A dsRNA of any one of E110-E219, the pharmaceutical composition of E220, the composition of E221, the vector of E222, or the cell of E223, for use in preventing or delaying progression of a nucleotide repeat expansion disorder in a subject
- E272. The dsRNA of E272, wherein the nucleotide repeat expansion disorder is a trinucleotide repeat expansion disorder.
- E274. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell for use of E272 or E273, 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.
- E275. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E273 or E274, wherein the trinucleotide repeat expansion disorder is Huntington's disease.
- E276. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E273 or E274, wherein the trinucleotide repeat expansion disorder is Friedrich's ataxia.
- E277. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of E273 or E274, wherein the trinucleotide repeat expansion disorder is myotonic dystrophy type 1
- E278. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of 273 or 274, wherein the trinucleotide repeat expansion disorder is a Spinocerebellar ataxia (SCA).
- E279. The dsRNA of E278, wherein the SCA is Spinocerebellar ataxia type 1 (SCA1).
- E280. The dsRNA of E278, wherein the SCA is Spinocerebellar ataxia type 10 (SCA10).
- E281. The dsRNA of E278, wherein the SCA is Spinocerebellar ataxia type 12 (SCA12).
- E282. The dsRNA of E278, wherein the SCA is Spinocerebellar ataxia type 17 (SCA17).
- E283. The dsRNA of E278, wherein the SCA is Spinocerebellar ataxia type 2 (SCA2).
- E284. The dsRNA of E278, wherein the SCA is Spinocerebellar ataxia type 3 (SCA3)/Machado-Joseph Disease.
- E285. The dsRNA of E278, wherein the SCA is Spinocerebellar ataxia type 45 (SCA45).
- E286. The dsRNA of E278, wherein the SCA is Spinocerebellar ataxia type 6 (SCA6).
- E287. The dsRNA of E278, wherein the SCA is Spinocerebellar ataxia type 7 (SCAT).
- E288. The dsRNA of E278, wherein the SCA is Spinocerebellar ataxia type 8 (SCAB).
- E289. The dsRNA, the pharmaceutical composition, the composition, the vector, or the cell of any one of E272-E288, 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.

METHODS FOR THE TREATMENT OF NUCLEOTIDE REPEAT EXPANSION DISORDERS ASSOCIATED WITH MSH3 ACTIVITY

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