Methods for treating trinucleotide repeat expansion disorders associated with MSH3 activity

Abstract

The present disclosure features useful compositions and methods for treating trinucleotide repeat expansion disorders, for example, in subjects in need of such treatment. In some embodiments, the compositions and methods described herein are useful in treating disorders associated with MSH3 activity. Some embodiments of the present disclosure relate to single-stranded oligonucleotides 10 to 30 linked nucleosides in length, comprising a region of at least 10 contiguous nucleobases having at least 80% complementarity to the MSH3 gene.

Description

[Technical Field]

[0001] Incorporation by reference of sequence listing The contents of the text file named "4398.008PC03_SL_ST25.txt", created on November 25, 2019, and having a size of 545,271 bytes, are hereby incorporated by reference in their entirety into this specification. [Background technology]

[0002] background Trinucleotide repeat expansion disorder is a genetic disorder caused by trinucleotide repeat expansion.Trinucleotide repeat expansion is a type of genetic mutation in which the nucleotide repeat in a certain gene or intron exceeds the normal stable threshold for that gene.Trinucleotide repeat can cause defective or toxic gene products, impair RNA transcription, and / or cause toxic effects by forming toxic mRNA transcripts.

[0003] 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 that result in a series of glutamine residues known as polyglutamine tracts; type 2 disorders are caused by heterogeneous expansions that are generally small in size; and type 3 disorders, such as fragile X syndrome, are characterized by large repeat expansions that are generally located outside the protein-coding region of genes. Trinucleotide repeat expansion disorders are characterized by a wide variety of symptoms, including progressive degeneration of neurons, which is common in type 1 disorders.

[0004] A subject with a trinucleotide repeat expansion disorder or a subject considered to be at risk of developing a trinucleotide repeat expansion disorder has a constitutive nucleotide expansion in the gene associated with the disease (i.e., the trinucleotide repeat expansion is present in the gene during embryogenesis). The constitutive trinucleotide repeat expansion can undergo expansion after embryogenesis (i.e., somatic trinucleotide repeat expansion). Both constitutive trinucleotide repeat expansion and somatic trinucleotide repeat expansion can be associated with the presence of the disease, the age at which the disease begins, and / or the rate of disease progression. Summary of the Invention [Means for solving the problem]

[0005] Summary of the Invention The present disclosure features useful compositions and methods for treating trinucleotide repeat expansion disorders, for example, in a subject in need of such treatment. In some embodiments, the compositions and methods described herein are useful in treating disorders associated with MSH3 activity.

[0006] Oligonucleotides Some embodiments of the present disclosure relate to single-stranded oligonucleotides 10 to 30 linked nucleosides in length, comprising a region of at least 10 contiguous nucleobases having at least 80% complementarity to the MSH3 gene. In some embodiments, the disclosure relates to single-stranded oligonucleotides 10 to 30 linked nucleosides in length, wherein the oligonucleotide comprises: (a) a DNA core sequence comprising linked deoxyribonucleosides; (b) a 5' flanking sequence comprising linked nucleosides; and (c) a 3' flanking sequence comprising linked nucleosides; the DNA core comprises a region of at least 10 contiguous nucleobases having at least 80% complementarity to the MSH3 gene and is positioned between the 5' flanking sequence and the 3' flanking sequence; the 5' flanking sequence and the 3' flanking sequence each comprise at least two linked nucleosides; and at least one nucleoside in each flanking sequence comprises an alternative nucleoside.

[0007] In some aspects, the present disclosure relates to a single-stranded oligonucleotide of 10 to 30 linked nucleosides in length for inhibiting expression of the human MSH3 gene in a cell, the oligonucleotide comprising a region of at least 10 contiguous nucleobases having at least 80% complementarity to the MSH3 gene. In some aspects, the disclosure relates to a single-stranded oligonucleotide 10 to 30 linked nucleosides in length for inhibiting expression of the human MSH3 gene in a cell, the oligonucleotide comprising: (a) a DNA core comprising linked deoxyribonucleosides; (b) a 5' flanking sequence comprising linked nucleosides; and (c) a 3' flanking sequence comprising linked nucleosides; the DNA core comprises a region of at least 10 contiguous nucleobases having at least 80% complementarity to the MSH3 gene and is positioned between the 5' flanking sequence and the 3' flanking sequence; the 5' flanking sequence and the 3' flanking sequence each comprise at least two linked nucleosides; and at least one nucleoside in each flanking sequence comprises an alternative nucleoside.

[0008] In some embodiments, the region of at least 10 nucleobases has at least 90% complementarity to the MSH3 gene. In some embodiments, the region of at least 10 nucleobases has at least 95% complementarity to the MSH3 gene.

[0009] In some embodiments, the region of at least 10 nucleobases is selected from the group consisting of 155 to 199, 355 to 385, 398 to 496, 559 to 589, 676 to 724, 762 to 810, 876 to 903, 912 to 974, 984 to 1047, 1054 to 1098, 1114 to 1179, 1200 to 1227, 1294 to 1337, 1392 to 1417, 1467 to 1493, 1517 to 1630, 1665 to 1747, 1768 to 1866, 2029 to 2063, 2087 to 2199, 2100 to 2129, 2200 to 2229, 2300 to 2329, 2400 to 2469, 2500 to 2520, 2600 to 2620, 2700 to 2720, 2800 to 2820, 2900 to 2920, 3000 to 3000, 3100 to 3120, 3200 to 3220, 3300 to 3320, 3400 to 3420, 3500 to 3520, 3600 to 3620, 3700 to 3720, 3800 to 3820, 3900 to 3920, 4000 to 4000, 4100 to 4100, 4200 to 4200 In some embodiments, the region of at least 10 nucleobases is complementary to a reference mRNA at one or more of positions 2262-2293, 2304-2330, 2371-2410, 2432-2458, 2494-2521, 2539-2647, 2679-2713, 2727-2753, 2767-2920, 2933-3000, 3046-3073, 31323245, 3266-3306, 3397-3484, 3528-3575, 3591-3617, 3753-3792, 3901-3936, 4074-4101, or 4281-4319. For the MSH3 gene corresponding to the sequence of NM_002439.4, 155 to 199, 359 to 385, 398 to 496, 559 to 589, 676 to 724, 762 to 810, 876 to 974, 984 to 1098, 1114 to 1179, 1200 to 1227, 1294 to 1337, 1392 to 1417, 1467 to 1493, 1517 to 1630, 1665 to 1747, 1834 to 1866, 2029 to 2056, 2093 to 2199, 2262 to 2293 of the MSH3 gene, It is complementary at one or more positions among 2304 to 2329, 2371 to 2410, 2433 to 2458, 2494 to 2521, 2539 to 2647, 2679 to 2713, 2727 to 2753, 2767 to 2920, 2933 to 3000, 3046 to 3072, 3132 to 3245, 3266 to 3303, 3397 to 3484, 3528 to 3575, 3591 to 3617, 3753 to 3792, 3901 to 3936, 4076 to 4101, or 4281 to 4319.In some embodiments, the region of at least 10 nucleobases is selected from the group consisting of 155 to 196, 359 to 385, 413 to 462, 559 to 589, 676 to 724, 762 to 810, 876 to 974, 984 to 1096, 1114 to 1179, 1200 to 1227, 1294 to 1337, 1467 to 1493, 1517 to 1630, 1665 to 1747, 1834 to 1866, 2029 to 2056, 2093 to 2199, 2100 to 2150, 2200 to 2250, 2300 to 2350, 2400 to 2450, 2500 to 2550, 2600 to 2650, 2700 to 2750, 2800 to 2850, 2900 to 2950, ​​3000 to 3000, 3100 to 3150, 3200 to 3250, 3300 to 3350, 3400 to 3450, 3500 to 3550, 3600 to 3650, 3700 to 3700, 3800 to 3800, 3900 to 3900, 4000 to 4000, 4100 to 4100, 4200 to 4200, 4300 to 4300, 4400 to 4400, 4500 to 45 It is complementary at one or more positions among 2265 to 2293, 2378 to 2410, 2433 to 2458, 2494 to 2521, 2539 to 2647, 2679 to 2712, 2727 to 2753, 2767 to 2919, 2934 to 3000, 3046 to 3071, 3144 to 3183, 3220 to 3245, 3397 to 3484, 3534 to 3575, 3591 to 3616, 3901 to 3931, or 4281 to 4306. In some embodiments, the region of at least 10 nucleobases is complementary to the MSH3 gene corresponding to the sequence of reference mRNA NM_002439.4 at one or more of positions 435-462, 559-584, 763-808, 876-902, 931-958, 1001-1083, 1114-1179, 1294-1337, 1544-1578, 1835-1863, 2031-2056, 2144-2169, 2543-2577, 2590-2615, 2621-2647, 2685-2711, 2769-2795, or 2816-2868 of the MSH3 gene. In some embodiments, the region of at least 10 nucleobases is complementary to the MSH3 gene corresponding to the sequence of reference mRNA NM_002439.4 at one or more of positions 876-902, 930-958, 1056-1081, 1114-1139, 1154-1179, 1310-1337, 1546-1571, 1836-1862, 2141-2199, 2267-2292, 2540-2580, 2620-2647, 2686-2711, 2769-2868, 2939-2976, 3144-3169, or 3399-3424 of the MSH3 gene.In some embodiments, the region of at least 10 nucleobases is complementary to the MSH3 gene corresponding to the sequence of reference mRNA NM_002439.4 at one or more of positions 984-1021, 1467-1493, 1722-1747, 1767-1802, 1833-1861, 2385-2410, 2554-2581, 2816-2845, 2861-2920, or 3151-3183 of the MSH3 gene.

[0010] In some embodiments, the oligonucleotide comprises any one of the nucleic acid base sequences of SEQ ID NOs: 6 to 2545. In some embodiments, the oligonucleotide comprises any one of the nucleic acid base sequences of SEQ ID NOs: 20, 22 to 29, 31 to 32, 77 to 78, 81 to 82, 115, 117, 130, 132 to 134, 144 to 145, 147, 167 to 168, 210, 212 to 215, 290 to 293, 295 to 296, 299 to 305, 309, 351 to 359, 361 to 362, 365 to 366, 368, 407 to 409, 432, 437 to 442, 444, 459 to 460, 479, 482 to 493, 497 to 498, 500 to 501, 503 to 512, 543 to 550, 552 ~560, 562, 582~585, 588~591, 603~604, 611, 613~616, 659, 661, 699~700, 702, 705~707, 724~725, 770~771, 812~816, 838~842, 845~852, 856, 883~885, 889, 893~897, 936, 940~941, 945, 948, 950, 955, 959~961, 965~968, 972~973, 999, 1007, 1016~1017, 1019, 1021~1022, 1036, 1040~1045, 1047, 1170, 1172-1173, 1211, 1216, 1222, 1235, 1240-1242, 1244-1249, 1251-1252, 1254-1259, 1268, 1316, 1318-1322, 1328-1329, 1373-1375, 1379-1383, 1386-1387, 1407-1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1541, 1565-1566, 1579, 1581-1582 89, 1591, 1600-1607, 1610, 1625, 1627-1629, 1631-1639, 1643, 1650-1660, 1663-1665, 1668-1675, 1713-1714, 1716-1722, 1724, 1727-1731, 1741, 1745-1747, 1751-1755, 1799-1801, 1859-1866, 1868-1869, 1894-1896, 1905-1908, 1954, 1964-1966, 1969, 2066-2070, 2075-2079, 2108,It contains any one of the nucleic acid base sequences 2138, 2143-2147, 2157-2160, 2193-2194, 2299-2300, 2312-2313, 2385, 2388, 2390-2395, 2416-2418, 2460, 2462, and 2463. In some embodiments, the oligonucleotide is selected from the group consisting of SEQ ID NOs: 20, 22, 25-29, 31-32, 81-82, 115, 130, 132-134, 144, 145, 147, 168, 210, 212-215, 290-293, 295-296, 299-305, 309, 351-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 459-460, 479, 482-493, 497-498, 500-501, 503-506, 508-509, 510-511, 512-513, 514-515, 516-517, 518-519, 520-521, 522-523, 524-525, 526-527, 528-529, 530-531, 532-533, 534-535, 536-537, 538-539, 540-541, 542-543, 544-545, 546-547, 548-549, 550-551, 552-553, 554-555, 556-557, 558-559, 559-560, 561-562, 563-564, 565-5 512, 543-550, 552-560, 562, 582-585, 589-591, 603-604, 611, 613-616, 659, 661, 699-700, 702, 705-707, 724, 770-771, 812-816, 838-842, 845-852, 856, 883-885, 889, 893-897, 936, 940-941, 945, 948, 950, 955, 959-961, 965-968, 972-973, 1041-1045, 1047, 1170, 1172, 1216, 1222, 1235, 1241-1242, 1244-1249, 1251-1252, 1254-1259, 1268, 1316, 1318-1319, 1321-1322, 1328, 1373, 1379-1383, 1386-1387, 1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1541, 1565-1566, 1579, 1581-1582, 1584- 1589, 1591, 1601-1607, 1610, 1625, 1627-1629, 1631-1638, 1650-1655, 1659, 1665, 1668-1675, 1713-1714, 1716-1722, 1727-1731, 1745, 1747, 1751-1755, 1799-1800, 1859, 1861-1862, 1865-1866, 1868-1869, 1895-1896, 1905-1908, 1954, 1964-1966, 2066-2070,It contains any one of the nucleic acid base sequences 2075 to 2079, 2108, 2138, 2144 to 2146, 2158 to 2160, 2193 to 2194, 2299, 2300, 2313, 2385, 2388, 2390 to 2392, 2394 to 2395, 2418, 2460, and 2462 to 2463. In some embodiments, the oligonucleotide is selected from the group consisting of SEQ ID NOs: 20, 25-29, 32, 81-82, 130, 133-134, 144-145, 147, 210, 212-213, 215, 290-293, 295-296, 299-304, 309, 351, 352-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 460, 479, 482-486, 488-492, 497-498, 500-501, 503-504, 505-506, 507-508, 509-510, 511-512, 513-514, 514-515, 515-516, 516-517, 517-518, 518-519, 520-521, 521-522, 522-523, 523-524, 524-525, 525-526, 526-527, 527-528, 528-529, 530-531, 532-533, 534-535, 535-536, 537-538, 538-539, 540-541, 542-543, 544-545, 546-547, 548-549, 506, 508-512, 544-550, 553-558, 560, 582-585, 589, 603-604, 611, 613-616, 659, 661, 699-700, 702, 705-707, 770-771, 812-816, 838-842, 845-851, 856, 883, 885, 889, 893, 895-897, 936, 940, 945, 961, 965-968, 972-973, 1041-1045, 1047, 1170, 1172, 1 216, 1222, 1235, 1244, 1246-1249, 1251-1252, 1254-1255, 1257-1259, 1268, 1319, 1321-1322, 1380-1381, 1386-1387, 1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1540, 1565-1566, 1579, 1581-1582, 1584-1589, 1591, 1601-1604, 1606-1607, 1610, 1625, 1627-1629, 1631-1638, 1651-1654, 1668, 1670-1674, 1714, 1717-1722, 1727-1731, 1745, 1751-1755, 1799, 1861, 1869, 1908, 1964, 1966, 2066-2069, 2075-2076, 2078-2079, 2108, 2144-2145, 2158-2160, 2193, 2385,2390 or 2460. In some embodiments, the oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 145, 147, 210, 352, 365, 366, 407, 408, 439-442, 444, 492, 500, 504, 511, 512, 544-547, 582, 604, 616, 699, 700, 702, 705-707, 839-842, 848, 1042-1045, 1172, 1255, 1454-1457, 1477-1499, 1538, 1539, 1581, 1582, 1606, 1607, 1610, or 1631-1633. In some embodiments, the oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 407, 408, 441, 442, 444, 545, 582, 616, 705-707, 841, 1043, 1044, 1252, 1255, 1268, 1321, 1451, 1454-1460, 1497-1499, 1538, 1539, 1581, 1582, 1587, 1601, 1602, 1606, 1607, 1610, 1631-1633, 1719, 1721, 1730, 1731, 1861, or 2068. In some embodiments, the oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 479, 482-491, 770, 771, 973, 998-1000, 1007, 1008, 1040-1043, 1387, 1454, 1456, 1459-1461, 1538, 1539, 1606, 1607, 1610, 1643-1665, 1668-1675, or 1862-1869.

[0011] In some embodiments, the nucleic acid base sequence of the oligonucleotide consists of any one of SEQ ID NOs: 6-2545. In some embodiments, the oligonucleotides are selected from the group consisting of SEQ ID NOs: 20, 22-29, 31-32, 77-78, 81-82, 115, 117, 130, 132-134, 144-145, 147, 167-168, 210, 212-215, 290-293, 295-296, 299-305, 309, 351-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 459-460, 479, 482-493, 497-498, 500-501, 503-512, 543-550, 552 ~560, 562, 582~585, 588~591, 603~604, 611, 613~616, 659, 661, 699~700, 702, 705~707, 724~725, 770~771, 812~816, 838~842, 845~852, 856, 883~885, 889, 893~897, 936, 940~941, 945, 948, 950, 955, 959~961, 965~968, 972~973, 999, 1007, 1016~1017, 1019, 1021~1022, 1036, 1040~1045, 1047, 1170, 1172-1173, 1211, 1216, 1222, 1235, 1240-1242, 1244-1249, 1251-1252, 1254-1259, 1268, 1316, 1318-1322, 1328-1329, 1373-1375, 1379-1383, 1386-1387, 1407-1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1541, 1565-1566, 1579, 1581-1582 89, 1591, 1600-1607, 1610, 1625, 1627-1629, 1631-1639, 1643, 1650-1660, 1663-1665, 1668-1675, 1713-1714, 1716-1722, 1724, 1727-1731, 1741, 1745-1747, 1751-1755, 1799-1801, 1859-1866, 1868-1869, 1894-1896, 1905-1908, 1954, 1964-1966, 1969, 2066-2070, 2075-2079, 2108,It consists of any one of the nucleic acid base sequences 2138, 2143-2147, 2157-2160, 2193-2194, 2299-2300, 2312-2313, 2385, 2388, 2390-2395, 2416-2418, 2460, 2462, and 2463. In some embodiments, the oligonucleotide is selected from the group consisting of SEQ ID NOs: 20, 22, 25-29, 31-32, 81-82, 115, 130, 132-134, 144, 145, 147, 168, 210, 212-215, 290-293, 295-296, 299-305, 309, 351-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 459-460, 479, 482-493, 497-498, 500-501, 503-506, 508-509, 510-511, 512-513, 514-515, 516-517, 518-519, 520-521, 522-523, 524-525, 526-527, 528-529, 530-531, 532-533, 534-535, 536-537, 538-539, 540-541, 542-543, 544-545, 546-547, 548-549, 550-551, 552-553, 554-555, 556-557, 558-559, 559-560, 561-562, 563-564, 565-5 512, 543-550, 552-560, 562, 582-585, 589-591, 603-604, 611, 613-616, 659, 661, 699-700, 702, 705-707, 724, 770-771, 812-816, 838-842, 845-852, 856, 883-885, 889, 893-897, 936, 940-941, 945, 948, 950, 955, 959-961, 965-968, 972-973, 1041-1045, 1047, 1170, 1172, 1216, 1222, 1235, 1241-1242, 1244-1249, 1251-1252, 1254-1259, 1268, 1316, 1318-1319, 1321-1322, 1328, 1373, 1379-1383, 1386-1387, 1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1541, 1565-1566, 1579, 1581-1582, 1584- 1589, 1591, 1601-1607, 1610, 1625, 1627-1629, 1631-1638, 1650-1655, 1659, 1665, 1668-1675, 1713-1714, 1716-1722, 1727-1731, 1745, 1747, 1751-1755, 1799-1800, 1859, 1861-1862, 1865-1866, 1868-1869, 1895-1896, 1905-1908, 1954, 1964-1966, 2066-2070,It consists of any one of the nucleic acid base sequences 2075-2079, 2108, 2138, 2144-2146, 2158-2160, 2193-2194, 2299, 2300, 2313, 2385, 2388, 2390-2392, 2394-2395, 2418, 2460, and 2462-2463. In some embodiments, the oligonucleotide is selected from the group consisting of SEQ ID NOs: 20, 25-29, 32, 81-82, 130, 133-134, 144-145, 147, 210, 212-213, 215, 290-293, 295-296, 299-304, 309, 351, 352-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 460, 479, 482-486, 488-492, 497-498, 500-501, 503-504, 505-506, 507-508, 509-510, 511-512, 513-514, 514-515, 515-516, 516-517, 517-518, 518-519, 520-521, 521-522, 522-523, 523-524, 524-525, 525-526, 526-527, 527-528, 528-529, 530-531, 532-533, 534-535, 535-536, 537-538, 538-539, 540-541, 542-543, 544-545, 546-547, 548-549, 506, 508-512, 544-550, 553-558, 560, 582-585, 589, 603-604, 611, 613-616, 659, 661, 699-700, 702, 705-707, 770-771, 812-816, 838-842, 845-851, 856, 883, 885, 889, 893, 895-897, 936, 940, 945, 961, 965-968, 972-973, 1041-1045, 1047, 1170, 1172, 1 216, 1222, 1235, 1244, 1246-1249, 1251-1252, 1254-1255, 1257-1259, 1268, 1319, 1321-1322, 1380-1381, 1386-1387, 1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1540, 1565-1566, 1579, 1581-1582, 1584-1589, 1591, 1601-1604, 1606-1607, 1610, 1625, 1627-1629, 1631-1638, 1651-1654, 1668, 1670-1674, 1714, 1717-1722, 1727-1731, 1745, 1751-1755, 1799, 1861, 1869, 1908, 1964, 1966, 2066-2069, 2075-2076, 2078-2079, 2108, 2144-2145, 2158-2160, 2193, 2385,2390 or 2460. In some embodiments, the oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 145, 147, 210, 352, 365, 366, 407, 408, 439-442, 444, 492, 500, 504, 511, 512, 544-547, 582, 604, 616, 699, 700, 702, 705-707, 839-842, 848, 1042-1045, 1172, 1255, 1454-1457, 1477-1499, 1538, 1539, 1581, 1582, 1606, 1607, 1610, or 1631-1633. In some embodiments, the oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 407, 408, 441, 442, 444, 545, 582, 616, 705-707, 841, 1043, 1044, 1252, 1255, 1268, 1321, 1451, 1454-1460, 1497-1499, 1538, 1539, 1581, 1582, 1587, 1601, 1602, 1606, 1607, 1610, 1631-1633, 1719, 1721, 1730, 1731, 1861, or 2068. In some embodiments, the oligonucleotide consists of any one of the nucleic acid base sequences of SEQ ID NOs: 479, 482 to 491, 770, 771, 973, 998 to 1000, 1007, 1008, 1040 to 1043, 1387, 1454, 1456, 1459 to 1461, 1538, 1539, 1606, 1607, 1610, 1643 to 1665, 1668 to 1675, or 1862 to 1869.

[0012] In some embodiments, the oligonucleotides exhibit at least 50% mRNA inhibition at an oligonucleotide concentration of 20 nM when compared to control cells, as determined using a cellular assay. In some embodiments, the oligonucleotides exhibit at least 60% mRNA inhibition at an oligonucleotide concentration of 20 nM when compared to control cells, as determined using a cellular assay. In some embodiments, the oligonucleotides exhibit at least 70% mRNA inhibition at an oligonucleotide concentration of 20 nM when compared to control cells, as determined using a cellular assay. In some embodiments, the oligonucleotides exhibit at least 85% mRNA inhibition at an oligonucleotide concentration of 20 nM when compared to control cells, as determined using a cellular assay. In some embodiments, the oligonucleotides exhibit at least 50% mRNA inhibition at 2 nM when compared to control cells, as determined using a cellular assay. In some embodiments, the oligonucleotides exhibit at least 60% mRNA inhibition at an oligonucleotide concentration of 2 nM when compared to control cells, as determined using a cellular assay. In some embodiments, the oligonucleotides exhibit at least 70% mRNA inhibition at a 2 nM oligonucleotide concentration compared to control cells as determined using a cellular assay. In some embodiments, the oligonucleotides exhibit at least 85% mRNA inhibition at a 2 nM oligonucleotide concentration compared to control cells as determined using a cellular assay.

[0013] The cellular assay may include transfecting mammalian cells, such as HEK293, NIH3T3, or HeLa, with the oligonucleotide using Lipofectamine 2000 (Invitrogen) and measuring mRNA levels compared to mammalian cells transfected with a mock oligonucleotide.

[0014] In some embodiments, the oligonucleotide comprises at least one alternative internucleoside linkage. In some embodiments, at least one alternative internucleoside linkage is a phosphorothioate internucleoside linkage. In some embodiments, at least one alternative internucleoside linkage is a 2'-alkoxy internucleoside linkage. In some embodiments, at least one alternative internucleoside linkage is an alkylphosphate internucleoside linkage.

[0015] In some embodiments, the oligonucleotide comprises at least one alternative nucleobase, hi some embodiments, the alternative nucleobase is 5'-methylcytosine, pseudouridine, or 5-methoxyuridine.

[0016] In some embodiments, the oligonucleotide comprises at least one alternative sugar moiety, hi some embodiments, the alternative sugar moiety is 2'-OMe or a bicyclic nucleic acid.

[0017] In some embodiments, the oligonucleotide further comprises a ligand conjugated to the 5' or 3' end of said oligonucleotide via a monovalent or branched divalent or trivalent linker.

[0018] In some embodiments, the oligonucleotide comprises a region complementary to at least 17 contiguous nucleotides of the MSH3 gene. In some embodiments, the oligonucleotide comprises a region complementary to at least 19 contiguous nucleotides of the MSH3 gene. In some embodiments, the oligonucleotide comprises a region complementary to 19-23 contiguous nucleotides of the MSH3 gene. In some embodiments, the oligonucleotide comprises a region complementary to 19 contiguous nucleotides of the MSH3 gene. In some embodiments, the oligonucleotide comprises a region complementary to 20 contiguous nucleotides of the MSH3 gene. In some embodiments, the oligonucleotide is about 15-25 nucleosides in length. In some embodiments, the oligonucleotide is 20 nucleosides in length.

[0019] Pharmaceutical compositions and methods of treatment using same In some aspects, the present application relates to pharmaceutical compositions comprising one or more of the oligonucleotides described herein and a pharmaceutically acceptable carrier or excipient.

[0020] In some aspects, the present application relates to a composition comprising one or more of the oligonucleotides described herein and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome.

[0021] In some aspects, the present application relates to a method of inhibiting transcription of MSH3 in a cell, comprising contacting the cell with one or more of the oligonucleotides described herein, a pharmaceutical composition of one or more of the oligonucleotides described herein, or a composition of one or more oligonucleotides and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, or liposomes described herein, for a time sufficient to effect degradation of mRNA transcripts of the MSH3 gene, thereby inhibiting expression of the MSH3 gene in the cell.

[0022] In some aspects, the present application relates to a method of treating, preventing, or delaying the progression of a trinucleotide repeat expansion disorder in a subject in need thereof, comprising contacting a cell with one or more of the oligonucleotides described herein, a pharmaceutical composition of one or more of the oligonucleotides described herein, or a composition of one or more oligonucleotides and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, or liposomes described herein, for a time sufficient to effect degradation of mRNA transcripts of the MSH3 gene, thereby inhibiting expression of the MSH3 gene in the cell.

[0023] In some aspects, the present application relates 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, comprising contacting the cell with one or more of the oligonucleotides described herein, a pharmaceutical composition of one or more of the oligonucleotides described herein, or a composition of one or more oligonucleotides and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, or liposomes described herein, for a time sufficient to effect degradation of mRNA transcripts of the MSH3 gene, thereby inhibiting expression of the MSH3 gene in the cell.

[0024] In some aspects, the present application relates to a method for inhibiting expression of the MSH3 gene in a cell, comprising contacting the cell with one or more of the oligonucleotides described herein, a pharmaceutical composition of one or more of the oligonucleotides described herein, or a composition of one or more oligonucleotides and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, or liposomes described herein, for a time sufficient to result in degradation of mRNA transcripts of the MSH3 gene, thereby inhibiting expression of the MSH3 gene in the cell, and maintaining the cell for a time sufficient to result in degradation of mRNA transcripts of the MSH3 gene, thereby inhibiting expression of the MSH3 gene in the cell.

[0025] In some aspects, the present application relates to a method of reducing trinucleotide repeat expansions in a cell, the method comprising contacting the cell with one or more of the oligonucleotides described herein, a pharmaceutical composition of one or more of the oligonucleotides described herein, or a composition of one or more oligonucleotides and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, or liposomes described herein, for a time sufficient to result in degradation of mRNA transcripts of the MSH3 gene, thereby inhibiting expression of the MSH3 gene in the cell.

[0026] 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.

[0027] In some embodiments, the subject is identified as having a trinucleotide repeat expansion disorder. In some embodiments, the trinucleotide repeat expansion disorder is a polyglutamine disease. In some embodiments, the polyglutamine disease is selected from the group consisting of dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-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 embodiments, the trinucleotide repeat expansion disorder is Huntington's disease.

[0028] In some embodiments, the trinucleotide repeat expansion disorder is a non-polyglutamine disease.In some embodiments, 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 embodiments, the trinucleotide repeat expansion disorder is Friedreich's ataxia.In some embodiments, the trinucleotide repeat expansion disorder is myotonic dystrophy type 1.

[0029] In some aspects, the present application relates to one or more of the oligonucleotides described herein, pharmaceutical compositions of one or more of the oligonucleotides described herein, or compositions of one or more of the oligonucleotides described herein and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, or liposomes, for use in the prevention or treatment of trinucleotide repeat expansion disorders. In some aspects, one or more of the oligonucleotides described herein, pharmaceutical compositions of one or more of the oligonucleotides described herein, or compositions of one or more of the oligonucleotides described herein and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, or liposomes, are administered intrathecally.

[0030] In some embodiments, one or more of the oligonucleotides described herein, a pharmaceutical composition of one or more of the oligonucleotides described herein, or a composition of one or more oligonucleotides and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, or liposomes described herein is administered intracerebroventricularly.

[0031] In some embodiments, one or more of the oligonucleotides described herein, a pharmaceutical composition of one or more of the oligonucleotides described herein, or a composition of one or more oligonucleotides and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, or liposomes described herein is administered intramuscularly.

[0032] In some aspects, the present application relates to a method of treating, preventing, or delaying the progression of a disorder in a subject in need thereof, wherein the subject is afflicted with a trinucleotide repeat expansion disorder, the method comprising administering to the subject one or more of the oligonucleotides described herein, a pharmaceutical composition of one or more of the oligonucleotides described herein, or a composition of one or more oligonucleotides and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, or liposomes described herein. In some aspects, the method of treating, preventing, or delaying the progression of a disorder in a subject further comprises administering an additional therapeutic agent. In some aspects, the additional therapeutic agent is another oligonucleotide that hybridizes to mRNA encoding the huntingtin gene.

[0033] In some embodiments, the methods of treating, preventing, or delaying the progression of a disorder in a subject delay the progression of the trinucleotide repeat expansion disorder by at least 120 days, e.g., 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 to expected progression.

[0034] In some aspects, the present application relates to one or more of the oligonucleotides described herein, pharmaceutical compositions of one or more of the oligonucleotides described herein, or compositions of one or more oligonucleotides described herein and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, or liposomes, for use in preventing or delaying the progression of a trinucleotide repeat expansion disorder in a subject.

[0035] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise indicated or implied from context, the following terms and phrases include the meanings provided below. Since the scope of the technology is limited only by the claims, definitions are provided to help describe particular embodiments and are not intended to limit the claimed technology. 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 herein shall control.

[0036] In this application, unless otherwise clear from the context, (i) the term "a" may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and / or"; and (iii) the terms "including" and "comprising" may be understood to encompass the listed elements or steps, whether presented alone or together with one or more additional elements or steps.

[0037] As used herein, the terms "about" and "approximately" refer to values ​​that are within 10% above or below the stated value. For example, the term "about 5 nM" indicates a range of 4.5 to 5.5 nM.

[0038] The term "at least" before a number or a series of numbers is understood to include the number adjacent to the term "at least", as well as all subsequent numbers and integers that can be logically included as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20 or 21 nucleotides have the specified property. When "at least" is present before a series of numbers or ranges, it is understood that "at least" can modify each number in the series or range. "At least" is also not limited to integers (for example, "at least" 5% includes 5.0%, 5.1%, 5.18%, regardless of the number of significant digits).

[0039] As used herein, " less than " or " less than " is understood logically from context as the value adjacent to this phrase and the theoretical lower limit or integer to zero.For example, the oligonucleotide that has " 3 or less mismatches with target sequence " has 3, 2, 1 or 0 mismatches with target sequence.When " less than " is present before a series of numbers or range, it is understood that " less than " can modify each number in the series of numbers or range.

[0040] As used herein, the term "administration" refers to the administration of a composition (e.g., a compound described herein or a preparation containing a compound) to a subject or system. Administration to an animal subject (e.g., to a human) can be by any suitable route, such as those described herein.

[0041] As used herein, "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 specifies the dosage and periodicity of administration of each agent so that the effects of the separate agents on the subject overlap. In some embodiments, delivery of two or more agents is simultaneous or concurrent, and the agents may be co-formulated. In some embodiments, two or more agents are not co-formulated but are administered in a sequential manner as part of a prescribed regimen. In some embodiments, the administration of two or more combined agents or treatments is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed with one agent or treatment delivered alone or in the absence of other agents or treatments. The effect of the two treatments can be partially additive, fully additive, or greater than additive (e.g., synergistic). The sequential or substantially simultaneous administration of each therapeutic agent can be achieved by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same or different routes. For example, one therapeutic agent in the combination can be administered by intravenous injection, while another therapeutic agent in the combination can be administered orally.

[0042] As used herein, the term "MSH3," unless otherwise specified, refers to MutS homolog 3, a DNA mismatch repair protein, having an amino acid sequence derived from any vertebrate or mammalian source, including, but not limited to, human, bovine, chicken, rodent, mouse, rat, pig, sheep, primate, monkey, and guinea pig. The term also refers to fragments and variants of native MSH3 that maintain at least one in vivo or in vitro activity of native MSH3. The term encompasses the unprocessed full-length precursor form of MSH3 as well as the mature form 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 SEQ ID NO:1. The term "MSH3" also refers to naturally occurring variants of the wild-type MSH3 protein, e.g., 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% or higher identity) to the amino acid sequence of wild-type human MSH3 set forth in NCBI Reference No. NP_002430.3 or 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 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 SEQ ID NO: 4. The nucleic acid sequence of an exemplary Macaca fascicularis (cynomolgus monkey) MSH3 gene is shown in NCBI reference number XM_005557283.2 or SEQ ID NO:5.

[0043] The term "MSH3" as used herein also refers to the naturally occurring DNA sequence variant of MSH3 gene, for example, the specific polypeptide that is expressed in cells due to single nucleotide polymorphism in MSH3 gene.A large number of SNPs in MSH3 gene have been identified, and can be found, for example, in NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp).Non-limiting examples of SNPs in MSH3 gene include: dbSNP accession numbers: rs1650697, rs70991108, rs10168, rs26279, rs26282, rs26779, rs26784, rs32989, rs33003, rs33008, rs33013, rs40139, rs181747, rs184967, rs245346, rs245397, rs249633, rs380691, rs408626, rs442767, rs836802, rs836808, rs863221, rs1105525, rs1428 rs6151627, rs6151640, rs6151662, rs6151670, rs6151735, rs6151838, rs7709909, rs7712332, rs10079641, rs12513549 and rs12522132.

[0044] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the MSH3 gene, including mRNA that is the product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence is at least sufficiently long to serve as a substrate for oligonucleotide-directed (e.g., antisense oligonucleotide (ASO)-directed) cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during transcription of the MSH3 gene. The target sequence can be, for example, about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or 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, or about 15-30 nucleotides. , 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-listed ranges and lengths are also contemplated.

[0045] "G", "C", "A", "T" and "U" generally refer to naturally occurring nucleotides, each containing guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it is understood that the term "nucleotide" can also refer to alternative nucleotides or substituted replacement moieties, as described in more detail below. Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be replaced by other moieties without substantially changing the base pairing properties of an oligonucleotide containing a nucleotide having such a replacement moiety. For example, without limitation, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine or uracil. Thus, a nucleotide containing uracil, guanine or adenine can be replaced in the nucleotide sequence of an oligonucleotide by, for example, a nucleotide containing inosine. In another example, adenine and cytosine at any position in an oligonucleotide can be replaced with guanine and uracil, respectively, to form a GU wobble base pair with a target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured herein.

[0046] The terms "nucleobase" and "base" include purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides that form hydrogen bonds during nucleic acid hybridization. The term nucleobase also encompasses alternative nucleobases that may 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, and alternative nucleobases. Such variants are described, for example, 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.

[0047] The term "nucleoside" refers to a monomeric unit of an oligonucleotide or polynucleotide having a nucleobase and a sugar moiety. Nucleosides can include naturally occurring nucleosides and 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.

[0048] The term "alternative nucleoside" refers to a nucleoside having an alternative sugar or an alternative nucleobase, such as those described herein.

[0049] In some aspects, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, e.g., a substituted purine or substituted pyrimidine, e.g., an "alternative nucleobase" selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 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.

[0050] Nucleobase moieties may be designated by a letter code for each corresponding nucleobase, e.g., A, T, G, C, or U, where each letter may include alternative nucleobases of equivalent function. In some embodiments, e.g., for gapmers, 5-methylcytosine LNA nucleosides may be used.

[0051] "Sugar" or "sugar moiety" includes naturally occurring sugars having a furanose ring. Sugar also includes "alternate sugars," defined as structures that can replace the furanose ring of a nucleoside. In some embodiments, the alternative sugar is a non-furanose (or 4'-substituted furanose) ring or ring system or open system. Such structures include simple variations compared to the natural furanose ring, e.g., a six-membered ring, or such structures can be more complex, as in the case of the acyclic systems used in peptide nucleic acids. Alternative sugars can include sugar surrogates in which the furanose ring is replaced with another ring system, e.g., a morpholino or hexitol ring system. Sugar moieties useful in preparing oligonucleotides having a motif include, but are not limited to, β-D-ribose, β-D-2'-deoxyribose, substituted sugars (e.g., 2', 5', and bis-substituted sugars), 4'-S-sugars (e.g., 4'-S-ribose, 4'-S-2'-deoxyribose, and 4'-S-2'-substituted ribose), bicyclic alternative sugars (e.g., bicyclic sugars derived from 2'-O-CH2-4' or 2'-O-(CH2)2-4' bridged ribose), and sugar surrogates (e.g., where the ribose ring is replaced with a morpholino or hexitol ring system). The type of heterocyclic base and internucleoside linkage used at each position is variable and not a factor in determining the motif. In most nucleosides with alternative sugar moieties, the heterocyclic nucleobase is generally maintained to allow hybridization.

[0052] As used herein, "nucleotide" refers to a monomeric unit of an oligonucleotide or polynucleotide, comprising a nucleoside and an internucleoside linkage. The internucleoside linkage can include a phosphate linkage. Similarly, "linked nucleosides" can be linked by a phosphate linkage. Many "alternative internucleoside linkages" are known in the art, including, but not limited to, phosphate, phosphorothioate, and boronophosphate linkages. Alternative nucleosides include bicyclic nucleosides (BNAs) (e.g., locked nucleosides (LNAs) and constrained ethyl (cEt) nucleosides), peptide nucleosides (PNAs), phosphotriesters, phosphorothioates, phosphoramidates, and other variants of the phosphate backbone of native nucleosides, including those described herein.

[0053] "Alternative nucleotide," as used herein, refers to a nucleotide having an internucleoside linkage that may include an alternative nucleoside or alternative sugar and alternative internucleoside linkage.

[0054] The terms "oligonucleotide" and "polynucleotide" as used herein are defined as molecules that comprise two or more covalently linked nucleosides, as generally understood by those skilled in the art.Such covalently linked nucleosides can also be referred to as nucleic acid molecules or oligomers.Oligonucleotides are generally produced in laboratories by solid-phase chemical synthesis and subsequent purification.When referring to the sequence of an oligonucleotide, reference is made to the sequence or order of the nucleic acid base moieties of covalently linked nucleotides or nucleosides, or their modification.Oligonucleotides can be artificial.For example, oligonucleotides can be chemically synthesized and purified or isolated. Oligonucleotides are also intended to include: (i) compounds in which one or more furanose moieties are replaced by furanose derivatives or any cyclic or acyclic structure that can be used as a covalent bond point for the base moiety; (ii) compounds in which one or more phosphodiester linkages are modified, as in the case of phosphoramidate or phosphorothioate linkages, or completely replaced by suitable linking moieties, as in the case of formacetal or riboacetal linkages; and / or (iii) compounds in which one or more linked furanose-phosphodiester linkages are replaced by any cyclic or acyclic structure that can be used as a covalent bond point for the base moiety. Oligonucleotides may contain one or more alternative nucleosides or nucleotides (including, for example, those described herein). It is also understood that oligonucleotides include compositions that lack sugar moieties or nucleobases but are still able to form pairings or hybridize with target sequences.

[0055] "Oligonucleotide" refers to a short polynucleotide (eg, of 100 or fewer linked nucleosides).

[0056] A "chimeric" oligonucleotide or "chimera," as used herein, is an oligonucleotide that contains two or more chemically distinct regions, each composed of at least one monomeric unit, i.e., in the case of oligonucleotides, a nucleotide or nucleoside. Chimeric oligonucleotides also include "gapmers."

[0057] The oligonucleotides can be of any length that allows for specific degradation of the desired target RNA via an RNase H-mediated pathway, and can be about 10-30 nucleosides in length, e.g., about 15-30 nucleosides in length, or about 18-20 nucleosides in length, e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides in length, e.g., 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, 2 The length may range from 0 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleosides. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated.

[0058] As used herein, the term "oligonucleotide comprising a nucleobase sequence" refers to an oligonucleotide comprising a chain of nucleotides or nucleosides described by a sequence referenced using standard nucleotide nomenclature.

[0059] The term "contiguous nucleobase region" refers to a region of an oligonucleotide that is complementary to a target nucleic acid. This term may be used interchangeably herein with the term "contiguous nucleotide sequence" or "contiguous nucleobase sequence." In some embodiments, all nucleotides of an oligonucleotide are present in a contiguous nucleotide or nucleoside region. In some embodiments, an oligonucleotide comprises a contiguous nucleotide region and may further comprise a nucleotide linker region that can be used to attach a functional group to a nucleotide(s) or nucleoside(s), for example, a contiguous nucleotide sequence. The nucleotide linker region may be complementary to a target nucleic acid. In some embodiments, all internucleoside linkages present between nucleotides in the contiguous nucleotide region are phosphorothioate internucleoside linkages. In some embodiments, the contiguous nucleotide region comprises one or more sugar-modified nucleosides.

[0060] The term "gapmer," as used herein, refers to an oligonucleotide comprising a region (gap or DNA core) of an RNase H-recruiting oligonucleotide flanked on the 5' and 3' sides by regions (wing or flanking sequences) that contain one or more affinity-enhancing alternative nucleosides. Various gapmer designs are described herein. Headmers and tailmers are oligonucleotides capable of recruiting RNase H where one of the flanks is missing, i.e., only one end of the oligonucleotide contains the affinity-enhancing alternative nucleoside. For headmers, the 3' flanking sequence is missing (i.e., the 5' flanking sequence contains the affinity-enhancing alternative nucleoside), and for tailmers, the 5' flanking sequence is missing (i.e., the 3' flanking sequence contains the affinity-enhancing alternative nucleoside). A "mixed flanking sequence gapmer" refers to a gapmer in which the flanking sequences contain at least one alternative nucleoside, e.g., at least one DNA nucleoside or at least one 2'-substituted alternative nucleoside, such as 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, 2'-F-ANA nucleoside(s), or a bicyclic nucleoside (e.g., a locked nucleoside or a constrained ethyl (cEt) nucleoside). In some embodiments, a mixed flanking sequence gapmer has one flanking sequence (e.g., 5' or 3') that contains the alternative nucleoside, and the other flanking sequence (3' or 5', respectively) contains the 2'-substituted alternative nucleoside(s).

[0061] A "linker" or "linking group" is a connection between two atoms that connects one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. The conjugate moiety can be attached to the oligonucleotide directly or via a linking moiety (e.g., a linker or tether). The linker functions to covalently connect a third region, e.g., the conjugate moiety, to the oligonucleotide (e.g., the end of region A or C). In some embodiments, the conjugate or oligonucleotide conjugate can include a linker region located between the oligonucleotide and the conjugate moiety. In some embodiments, the linker between the conjugate and the oligonucleotide is biocleavable. Biocleavable linkers containing phosphodiesters are described in more detail in WO2014 / 076195, which is hereby incorporated by reference.

[0062] As used herein, 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 containing the first nucleotide or nucleoside sequence to hybridize and form a duplex structure with an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions, as understood by those skilled in the art. Such conditions may be, for example, stringent conditions, where stringent conditions may include the following: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 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 such as those encountered inside an organism, may be used. Those skilled in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences according to the ultimate application of the hybridized nucleotide or nucleoside.

[0063] "Complementary" sequences, as used herein, may include or be formed entirely of non-Watson-Crick base pairs and / or base pairs formed from non-natural and alternative nucleotides or nucleosides, so long as the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogstein base pairing. As described herein, a complementary sequence between an oligonucleotide and a target sequence includes base pairing of an oligonucleotide or polynucleotide containing a first nucleotide or nucleoside sequence to an oligonucleotide or polynucleotide containing a second nucleotide or nucleoside sequence throughout the entire length of one or both nucleotide or nucleoside sequences. Such sequences may be referred to herein as "fully complementary" with respect to each other. However, when a first sequence is referred to herein as "substantially complementary" with respect to a second sequence, the two sequences may be perfectly complementary or may form one or more, but generally no more than 5, 4, 3, or 2, mismatched base pairs upon hybridization for up to a 30 base pair duplex while retaining the ability to hybridize under conditions most appropriate for their ultimate application, e.g., inhibition of gene expression via the RNase H-mediated pathway. "Substantially complementary" can refer to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding MSH3). For example, a polynucleotide is complementary to at least a portion of an MSH3 mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding MSH3.

[0064] As used herein, the term "region of complementarity" refers to a region on an oligonucleotide that is substantially complementary to all or a portion of a gene, primary transcript, sequence (e.g., a target sequence, e.g., an MSH3 nucleotide sequence), or processed mRNA, such that expression of the endogenous gene (e.g., MSH3) is disrupted. If the region of complementarity is not perfectly complementary to the target sequence, mismatches can occur in the internal or terminal regions of the molecule. Generally, mismatches are most tolerated in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' end of the oligonucleotide.

[0065] As used herein, "an agent that reduces the level and / or activity of MSH3" refers to any polynucleotide agent (e.g., an oligonucleotide, e.g., an ASO) that reduces the level of or inhibits the expression of MSH3 in a cell or subject. The phrase "inhibiting the expression of MSH3," as used herein, includes the inhibition of expression of any MSH3 gene (e.g., the mouse MSH3 gene, rat MSH3 gene, monkey MSH3 gene, or human MSH3 gene, etc.), as well as variants or mutants of the MSH3 gene that encode the MSH3 protein. Thus, the MSH3 gene may be a wild-type MSH3 gene, a mutant MSH3 gene, or a transgenic MSH3 gene in the context of a genetically engineered cell, group of cells, or organism.

[0066] "Reducing the activity of MSH3" means decreasing the level of activity associated with MSH3 (e.g., by reducing the amount of trinucleotide repeats in a gene associated with a trinucleotide repeat expansion disorder associated with MSH3 activity). The level of MSH3 activity can be measured using any method known in the art (e.g., by directly sequencing the gene associated with a trinucleotide repeat expansion disorder to measure the level of trinucleotide repeats).

[0067] "Reducing the level of MSH3" means decreasing the level of MSH3 in a cell or a subject, for example, by administering an oligonucleotide to the cell or subject. The level of MSH3 can be measured using any method known in the art (e.g., by measuring the level of MSH3 mRNA or the level of MSH3 protein in the cell or subject).

[0068] "Modulating the activity of a MutSβ heterodimer containing MSH3" means altering the level of an activity associated with a MutSβ heterodimer or an associated downstream effect. The activity level of a MutSβ heterodimer can be measured using any method known in the art.

[0069] As used herein, the term "inhibitor" refers to any agent that reduces the level and / or activity of a protein (e.g., MSH3). Non-limiting examples of inhibitors include polynucleotides (e.g., oligonucleotides, e.g., ASOs). The term "inhibit" as used herein is used interchangeably with "reduce," "silencing," "downregulate," "suppress," and other similar terms, and includes any level of inhibition.

[0070] The phrase "contacting a cell with an oligonucleotide", for example, an oligonucleotide, as used herein, includes contacting a cell by any possible means. Contacting a cell with an oligonucleotide includes contacting a cell with an oligonucleotide in vitro or contacting a cell with an oligonucleotide in vivo. Contacting can be performed directly or indirectly. Thus, for example, the oligonucleotide can be physically contacted with the cell by the individual who performs the method, or alternatively, the oligonucleotide agent can be placed in a situation that allows it to subsequently contact with the cell or subsequently contacts with the cell.

[0071] Contacting cells in vitro can be carried out, for example, by incubating cells with oligonucleotide.Contacting cells in vivo can be carried out, for example, by injecting oligonucleotide into the tissue where cells are located or nearby, or by injecting oligonucleotide drug into another area, for example, bloodstream or subcutaneous space, so that the drug subsequently reaches the tissue where contacted cells are located.For example, oligonucleotide can contain and / or be coupled to a ligand, such as GalNAc3, that directs oligonucleotide to target site, for example, liver.Contacting in vitro and in vivo methods can also be combined.For example, cell can be contacted with oligonucleotide in vitro, and then be transplanted into subject.

[0072] In one embodiment, contacting a cell with an oligonucleotide includes "introducing" or "delivering an oligonucleotide into a cell" by promoting or causing uptake or absorption into the cell. Absorption or uptake of the ASO can occur through spontaneous diffusion processes or active cellular processes, or by auxiliary agents or devices. Introduction of an oligonucleotide into a cell can be in vitro and / or in vivo. For example, for in vivo introduction, the oligonucleotide 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 known in the art.

[0073] As used herein, " lipid nanoparticles " or " LNP " refers to vesicles that contain a lipid layer that encapsulates pharmaceutically active molecules, such as nucleic acid molecules, for example, oligonucleotides. LNP refers to stable nucleic acid-lipid particles. LNP typically contains cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (for example, PEG-lipid conjugates). LNP is described in, for example, U.S. Patent No. 6,858,225; U.S. Patent No. 6,815,432; U.S. Patent No. 8,158,601; and U.S. Patent No. 8,058,069, the entire contents of which are hereby incorporated by reference herein.

[0074] As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, for example, one bilayer or multiple bilayers. Liposomes include unilamellar and multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the oligonucleotide composition. The lipophilic material may contain the oligonucleotide composition in some cases, but typically separates the aqueous interior from the aqueous exterior, which does not contain the oligonucleotide composition. Liposomes also include "sterically stabilized" liposomes, which, as used herein, refers to liposomes that contain one or more special lipids that, when incorporated into liposomes, result in enhanced circulation life compared to liposomes that lack such special lipids.

[0075] A "micelle" is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all of the hydrophobic portions of the molecule face inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The reverse arrangement exists when the environment is hydrophobic.

[0076] The term "antisense," as used herein, refers to a nucleic acid comprising an oligonucleotide or polynucleotide sufficiently complementary to all or a portion of a gene, primary transcript, or processed mRNA so as to disrupt expression of an endogenous gene (e.g., MSH3). A "complementary" polynucleotide is one capable of base-pairing according to the standard Watson-Crick complementarity rules. Specifically, purines base-pair with pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with 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.

[0077] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of an agent that reduces MSH3 level and / or activity (e.g., in a cell or a subject) described herein refer to an amount sufficient to produce a beneficial or desired result, including a clinical result, when administered to a subject, including a human; therefore, "effective amount" or its synonyms will depend on the context in which it is applied. For example, in the context of treating a trinucleotide repeat expansion disorder, this would be the amount of an agent that reduces MSH3 level and / or activity sufficient to achieve a therapeutic response compared to the response obtained without administration of the agent that reduces MSH3 level and / or activity. The amount of a given agent that reduces MSH3 level and / or activity described herein that corresponds to such an amount will vary depending on various factors, such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, identity of the subject (e.g., age, sex, and / or weight), or host being treated, 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 that produces a beneficial or desired result in a subject 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 in the art by routine methods known in the art. Dosage regimens can be adjusted to provide an optimal therapeutic response.

[0078] "Prophylactically effective amount," as used herein, is intended to include an amount of oligonucleotide that, when administered to a subject with a trinucleotide repeat expansion disorder or a subject predisposed to having a trinucleotide repeat expansion disorder, is sufficient to prevent or ameliorate disease or one or more symptoms of the disease. Alleviating disease includes slowing the course of the disease or reducing the severity of subsequent disease. A "prophylactically effective amount" can vary depending on the oligonucleotide, how the agent is administered, the degree of risk of disease, and medical history, age, weight, family history, genetic makeup, type of previous or concomitant treatment, if any, and other individual characteristics of the patient being treated. A prophylactically effective amount may refer, for example, to an amount of an agent described herein that reduces the level and / or activity of MSH3 (e.g., in a cell or a subject), or may refer to an amount that, when administered to a subject, including a human, is sufficient to delay the onset of one or more of the trinucleotide repeat disorders described herein by at least 120 days, e.g., 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, relative to the predicted onset.

[0079] A "therapeutically effective amount" or a "prophylactically effective amount" also includes an amount of oligonucleotide (administered in either a single dose or multiple doses) that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The oligonucleotides used in the methods herein can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0080] As used herein, the term "region of complementarity" refers to a region on an oligonucleotide that is substantially complementary to all or a portion of a gene, primary transcript, sequence (e.g., a target sequence, e.g., an MSH3 nucleotide sequence), or processed mRNA, such that expression of the endogenous gene (e.g., MSH3) is disrupted. If the region of complementarity is not perfectly complementary to the target sequence, mismatches can occur in the internal or terminal regions of the molecule. Generally, mismatches are most tolerated in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' end of the oligonucleotide.

[0081] An "amount effective to reduce trinucleotide repeat expansion" of a particular gene refers to an amount of an agent described herein that reduces the level and / or activity of MSH3 (e.g., in a cell or a subject), or an amount that, when administered to a subject, including a human, is sufficient to reduce trinucleotide repeat expansion of a particular gene (e.g., a gene associated with a trinucleotide repeat expansion disorder described herein).

[0082] As used herein, the term "subject identified as having a trinucleotide repeat expansion disorder" refers to a subject identified as having a molecular or pathological state, disease or condition of or associated with a trinucleotide repeat expansion disorder, such as identification of a trinucleotide repeat expansion disorder or a symptom thereof, or identification of a subject having or suspected of having a trinucleotide repeat expansion disorder that may benefit from a particular treatment regimen.

[0083] 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 of a subject, which exceed the normal stable threshold for that gene or intron.Nucleotide repeats are common in the human genome and are not usually associated with disease.However, in some cases, the number of repeats may expand beyond the stable threshold, resulting in disease, and the severity of symptoms generally correlates with the number of repeats.Trinucleotide repeat expansion disorders include "polyglutamine" and "non-polyglutamine" disorders.

[0084] "Determining the level of a protein" refers to detecting the protein or the mRNA encoding the protein, either directly or indirectly, by methods known in the art. "Directly determining" means performing a process to obtain a physical entity or value (e.g., performing an assay or test on a sample, or "analyzing a sample," as that term is defined herein). "Indirectly determining" refers to receiving a physical entity or value from another party or source (e.g., a third-party laboratory that directly obtained the physical entity or value). The methods for measuring 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, fluorescence 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, and assays based on protein properties, including but not limited to enzyme activity, or interaction with other protein partners.Methods for measuring mRNA level are known in the art.

[0085] With respect to a reference polynucleotide or polypeptide sequence, "percent (%) sequence identity" is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence after aligning the sequences and, if necessary, introducing gaps (DNA core sequences) to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways within the skill of those 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 necessary to achieve maximum alignment across the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to ("to," "with," or "against") a given nucleic acid or amino acid sequence B (alternatively, this can be expressed as a given nucleic acid or amino acid sequence A having a certain percent sequence identity to ("to," "with," or "against") a given nucleic acid or amino acid sequence B) is calculated as follows: 100 x (ratio 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 Y is the total number of nucleic acids in B. It is understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0086] "Level" refers to the level or activity of a protein, or of mRNA encoding a protein (e.g., MSH3), optionally compared to a reference. The reference can be any useful reference, as defined herein. A "decreased level" or "increased level" of a protein refers to a decrease or increase in protein level compared to a reference (e.g., 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 greater decrease or increase); a decrease or increase of about 10%, about 15%, about 20%, about 300%, about 400%, about 500% or greater compared to a reference. "A decrease or increase of greater than about 20%, about 50%, about 75%, about 100%, or about 200%; a decrease or increase of 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 of greater 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. Protein levels can be expressed in mass / volume (e.g., g / dL, mg / mL, μg / mL, or ng / mL) or as a percentage compared to the total protein or mRNA in the sample.

[0087] The term " pharmaceutical composition " as used herein refers to the composition comprising the compound described herein, which is formulated with pharmaceutically acceptable excipients, and can be manufactured or sold as part of the therapeutic regimen for treating disease in mammals, according to the approval of government regulatory agencies.The pharmaceutical composition can be formulated for oral administration (for example, tablet, capsule, caplet, gel cap or syrup) in unit dosage form; for external administration (for example, as cream, gel, lotion or ointment); for intravenous administration (for example, as a sterile solution without particulate embolism and in a solvent system suitable for intravenous use); for intrathecal injection; for intraventricular injection; for intraparenchymal injection; or any other pharmaceutically acceptable formulation.

[0088] As used herein, "pharmaceutically acceptable excipient" refers to any component other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving an active compound) that has substantially non-toxic and non-inflammatory properties in patients. Excipients may include, for example, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), film-forming agents or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0089] As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt of any compound described herein. For example, any pharmaceutically acceptable salt of any compound described herein includes salts that are within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, or allergic response, and 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 Pharmaceutical Salts: Properties, Selection, and Use, (Eds. PH Stahl and CG Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein, or can be prepared separately by reacting free base groups with a suitable organic acid.

[0090] The compounds described herein may have ionizable groups so that they can be prepared as pharmaceutically acceptable salts.These salts may be acid addition salts involving inorganic or organic acids, or salts may be prepared from inorganic or organic bases in the case of the compounds described herein in acid form.Frequently, 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 preparing suitable salts are well known in the art.Salts may 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, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, and 2-hydroxyethanesulfonate. Salts include 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. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0091] "Reference" refers to any useful reference used to compare the level or activity of a protein or mRNA. A reference can be any sample, standard, standard curve, or level used for comparison purposes. A reference can be a normal reference sample or reference standard or level. A "reference sample" can be, for example, a control, e.g., a predetermined negative control value, e.g., a "normal control" or a previous sample taken from the same subject; a sample from a normal healthy subject, e.g., normal cells or normal tissue; a sample (e.g., cell or tissue) from a subject without a disease; a sample from a subject diagnosed with a disease but not yet treated with a compound described herein; a sample from a subject treated with a compound described herein; or a sample of a known normal concentration of purified protein (e.g., any of those described herein). A "reference standard or level" refers to a value or number derived from a reference sample. A "normal control value" is a predetermined value indicating a 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 ("below X"), or a low threshold ("above X"). A subject having a measured value within the normal control value for a particular biomarker is typically referred to as "within the normal range" for that biomarker. The normal reference standard or level can be a value or number derived from a normal subject without a disease or disorder (e.g., a trinucleotide repeat expansion disorder); or a subject treated with a compound described herein. In some embodiments, 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 general health. A standard curve of purified protein levels within the normal reference range, for example, any of those described herein, can be used as a reference.

[0092] As used herein, the term "subject" refers to any living organism to which a composition can be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals, such as mice, rats, rabbits, non-human primates, and humans). A subject may be a human or animal that may be seeking or in need of treatment, may be requesting treatment, may be undergoing treatment, may be undergoing treatment in the future, or is receiving the care of a skilled professional for a particular disease or condition.

[0093] As used herein, the terms "treat," "treated," and "treating" refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (reduce) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; a decrease in the extent of the condition, disorder, or disease; a stabilized (i.e., not worsening) state of the condition, disorder, or disease; a delay in the onset or slowing of the progression of the condition, disorder, or disease; remission, or alleviation (whether partial or complete), of the condition, disorder, or disease state, whether detectable or undetectable; amelioration of at least one measurable physical parameter not necessarily discernible by the patient; or an enhancement or amelioration of the condition, disorder, or disease. Treating includes eliciting a clinically significant response without excessive levels of side effects. Treating also includes prolonging survival compared to expected survival if not receiving treatment.

[0094] As used herein, the terms "variant" and "derivative" are used interchangeably and refer to naturally occurring, synthetic, and semi-synthetic analogs of compounds, peptides, proteins, or other substances described herein. Variants or derivatives of compounds, peptides, proteins, or other substances described herein may retain or improve the biological activity of the original material.

[0095] The details of one or more embodiments are set forth in the description below. Other features, objects, and advantages will be apparent from the description and the claims. In certain embodiments, for example, the following are provided: (Item 1) A single-stranded oligonucleotide of 10 to 30 linked nucleosides in length, comprising a region of at least 10 contiguous nucleobases having at least 80% complementarity to the MSH3 gene. (Item 2) the oligonucleotide is (a) a DNA core sequence containing linked deoxyribonucleosides; (b) a 5' flanking sequence comprising linked nucleosides; and (c) 3' flanking sequence containing linked nucleosides Including, 2. The oligonucleotide of claim 1, wherein the DNA core comprises a region of at least 10 contiguous nucleobases having at least 80% complementarity to the MSH3 gene and is positioned between the 5' flanking sequence and the 3' flanking sequence; the 5' flanking sequence and the 3' flanking sequence each comprise at least two linked nucleosides; and at least one nucleoside in each flanking sequence comprises an alternative nucleoside. (Item 3) A single-stranded oligonucleotide of 10 to 30 linked nucleosides in length for inhibiting expression of the human MSH3 gene in a cell, the oligonucleotide comprising a region of at least 10 consecutive nucleobases having at least 80% complementarity to the MSH3 gene. (Item 4) the oligonucleotide is (a) A DNA core containing linked deoxyribonucleosides; (b) a 5' flanking sequence comprising linked nucleosides; and (c) 3' flanking sequence containing linked nucleosides Including, 4. The oligonucleotide of item 3, wherein the DNA core comprises a region of at least 10 contiguous nucleobases having at least 80% complementarity to the MSH3 gene and is positioned between the 5' flanking sequence and the 3' flanking sequence; the 5' flanking sequence and the 3' flanking sequence each comprise at least two linked nucleosides; and at least one nucleoside in each flanking sequence comprises an alternative nucleoside. (Item 5) 5. The oligonucleotide of any one of items 1 to 4, wherein said region of at least 10 nucleobases has at least 90% complementarity to the MSH3 gene. (Item 6) 6. The oligonucleotide of any one of items 1 to 5, wherein said region of at least 10 nucleobases has at least 95% complementarity to the MSH3 gene. (Item 7) The region of at least 10 nucleic acid bases is selected from the group consisting of 155 to 199, 355 to 385, 398 to 496, 559 to 589, 676 to 724, 762 to 810, 876 to 903, 912 to 974, 984 to 1047, 1054 to 1098, 1114 to 1179, 1200 to 1227, 1294 to 1337, 1392 to 1417, 1467 to 1493, 1517 to 1630, 1665 to 1747, 1768 to 1866, 2029 to 2063, 2087 to 2199, 2262 to 2293, and 2304 of the MSH3 gene, relative to the sequence of reference mRNA NM_002439.4. 7. The oligonucleotide of any one of items 1 to 6, wherein the oligonucleotide is complementary at one or more positions among positions 1 to 2330, 2371 to 2410, 2432 to 2458, 2494 to 2521, 2539 to 2647, 2679 to 2713, 2727 to 2753, 2767 to 2920, 2933 to 3000, 3046 to 3073, 31323245, 3266 to 3306, 3397 to 3484, 3528 to 3575, 3591 to 3617, 3753 to 3792, 3901 to 3936, 4074 to 4101, or 4281 to 4319. (Item 8) The region of at least 10 nucleic acid bases is selected from the group consisting of 155 to 199, 359 to 385, 398 to 496, 559 to 589, 676 to 724, 762 to 810, 876 to 974, 984 to 1098, 1114 to 1179, 1200 to 1227, 1294 to 1337, 1392 to 1417, 1467 to 1493, 1517 to 1630, 1665 to 1747, 1834 to 1866, 2029 to 2056, 2093 to 2199, 2262 to 2293, 2304 to 2329, 237 7. The oligonucleotide of any one of items 1 to 6, which is complementary at one or more positions among positions 1 to 2410, 2433 to 2458, 2494 to 2521, 2539 to 2647, 2679 to 2713, 2727 to 2753, 2767 to 2920, 2933 to 3000, 3046 to 3072, 3132 to 3245, 3266 to 3303, 3397 to 3484, 3528 to 3575, 3591 to 3617, 3753 to 3792, 3901 to 3936, 4076 to 4101, or 4281 to 4319. (Item 9) The region of at least 10 nucleic acid bases is selected from the group consisting of 155 to 196, 359 to 385, 413 to 462, 559 to 589, 676 to 724, 762 to 810, 876 to 974, 984 to 1096, 1114 to 1179, 1200 to 1227, 1294 to 1337, 1467 to 1493, 1517 to 1630, 1665 to 1747, 1834 to 1866, 2029 to 2056, 2093 to 2199, 2265 to 2293, 237 7. The oligonucleotide of any one of items 1 to 6, wherein the oligonucleotide is complementary at one or more positions among positions 8 to 2410, 2433 to 2458, 2494 to 2521, 2539 to 2647, 2679 to 2712, 2727 to 2753, 2767 to 2919, 2934 to 3000, 3046 to 3071, 3144 to 3183, 3220 to 3245, 3397 to 3484, 3534 to 3575, 3591 to 3616, 3901 to 3931, or 4281 to 4306. (Item 10) 7. The oligonucleotide of any one of items 1 to 6, wherein the region of at least 10 nucleobases is complementary to the MSH3 gene corresponding to the sequence of reference mRNA NM_002439.4 at one or more of positions 435 to 462, 559 to 584, 763 to 808, 876 to 902, 931 to 958, 1001 to 1083, 1114 to 1179, 1294 to 1337, 1544 to 1578, 1835 to 1863, 2031 to 2056, 2144 to 2169, 2543 to 2577, 2590 to 2615, 2621 to 2647, 2685 to 2711, 2769 to 2795, or 2816 to 2868 of the MSH3 gene. (Item 11) 7. The oligonucleotide of any one of items 1 to 6, wherein the region of at least 10 nucleobases is complementary to the MSH3 gene corresponding to the sequence of reference mRNA NM_002439.4 at one or more of positions 876 to 902, 930 to 958, 1056 to 1081, 1114 to 1139, 1154 to 1179, 1310 to 1337, 1546 to 1571, 1836 to 1862, 2141 to 2199, 2267 to 2292, 2540 to 2580, 2620 to 2647, 2686 to 2711, 2769 to 2868, 2939 to 2976, 3144 to 3169, or 3399 to 3424 of the MSH3 gene. (Item 12) 7. The oligonucleotide of any one of items 1 to 6, wherein the region of at least 10 nucleobases is complementary to the MSH3 gene corresponding to the sequence of reference mRNA NM_002439.4 at one or more of positions 984 to 1021, 1467 to 1493, 1722 to 1747, 1767 to 1802, 1833 to 1861, 2385 to 2410, 2554 to 2581, 2816 to 2845, 2861 to 2920, or 3151 to 3183 of the MSH3 gene. (Item 13) 7. The oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleic acid base sequences of SEQ ID NOs: 6 to 2545. (Item 14) SEQ ID NOs: 20, 22-29, 31-32, 77-78, 81-82, 115, 117, 130, 132-134, 144-145, 147, 167-168, 210, 212-215, 290-293, 295-296, 299-305, 309, 351-359, 361-363 62, 365-366, 368, 407-409, 432, 437-442, 444, 459-460, 479, 482-493, 497-498, 500-501, 503-512, 543-550, 552-560, 562, 582-585, 588-591, 603-60 4, 611, 613-616, 659, 661, 699-700, 702, 705-707, 724-725, 770-771, 812-816, 838-842, 845-852, 856, 883-885, 889, 893-897, 936, 940-941, 945, 94 8, 950, 955, 959-961, 965-968, 972-973, 999, 1007, 1016-1017, 1019, 1021-1022, 1036, 1040-1045, 1047, 1170, 1172-1173, 1211, 1216, 1222, 1235, 12 40~1242, 1244~1249, 1251~1252, 1254~1259, 1268, 1316, 1318~1322, 1328~1329, 1373~1375, 1379~1383, 1386~1387, 1407~1408, 1433~1435, 1450~1451 451, 1454~1461, 1476~1477, 1496~1499, 1532, 1538~1541, 1565~1566, 1579, 1581~1589, 1591, 1600~1607, 1610, 1625, 1627~1629, 1631~1639, 1643, 1 650~1660, 1663~1665, 1668~1675, 1713~1714, 1716~1722, 1724, 1727~1731, 1741, 1745~1747, 1751~1755, 1799~1801, 1859~1866, 1868~1869, 1894~ 1896, 1905-1908, 1954, 1964-1966, 1969, 2066-2070, 2075-2079, 2108, 2138, 2143-2147, 2157-2160, 2193-2194, 2299-2300, 2312-2313, 2385, 2388,7. The oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleic acid base sequences 2390 to 2395, 2416 to 2418, 2460, 2462, and 2463. (Item 15) SEQ ID NOs: 20, 22, 25-29, 31-32, 81-82, 115, 130, 132-134, 144, 145, 147, 168, 210, 212-215, 290-293, 295-296, 299-305, 309, 351-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 459-460, 479, 482-493, 497-498, 500-501, 503-506, 508-512, 543-550, 552-560, 562, 582-585, 589-591, 603-604, 6 11, 613-616, 659, 661, 699-700, 702, 705-707, 724, 770-771, 812-816, 838-842, 845-852, 856, 883-885, 889, 893-897, 936, 940-941, 945, 948, 950, 9 55, 959-961, 965-968, 972-973, 1041-1045, 1047, 1170, 1172, 1216, 1222, 1235, 1241-1242, 1244-1249, 1251-1252, 1254-1259, 1268, 1316, 1318-1319 9, 1321-1322, 1328, 1373, 1379-1383, 1386-1387, 1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1541, 1565-1566, 15 79, 1581-1582, 1584-1589, 1591, 1601-1607, 1610, 1625, 1627-1629, 1631-1638, 1650-1655, 1659, 1665, 1668-1675, 1713-1714, 1716-1722, 1727-1728 31, 1745, 1747, 1751-1755, 1799-1800, 1859, 1861-1862, 1865-1866, 1868-1869, 1895-1896, 1905-1908, 1954, 1694-1966, 2066-2070, 2075-2079, 2108, 2138, 2144-2146, 2158-2160, 2193-2194, 2299, 2300, 2313, 2385, 2388, 2390-2392, 2394-2395, 2418, 2460, or 2462-2463,The oligonucleotide according to any one of items 1 to 6. (Item 16) SEQ ID NOs: 20, 25-29, 32, 81-82, 130, 133-134, 144-145, 147, 210, 212-213, 215, 290-293, 295-296, 299-304, 309, 351, 352-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 460, 479, 482-486, 488-492, 497-498, 500-501, 503-506, 508-512, 544-550, 553-558, 5 60, 582-585, 589, 603-604, 611, 613-616, 659, 661, 699-700, 702, 705-707, 770-771, 812-816, 838-842, 845-851, 856, 883, 885, 889, 893, 895-897, 936, 940, 945, 961, 965-968, 972-973, 1041-1045, 1047, 1170, 1172, 1216, 1222, 1235, 1244, 1246-1249, 1251-1252 , 1254-1255, 1257-1259, 1268, 1319, 1321-1322, 1380-1381, 1386-1387, 1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1540, 1565-1566, 1579, 1581-1582, 1584-1589, 1591, 1601-1604, 1606-1607, 1610, 1625, 1627-1629, 1631-163 8, 1651 to 1654, 1668, 1670 to 1674, 1714, 1717 to 1722, 1727 to 1731, 1745, 1751 to 1755, 1799, 1861, 1869, 1908, 1964, 1966, 2066 to 2069, 2075 to 2076, 2078 to 2079, 2108, 2144 to 2145, 2158 to 2160, 2193, 2385, 2390, or 2460. (Item 17) 7. The oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleic acid base sequences of SEQ ID NOs: 145, 147, 210, 352, 365, 366, 407, 408, 439 to 442, 444, 492, 500, 504, 511, 512, 544 to 547, 582, 604, 616, 699, 700, 702, 705 to 707, 839 to 842, 848, 1042 to 1045, 1172, 1255, 1454 to 1457, 1477 to 1499, 1538, 1539, 1581, 1582, 1606, 1607, 1610, and 1631 to 1633. (Item 18) 7. The oligonucleotide of any one of items 1 to 6, comprising any one of the nucleobase sequences of SEQ ID NOs: 407, 408, 441, 442, 444, 545, 582, 616, 705 to 707, 841, 1043, 1044, 1252, 1255, 1268, 1321, 1451, 1454 to 1460, 1497 to 1499, 1538, 1539, 1581, 1582, 1587, 1601, 1602, 1606, 1607, 1610, 1631 to 1633, 1719, 1721, 1730, 1731, 1861, or 2068. (Item 19) 7. The oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleic acid base sequences of SEQ ID NOs: 479, 482 to 491, 770, 771, 973, 998 to 1000, 1007, 1008, 1040 to 1043, 1387, 1454, 1456, 1459 to 1461, 1538, 1539, 1606, 1607, 1610, 1643 to 1665, 1668 to 1675, and 1862 to 1869. (Item 20) 7. The oligonucleotide according to any one of items 1 to 6, wherein the nucleic acid base sequence of the oligonucleotide consists of any one of SEQ ID NOs: 6 to 2545. (Item 21) SEQ ID NOs: 20, 22-29, 31-32, 77-78, 81-82, 115, 117, 130, 132-134, 144-145, 147, 167-168, 210, 212-215, 290-293, 295-296, 299-305, 309, 351-359, 361-363 62, 365-366, 368, 407-409, 432, 437-442, 444, 459-460, 479, 482-493, 497-498, 500-501, 503-512, 543-550, 552-560, 562, 582-585, 588-591, 603-60 4, 611, 613-616, 659, 661, 699-700, 702, 705-707, 724-725, 770-771, 812-816, 838-842, 845-852, 856, 883-885, 889, 893-897, 936, 940-941, 945, 94 8, 950, 955, 959-961, 965-968, 972-973, 999, 1007, 1016-1017, 1019, 1021-1022, 1036, 1040-1045, 1047, 1170, 1172-1173, 1211, 1216, 1222, 1235, 12 40~1242, 1244~1249, 1251~1252, 1254~1259, 1268, 1316, 1318~1322, 1328~1329, 1373~1375, 1379~1383, 1386~1387, 1407~1408, 1433~1435, 1450~1451 451, 1454~1461, 1476~1477, 1496~1499, 1532, 1538~1541, 1565~1566, 1579, 1581~1589, 1591, 1600~1607, 1610, 1625, 1627~1629, 1631~1639, 1643, 1 650~1660, 1663~1665, 1668~1675, 1713~1714, 1716~1722, 1724, 1727~1731, 1741, 1745~1747, 1751~1755, 1799~1801, 1859~1866, 1868~1869, 1894~ 1896, 1905-1908, 1954, 1964-1966, 1969, 2066-2070, 2075-2079, 2108, 2138, 2143-2147, 2157-2160, 2193-2194, 2299-2300, 2312-2313, 2385, 2388,7. The oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleic acid base sequences 2390 to 2395, 2416 to 2418, 2460, 2462, and 2463. (Item 22) SEQ ID NOs: 20, 22, 25-29, 31-32, 81-82, 115, 130, 132-134, 144, 145, 147, 168, 210, 212-215, 290-293, 295-296, 299-305, 309, 351-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 459-460, 479, 482-493, 497-498, 500-501, 503-506, 508-512, 543-550, 552-560, 562, 582-585, 589-591, 603-604, 6 11, 613-616, 659, 661, 699-700, 702, 705-707, 724, 770-771, 812-816, 838-842, 845-852, 856, 883-885, 889, 893-897, 936, 940-941, 945, 948, 950, 9 55, 959-961, 965-968, 972-973, 1041-1045, 1047, 1170, 1172, 1216, 1222, 1235, 1241-1242, 1244-1249, 1251-1252, 1254-1259, 1268, 1316, 1318-1319 9, 1321-1322, 1328, 1373, 1379-1383, 1386-1387, 1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1541, 1565-1566, 15 79, 1581-1582, 1584-1589, 1591, 1601-1607, 1610, 1625, 1627-1629, 1631-1638, 1650-1655, 1659, 1665, 1668-1675, 1713-1714, 1716-1722, 1727-1728 31, 1745, 1747, 1751-1755, 1799-1800, 1859, 1861-1862, 1865-1866, 1868-1869, 1895-1896, 1905-1908, 1954, 1694-1966, 2066-2070, 2075-2079, 2108, 2138, 2144-2146, 2158-2160, 2193-2194, 2299, 2300, 2313, 2385, 2388, 2390-2392, 2394-2395, 2418, 2460, or 2462-2463,The oligonucleotide according to any one of items 1 to 6. (Item 23) SEQ ID NOs: 20, 25-29, 32, 81-82, 130, 133-134, 144-145, 147, 210, 212-213, 215, 290-293, 295-296, 299-304, 309, 351, 352-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 460, 479, 482-486, 488-492, 497-498, 500-501, 503-506, 508-512, 544-550, 553-558, 5 60, 582-585, 589, 603-604, 611, 613-616, 659, 661, 699-700, 702, 705-707, 770-771, 812-816, 838-842, 845-851, 856, 883, 885, 889, 893, 895-897, 936, 940, 945, 961, 965-968, 972-973, 1041-1045, 1047, 1170, 1172, 1216, 1222, 1235, 1244, 1246-1249, 1251-1252, 1254-1255, 1257-1259, 1268, 1319, 1321-1322, 1380-1381, 1386-1387, 1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1540, 1565-1566, 1579, 1581-1582, 1584-1589, 1591, 1601-1604, 1606-1607, 1610, 1625, 1627-1629, 1631-1638 7. The oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleic acid base sequences selected from the group consisting of 1651 to 1654, 1668, 1670 to 1674, 1714, 1717 to 1722, 1727 to 1731, 1745, 1751 to 1755, 1799, 1861, 1869, 1908, 1964, 1966, 2066 to 2069, 2075 to 2076, 2078 to 2079, 2108, 2144 to 2145, 2158 to 2160, 2193, 2385, 2390, and 2460. (Item 24) 7. The oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleic acid base sequences of SEQ ID NOs: 145, 147, 210, 352, 365, 366, 407, 408, 439 to 442, 444, 492, 500, 504, 511, 512, 544 to 547, 582, 604, 616, 699, 700, 702, 705 to 707, 839 to 842, 848, 1042 to 1045, 1172, 1255, 1454 to 1457, 1477 to 1499, 1538, 1539, 1581, 1582, 1606, 1607, 1610, and 1631 to 1633. (Item 25) 7. The oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleic acid base sequences of SEQ ID NOs: 407, 408, 441, 442, 444, 545, 582, 616, 705 to 707, 841, 1043, 1044, 1252, 1255, 1268, 1321, 1451, 1454 to 1460, 1497 to 1499, 1538, 1539, 1581, 1582, 1587, 1601, 1602, 1606, 1607, 1610, 1631 to 1633, 1719, 1721, 1730, 1731, 1861, and 2068. (Item 26) 7. The oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleic acid base sequences of SEQ ID NOs: 479, 482 to 491, 770, 771, 973, 998 to 1000, 1007, 1008, 1040 to 1043, 1387, 1454, 1456, 1459 to 1461, 1538, 1539, 1606, 1607, 1610, 1643 to 1665, 1668 to 1675, and 1862 to 1869. (Item 27) 27. The oligonucleotide of any one of items 1 to 26, which exhibits at least 50% mRNA inhibition at an oligonucleotide concentration of 20 nM when compared to control cells, as determined using a cellular assay. (Item 28) 27. The oligonucleotide of any one of items 1 to 26, which exhibits at least 60% mRNA inhibition at an oligonucleotide concentration of 20 nM when compared to control cells, as determined using a cellular assay. (Item 29) 27. The oligonucleotide of any one of items 1 to 26, which exhibits at least 70% mRNA inhibition at an oligonucleotide concentration of 20 nM when compared to control cells, as determined using a cellular assay. (Item 30) 27. The oligonucleotide of any one of items 1 to 26, which exhibits at least 85% mRNA inhibition at an oligonucleotide concentration of 20 nM when compared to control cells, as determined using a cellular assay. (Item 31) 27. The oligonucleotide of any one of items 1 to 26, which exhibits at least 50% mRNA inhibition at 2 nM when compared to control cells as determined using a cellular assay. (Item 32) 27. The oligonucleotide of any one of items 1 to 26, which exhibits at least 60% mRNA inhibition at an oligonucleotide concentration of 2 nM when compared to control cells, as determined using a cellular assay. (Item 33) 27. The oligonucleotide of any one of items 1 to 26, which exhibits at least 70% mRNA inhibition at an oligonucleotide concentration of 2 nM when compared to control cells, as determined using a cellular assay. (Item 34) 27. The oligonucleotide of any one of items 1 to 26, which exhibits at least 85% mRNA inhibition at an oligonucleotide concentration of 2 nM when compared to control cells, as determined using a cellular assay. (Item 35) 35. The oligonucleotide according to any one of items 1 to 34, comprising at least one alternative internucleoside linkage. (Item 36) 36. The oligonucleotide of claim 35, wherein the at least one alternative internucleoside linkage is a phosphorothioate internucleoside linkage. (Item 37) 36. The oligonucleotide of claim 35, wherein the at least one alternative internucleoside linkage is a 2'-alkoxy internucleoside linkage. (Item 38) 36. The oligonucleotide of claim 35, wherein the at least one alternative internucleoside linkage is an alkylphosphate internucleoside linkage. (Item 39) 39. The oligonucleotide of any one of items 1 to 38, comprising at least one alternative nucleobase. (Item 40) 40. The oligonucleotide of claim 39, wherein the alternative nucleobase is 5'-methylcytosine, pseudouridine or 5-methoxyuridine. (Item 41) 41. The modified oligonucleotide of any one of items 1 to 40, comprising at least one alternative sugar moiety. (Item 42) 42. The modified oligonucleotide of claim 41, wherein the alternative sugar moiety is 2'-OMe or a bicyclic nucleic acid. (Item 43) 43. The oligonucleotide of any one of items 1 to 42, further comprising a ligand conjugated to the 5' or 3' end of the oligonucleotide via a monovalent or branched divalent or trivalent linker. (Item 44) 44. The oligonucleotide of any one of items 1 to 43, comprising a region complementary to at least 17 consecutive nucleotides of the MSH3 gene. (Item 45) 44. The oligonucleotide of any one of items 1 to 43, comprising a region complementary to at least 19 consecutive nucleotides of the MSH3 gene. (Item 46) 44. The oligonucleotide of any one of items 1 to 43, comprising a region complementary to 19 to 23 consecutive nucleotides of the MSH3 gene. (Item 47) 44. The oligonucleotide of any one of items 1 to 43, comprising a region complementary to 19 consecutive nucleotides of the MSH3 gene. (Item 48) 44. The oligonucleotide of any one of items 1 to 43, comprising a region complementary to 20 consecutive nucleotides of the MSH3 gene. (Item 49) 44. The oligonucleotide of any one of items 1 to 43, which is about 15 to 25 nucleosides in length. (Item 50) 44. The oligonucleotide of any one of items 1 to 43, which is 20 nucleosides in length. (Item 51) 51. A pharmaceutical composition comprising one or more of the oligonucleotides according to any one of items 1 to 50 and a pharmaceutically acceptable carrier or excipient. (Item 52) 51. A composition comprising one or more of the oligonucleotides according to any one of items 1 to 50 and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle or a liposome. (Item 53) 52. A method for inhibiting transcription of MSH3 in a cell, comprising contacting the cell with one or more of the oligonucleotides of any one of items 1 to 50, the pharmaceutical composition of item 51, or the composition of item 52, for a time sufficient to result in degradation of mRNA transcripts of the MSH3 gene, thereby inhibiting expression of the MSH3 gene in the cell. (Item 54) 52. A method of treating, preventing, or delaying the progression of a trinucleotide repeat expansion disorder in a subject in need thereof, comprising administering to the subject one or more of the oligonucleotides of any one of items 1 to 50, the pharmaceutical composition of item 51, or the composition of item 52. (Item 55) 52. 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, comprising contacting the cell with one or more of the oligonucleotides of any one of items 1 to 50, the pharmaceutical composition of item 51, or the composition of item 52. (Item 56) 52. A method for inhibiting expression of the MSH3 gene in a cell, comprising contacting the cell with one or more of the oligonucleotides of any one of items 1 to 50, the pharmaceutical composition of item 51, or the composition of item 52, and maintaining the cell for a time sufficient to obtain degradation of mRNA transcripts of the MSH3 gene, thereby inhibiting expression of the MSH3 gene in the cell. (Item 57) 52. A method for reducing trinucleotide repeat expansions in a cell, comprising contacting the cell with one or more of the oligonucleotides described in any one of items 1 to 50, the pharmaceutical composition of item 51, or the composition of item 52. (Item 58) 58. The method of item 56 or 57, wherein the cell is in a subject. (Item 59) 59. The method of any one of items 54, 55 and 58, wherein the subject is a human. (Item 60) 59. The method of any one of items 54 to 58, wherein the cell is a cell of the central nervous system or a muscle cell. (Item 61) 61. The method of any one of paragraphs 54, 55, and 58-60, wherein the subject is identified as having a trinucleotide repeat expansion disorder. (Item 62) 62. The method of any one of items 54, 55, and 57 to 61, wherein the trinucleotide repeat expansion disorder is a polyglutamine disease. (Item 63) 63. The method of item 62, wherein the polyglutamine disease is selected from the group consisting of dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-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. (Item 64) 62. The method of any one of items 54 to 61, wherein the trinucleotide repeat expansion disorder is a non-polyglutamine disease. (Item 65) 65. The method of item 64, 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. (Item 66) 51. The one or more oligonucleotides according to any one of items 1 to 50, the pharmaceutical composition according to item 51, or the composition according to item 52, for use in the prevention or treatment of a trinucleotide repeat expansion disorder. (Item 67) 69. The oligonucleotide, pharmaceutical composition, or composition for use according to item 68, wherein the trinucleotide repeat expansion disorder is selected from the group consisting of dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-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. (Item 68) 68. The oligonucleotide, pharmaceutical composition or composition for use according to item 66 or 67, wherein the trinucleotide repeat expansion disorder is Huntington's disease. (Item 69) 68. The oligonucleotide, pharmaceutical composition or composition according to item 66 or 67, wherein the trinucleotide repeat expansion disorder is Friedreich's ataxia. (Item 70) 68. The oligonucleotide, pharmaceutical composition or composition for use according to item 66 or 67, wherein the trinucleotide repeat expansion disorder is myotonic dystrophy type 1. (Item 71) 71. The oligonucleotide, pharmaceutical composition or composition of any one of items 66 to 70, wherein the modified oligonucleotide, pharmaceutical composition or composition is administered intrathecally. (Item 72) 71. The oligonucleotide, pharmaceutical composition or composition of any one of items 66 to 70, wherein the modified oligonucleotide, pharmaceutical composition or composition is administered intracerebroventricularly. (Item 73) 71. The oligonucleotide, pharmaceutical composition or composition according to any one of items 66 to 70, which is administered intramuscularly. (Item 74) 52. A method of treating, preventing, or delaying the progression of a disorder in a subject in need thereof, wherein the subject is afflicted with a trinucleotide repeat expansion disorder, the method comprising administering to the subject one or more of the oligonucleotides of any one of items 1 to 50, the pharmaceutical composition of item 51, or the composition of item 52. (Item 75) 75. The method of claim 74, further comprising administering an additional therapeutic agent. (Item 76) 76. The method of claim 75, wherein the additional therapeutic agent is another oligonucleotide that hybridizes to mRNA encoding the huntingtin gene. (Item 77) 52. A method of preventing or delaying the progression of a trinucleotide repeat expansion disorder in a subject, comprising administering to the subject one or more of the oligonucleotides of any one of items 1 to 50, the pharmaceutical composition of item 51, or the composition of item 52, in an amount effective to delay the progression of the trinucleotide repeat expansion disorder in the subject. (Item 78) 78. The method of item 77, wherein the trinucleotide repeat expansion disorder is selected from the group consisting of dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-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. (Item 79) 79. The method of item 77 or 78, wherein the trinucleotide repeat expansion disorder is Huntington's disease. (Item 80) 79. The method of item 77 or 78, wherein the trinucleotide repeat expansion disorder is Friedreich's ataxia. (Item 81) 79. The method of item 77 or 78, wherein the trinucleotide repeat expansion disorder is myotonic dystrophy type 1. (Item 82) 79. The method of item 77 or 78, further comprising administering an additional therapeutic agent. (Item 83) 83. The method of claim 82, wherein the additional therapeutic agent is an oligonucleotide that hybridizes to mRNA encoding the huntingtin gene. (Item 84) 84. The method of any one of paragraphs 77 to 83, wherein the progression of the trinucleotide repeat expansion disorder is delayed by at least 120 days, e.g., 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, compared to expected progression. (Item 85) 51. The one or more oligonucleotides of any one of items 1 to 50, the pharmaceutical composition of item 51, or the composition of item 52, for use in preventing or delaying the progression of a trinucleotide repeat expansion disorder in a subject. (Item 86) 86. The oligonucleotide, pharmaceutical composition, or composition according to Item 85, wherein the trinucleotide repeat expansion disorder is selected from the group consisting of dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-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. (Item 87) 87. The oligonucleotide, pharmaceutical composition or composition according to item 85 or 86, wherein the trinucleotide repeat expansion disorder is Huntington's disease. (Item 88) 87. The oligonucleotide, pharmaceutical composition or composition according to item 85 or 86, wherein the trinucleotide repeat expansion disorder is Friedreich's ataxia. (Item 89) 87. The oligonucleotide, pharmaceutical composition or composition according to item 85 or 86, wherein the trinucleotide repeat expansion disorder is myotonic dystrophy type 1. (Item 90) 90. The oligonucleotide, pharmaceutical composition or composition of any one of items 85 to 89, wherein the progression of the trinucleotide repeat expansion disorder is delayed by at least 120 days, such as 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 compared to expected progression. [Brief explanation of the drawings]

[0096] [Figure 1] Figure 1 is a distribution plot showing somatic outgrowth of the human HTT transgene in the striatum as measured by the instability index in 4-, 8-, 12-, and 16-week-old R6 / 2 mice (four males and four females per age group). Bars indicate the mean, and error bars indicate the standard deviation. [Figure 2] Figure 2 is a distribution plot showing somatic outgrowth of the human HTT transgene in the cerebellum as measured by the instability index in 4-, 8-, 12-, and 16-week-old R6 / 2 mice (4 males and 4 females per age group). DETAILED DESCRIPTION OF THE INVENTION

[0097] Detailed Description The present inventors have found that inhibiting or depleting MSH3 levels and / or activity in cells is effective in treating trinucleotide repeat expansion disorders. Thus, useful compositions and methods for treating trinucleotide repeat expansion disorders, for example, in subjects in need of such treatment, are provided herein.

[0098] I. Trinucleotide Repeat Expansion Disorders Trinucleotide repeat expansion disorders are a family of genetic disorders characterized by pathogenic expansion of repeat regions within genomic regions, in which the number of repeats exceeds the normal stable threshold number of the gene and expands into the pathological range.

[0099] Trinucleotide repeat expansion disorders can generally be categorized as "polyglutamine" or "non-polyglutamine." Polyglutamine disorders, including Huntington's disease (HD) and some spinocerebellar ataxias, are caused by CAG (glutamine) repeats in the protein-coding regions of specific genes. Non-polyglutamine disorders are more heterogeneous and can be caused by CAG trinucleotide repeat expansions in non-coding regions, such as in myotonic dystrophy, or by expansions of trinucleotide repeats other than CAG, which can be in coding or non-coding regions, such as the CGG repeat expansion responsible for fragile X syndrome.

[0100] 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 within the same individual. Repeat expansion is thought to be caused by polymerase "slipping" during DNA replication. Tandem repeats in DNA sequences can "loop out" while maintaining complementary base pairing between the parent and daughter strands. When a loop structure is formed from the daughter strand, the number of repeats increases.

[0101] Conversely, when loop structures are formed from parental strands, the number of repeats decreases. Expansion appears to be more common than reduction. In general, the length of repeat expansion is negatively correlated with prognosis; longer repeats are correlated with earlier onset age and worsening disease severity. Therefore, trinucleotide repeat expansion disorders are prone to "anticipation," which means that the severity and / or onset age of symptoms worsens through successive generations of affected families due to the expansion of these repeats from one generation to the next.

[0102] Trinucleotide repeat expansion disorders are well known in the art. Exemplary trinucleotide repeat expansion disorders and the genetic trinucleotide repeats commonly associated therewith are included in Table 1.

[0103] [Table 1-1] [Table 1-2]

[0104] Proteins associated with trinucleotide repeat expansion disorders are typically selected based on their experimental association with trinucleotide repeat expansion disorders. For example, the production rate or circulating concentration of a protein associated with trinucleotide repeat expansion disorders may be elevated or suppressed in a population with a trinucleotide repeat expansion disorder compared to a population lacking the 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, proteins associated with trinucleotide repeat expansion disorders can be identified by obtaining gene expression profiles of protein-encoding genes 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).

[0105] II. Evidence for the involvement of the mismatch repair pathway in trinucleotide repeat expansion There is increasing evidence that DNA repair pathways, particularly mismatch repair (MMR), are involved in trinucleotide repeat expansions. A recent genome-wide association (GWA) study identified loci harboring genetic variants that alter the age of neurological onset of Huntington's disease (HD) (GEM-HD Consortium, Cell. 2015 Jul 30;162(3):516-26). This study identified MLH1, the human homolog of the E. coli DNA mismatch repair gene mutL. ​​Subsequent GWA studies in patients with polyglutamine diseases found significant associations of age of onset with DNA repair genes as a group for all polyglutamine diseases (HD and SCA) combined, as well as with specific SNPs in FAN1 and PMS2 (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 key 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). Somatic expansion was found to be reduced in transgenic mice lacking another member of the mismatch repair pathway, 8-oxo-guanine glycosylase (OGG1), implicating OGG1 in expansion as well (Kovtun IV et al. (2007) Nature 447, 447-452). However, another study found that human subjects containing the Ser326Cys polymorphism in hOGG1, which results in reduced OGG1 activity, developed increased mutant huntingtin (Coppede et al., (2009) Toxicol., 278: 199-203).Similarly, complete inactivation of Fan1, another component of the DNA repair pathway, in a mouse HD model results in somatic CAG expansions (Long et al. (2018) J. Hum Genet., 103: 1-9). Another component of the mismatch repair pathway, MSH3, has been reported to be involved in somatic expansions: polymorphisms in Msh3 were associated with somatic instability of expanded CTG trinucleotide repeats in patients with myotonic dystrophy type 1 (DM1) (Morales et al., (2016) DNA Repair 40: 57-66). Furthermore, natural polymorphisms in Msh3 and Mlh1 were found to mediate mouse strain-specific differences in CTG·CAG repeat instability (Pinto et al. (2013) ibid; Tome et al., (2013) PLoS Genet. 9 e1003280). Further evidence for the involvement of Msh2 and Msh3 in expanded repeats was reported in a study in which short hairpin RNA (shRNA) knockdown of either MSH2 or MSH3 slowed GAA trinucleotide repeat expansion of the Friedreich ataxia (FRDA) gene in fibroblasts derived from FRDA patients, 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). Despite some inconsistent results presented above, strong evidence exists that the MMR pathway plays some role in trinucleotide repeat expansion in various disorders. Furthermore, they were the first to recognize that inhibition of the MMR pathway provides a treatment or prevention of these repeat expansion disorders; however, no therapies are currently available or in development that modulate MMR with the intent of treating or preventing these repeat expansion disorders.

[0106] III. Oligonucleotide Drugs Agents described herein that reduce the level and / or activity of MSH3 in a cell can be, for example, polynucleotides, e.g., oligonucleotides, that reduce the level of MSH3-associated activity or associated downstream effects, or that reduce the level of MSH3 in a cell or subject.

[0107] In some embodiments, the agent that reduces the level and / or activity of MSH3 is a polynucleotide. In some embodiments, the polynucleotide is a single-stranded oligonucleotide, e.g., acting via an RNase H-mediated pathway. Oligonucleotides include DNA and DNA / RNA chimeric molecules, typically about 10-30 nucleotides in length, that recognize polynucleotide target sequences or sequence portions through hydrogen bonding interactions with the nucleotide bases of the target sequence (e.g., MSH3). The oligonucleotide molecule can reduce the expression level (e.g., protein level or mRNA level) of MSH3. For example, the oligonucleotide includes an oligonucleotide that targets full-length MSH3. In some embodiments, the oligonucleotide molecule recruits RNase H enzyme, resulting in target mRNA degradation.

[0108] In some embodiments, the oligonucleotide decreases the level and / or activity of a positive regulator of function. In other embodiments, the oligonucleotide increases the level and / or activity of an inhibitor of a positive regulator of function. In some embodiments, the oligonucleotide increases the level and / or activity of a negative regulator of function.

[0109] In some embodiments, the oligonucleotide decreases the level and / or activity or function of MSH3. In some embodiments, the oligonucleotide inhibits expression of MSH3. In other embodiments, the oligonucleotide increases the degradation of MSH3 and / or decreases the stability (i.e., half-life) of MSH3. The oligonucleotide can be chemically synthesized.

[0110] Oligonucleotides include those having a region of complementarity (e.g., a contiguous nucleobase region) complementary to at least a portion of an mRNA formed upon expression of the 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 oligonucleotide can inhibit expression of the MSH3 gene (e.g., a human, primate, non-primate, or avian MSH3 gene) by at least about 10%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence analysis using Western blotting or flow cytometry techniques.

[0111] Similarly, the region of complementarity to the target sequence can be between 10 and 30 linked nucleosides in length, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or 10-29, 10-28, 10-27, 10-26, 10- 25, 10-24, 10-23, 10-22, 10-21, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15 ~19, 15~18, 15~17, 18~30, 18~29, 18~28, 18~27, 18~26, 18~25, 18~24, 18~23, 18~22, 18~21, 18~20, 19~30, 19~29, 19~28, 19~27, 19~26, 19~25, 19~24, 19~23, 19~22, 19~21, The linked nucleoside length can be between 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. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated.

[0112] Oligonucleotides can be synthesized by standard methods known in the art, for example, by use of an automated DNA synthesizer such as those commercially available from Biosearch, Applied Biosystems, Inc., etc., as discussed further below.

[0113] Oligonucleotide compounds can be prepared using solution phase or solid phase organic synthesis, or both. Organic synthesis offers the advantage that oligonucleotides containing unnatural or alternative nucleotides can be easily prepared. Single-stranded oligonucleotides can be prepared using solution phase or solid phase organic synthesis, or both.

[0114] In one embodiment, the oligonucleotide comprises a region of at least 10 contiguous nucleobases that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) complementary to at least 10 contiguous nucleotides of the MSH3 gene. In some embodiments, the oligonucleotide comprises a sequence that is complementary to at least 17 contiguous nucleotides, 19-23 contiguous nucleotides, 19 contiguous nucleotides, or 20 contiguous nucleotides of the MSH3 gene. The oligonucleotide sequence may be selected from the group of sequences provided in any one of SEQ ID NOS: 6-2545.

[0115] In one embodiment, the sequence is substantially complementary to the sequence of an mRNA produced upon expression of the MSH3 gene. In some embodiments, the region of at least 10 nucleobases is substantially complementary to the sequence of an mRNA produced upon expression of the MSH3 gene. For the MSH3 gene corresponding to the sequence of NM_002439.4, the following sequences were identified: 155–199, 355–385, 398–496, 559–589, 676–724, 762–810, 876–903, 912–974, 984–1047, 1054–1098, 1114–1179, 1200–1227, 1294–1337, 1392–1417, 1467–1493, 1517–1630, 1665–1747, 1768–1866, 2029–2063, 2087–2199, 2229–2309, 2320–2332, 2400–2422, 2423–2500, 2516–2600, 2620–2700, 2720–2800, 2820–2900, 2920–3000, 3000–3100, 3100–3200, 3200–3300, 3300–3400, 3400–3500, 3500–3600, 3600–3700, 3700–3800, 3800–3900, 4000–4100, 4100–4200, 4300–4400, 4400–4500, 4500–4600, 4600–4700, 4700–4800, 480 In one embodiment, the region of at least 10 nucleobases is complementary to a reference mRNA at one or more of positions 62-2293, 2304-2330, 2371-2410, 2432-2458, 2494-2521, 2539-2647, 2679-2713, 2727-2753, 2767-2920, 2933-3000, 3046-3073, 3132-3245, 3266-3306, 3397-3484, 3528-3575, 3591-3617, 3753-3792, 3901-3936, 4074-4101, and 4281-4319. For the MSH3 gene corresponding to the sequence of NM_002439.4, the sequences of 155–199, 359–385, 398–496, 559–589, 676–724, 762–810, 876–974, 984–1098, 1114–1179, 1200–1227, 1294–1337, 1392–1417, 1467–1493, 1517–1630, 1665–1747, 1834–1866, 2029–2056, 2093–2199, 2262–2293, and 2302–2312 were identified. It is complementary at one or more positions among 304 to 2329, 2371 to 2410, 2433 to 2458, 2494 to 2521, 2539 to 2647, 2679 to 2713, 2727 to 2753, 2767 to 2920, 2933 to 3000, 3046 to 3072, 3132 to 3245, 3266 to 3303, 3397 to 3484, 3528 to 3575, 3591 to 3617, 3753 to 3792, 3901 to 3936, 4076 to 4101, and 4281 to 4319.In one embodiment, the region of at least 10 nucleobases is selected from the group consisting of 155-196, 359-385, 413-462, 559-589, 676-724, 762-810, 876-974, 984-1096, 1114-1179, 1200-1227, 1294-1337, 1467-1493, 1517-1630, 1665-1747, 1834-1866, 2029-2056, 2093-2199, 2100-2129, 2200-2229, 2300-2329, 2400-2450, 2500-2520, 2600-2620, 2700-2720, 2800-2820, 2900-2920, 3000-3000, 3100-3129, 3200-3220, 3300-3320, 3400-3420, 3500-3520, 3600-3620, 3700-3720, 3800-3800, 3900-4000, 41000-4100, 4200-4200, 4300-4300, 4400-4400, 4500-4500, 4600-4600, 4700-4700, 4800-4800, 4900-4900, 5000 It is complementary at one or more positions among 265 to 2293, 2378 to 2410, 2433 to 2458, 2494 to 2521, 2539 to 2647, 2679 to 2712, 2727 to 2753, 2767 to 2919, 2934 to 3000, 3046 to 3071, 3144 to 3183, 3220 to 3245, 3397 to 3484, 3534 to 3575, 3591 to 3616, 3901 to 3931, and 4281 to 4306. In one embodiment, the region of at least 10 nucleobases is complementary to the MSH3 gene corresponding to the sequence of reference mRNA NM_002439.4 at one or more of positions 435-462, 559-584, 763-808, 876-902, 931-958, 1001-1083, 1114-1179, 1294-1337, 1544-1578, 1835-1863, 2031-2056, 2144-2169, 2543-2577, 2590-2615, 2621-2647, 2685-2711, 2769-2795, and 2816-2868 of the MSH3 gene. In one embodiment, a region of at least 10 nucleobases is complementary to the MSH3 gene corresponding to the sequence of reference mRNA NM_002439.4 at one or more of positions 876-902, 930-958, 1056-1081, 1114-1139, 1154-1179, 1310-1337, 1546-1571, 1836-1862, 2141-2199, 2267-2292, 2540-2580, 2620-2647, 2686-2711, 2769-2868, 2939-2976, 3144-3169, and 3399-3424 of the MSH3 gene.In one embodiment, the region of at least 10 nucleobases is complementary to the MSH3 gene corresponding to the sequence of reference mRNA NM_002439.4 at one or more of positions 984-1021, 1467-1493, 1722-1747, 1767-1802, 1833-1861, 2385-2410, 2554-2581, 2816-2845, 2861-2920, and 3151-3183 of the MSH3 gene.

[0116] In one embodiment, the oligonucleotide comprises the nucleic acid base sequence of any one of SEQ ID NOs: 6-2545. In one embodiment, the oligonucleotide is selected from the group consisting of SEQ ID NOs: 20, 22-29, 31-32, 77-78, 81-82, 115, 117, 130, 132-134, 144-145, 147, 167-168, 210, 212-215, 290-293, 295-296, 299-305, 309, 351-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 459-460, 479, 482-493, 497-498, 500-501, 503-512, 543-550, 552-56 0, 562, 582-585, 588-591, 603-604, 611, 613-616, 659, 661, 699-700, 702, 705-707, 724-725, 770-771, 812-816, 838-842, 845-852, 856, 883-885, 889, 893-897, 936, 940-941, 945, 948, 950, 955, 959-961, 965-968, 972-973, 999, 1007, 1016-1017, 1019, 1021-1022, 1036, 1040-1045, 104 7, 1170, 1172-1173, 1211, 1216, 1222, 1235, 1240-1242, 1244-1249, 1251-1252, 1254-1259, 1268, 1316, 1318-1322, 1328-1329, 1373-1375, 1379-1383, 1386-1387, 1407-1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1541, 1565-1566, 1579, 1581-1589, 1 591, 1600-1607, 1610, 1625, 1627-1629, 1631-1639, 1643, 1650-1660, 1663-1665, 1668-1675, 1713-1714, 1716-1722, 1724, 1727-1731, 1741, 1745-1747, 1751-1755, 1799-1801, 1859-1866, 1868-1869, 1894-1896, 1905-1908, 1954, 1964-1966, 1969, 2066-2070, 2075-2079, 2108, 2138,It contains any one of the nucleic acid base sequences 2143-2147, 2157-2160, 2193-2194, 2299-2300, 2312-2313, 2385, 2388, 2390-2395, 2416-2418, 2460, 2462 and 2463. In one embodiment, the oligonucleotide is selected from the group consisting of SEQ ID NOs: 20, 22, 25-29, 31-32, 81-82, 115, 130, 132-134, 144, 145, 147, 168, 210, 212-215, 290-293, 295-296, 299-305, 309, 351-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 459-460, 479, 482-493, 497-498, 500-501, 503-506, 508-51 2, 543-550, 552-560, 562, 582-585, 589-591, 603-604, 611, 613-616, 659, 661, 699-700, 702, 705-707, 724, 770-771, 812-816, 838-842, 845-852, 856, 883-885, 889, 893-897, 936, 940-941, 945, 948, 950, 955, 959-961, 965-968, 972-973, 1041-1045, 1047, 1170, 1 172, 1216, 1222, 1235, 1241-1242, 1244-1249, 1251-1252, 1254-1259, 1268, 1316, 1318-1319, 1321-1322, 1328, 1373, 1379-1383, 1386-1387, 1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1541, 1565-1566, 1579, 1581-1582, 1584- 1589, 1591, 1601-1607, 1610, 1625, 1627-1629, 1631-1638, 1650-1655, 1659, 1665, 1668-1675, 1713-1714, 1716-1722, 1727-1731, 1745, 1747, 1751-1755, 1799-1800, 1859, 1861-1862, 1865-1866, 1868-1869, 1895-1896, 1905-1908, 1954, 1964-1966, 2066-2070,It contains any one of the nucleic acid base sequences 2075-2079, 2108, 2138, 2144-2146, 2158-2160, 2193-2194, 2299, 2300, 2313, 2385, 2388, 2390-2392, 2394-2395, 2418, 2460, 2462, and 2463. In one embodiment, the oligonucleotide is selected from the group consisting of SEQ ID NOs: 20, 25-29, 32, 81-82, 130, 133-134, 144-145, 147, 210, 212-213, 215, 290-293, 295-296, 299-304, 309, 351, 352-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 460, 479, 482-486, 488-492, 497-498, 500-501, 503-504, 505-506, 507-508, 509-510, 511-512, 513-514, 514-515, 515-516, 516-517, 517-518, 518-519, 520-521, 521-522, 522-523, 523-524, 524-525, 525-526, 526-527, 527-528, 528-529, 530-531, 532-533, 534-535, 535-536, 537-538, 538-539, 540-541, 542-543, 544, 545-546, 546-547, 547 6, 508-512, 544-550, 553-558, 560, 582-585, 589, 603-604, 611, 613-616, 659, 661, 699-700, 702, 705-707, 770-771, 812-816, 838-842, 845-851, 856, 883, 885, 889, 893, 895-897, 936, 940, 945, 961, 965-968, 972-973, 1041-1045, 1047, 1170, 1172, 12 16, 1222, 1235, 1244, 1246-1249, 1251-1252, 1254-1255, 1257-1259, 1268, 1319, 1321-1322, 1380-1381, 1386-1387, 1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1540, 1565-1566, 1579, 1581-1582, 1584-1589, 1591, 1 601~1604, 1606~1607, 1610, 1625, 1627~1629, 1631~1638, 1651~1654, 1668, 1670~1674, 1714, 1717~1722, 1727~1731, 1745, 1751~1755, 1799, 1861, 1869, 1908, 1964, 1966, 2066~2069, 2075~2076, 2078~2079, 2108, 2144~2145, 2158~2160, 2193, 2385,2390 and 2460. In one embodiment, the oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 145, 147, 210, 352, 365, 366, 407, 408, 439-442, 444, 492, 500, 504, 511, 512, 544-547, 582, 604, 616, 699, 700, 702, 705-707, 839-842, 848, 1042-1045, 1172, 1255, 1454-1457, 1477-1499, 1538, 1539, 1581, 1582, 1606, 1607, 1610 and 1631-1633. In one embodiment, the oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 407, 408, 441, 442, 444, 545, 582, 616, 705-707, 841, 1043, 1044, 1252, 1255, 1268, 1321, 1451, 1454-1460, 1497-1499, 1538, 1539, 1581, 1582, 1587, 1601, 1602, 1606, 1607, 1610, 1631-1633, 1719, 1721, 1730, 1731, 1861 and 2068. In one embodiment, the oligonucleotide comprises any one of the nucleic acid base sequences of SEQ ID NOs: 479, 482-491, 770, 771, 973, 998-1000, 1007, 1008, 1040-1043, 1387, 1454, 1456, 1459-1461, 1538, 1539, 1606, 1607, 1610, 1643-1665, 1668-1675, and 1862-1869.

[0117] In some embodiments, the nucleic acid base sequence of the oligonucleotide consists of any one of SEQ ID NOs: 6-2545. In one embodiment, the oligonucleotide is selected from the group consisting of SEQ ID NOs: 20, 22-29, 31-32, 77-78, 81-82, 115, 117, 130, 132-134, 144-145, 147, 167-168, 210, 212-215, 290-293, 295-296, 299-305, 309, 351-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 459-460, 479, 482-493, 497-498, 500-501, 503-512, 543-550, 552-553, 554-555, 555-556, 556-557, 557-558, 558-559, 560-561, 561-562, 562-563, 563-564, 564-565, 565-566, 566-567, 567-568, 568-569, 570-571, 572-573, 574-575, 576-577, 578-579, 579-580, 581-582, 582-583, 583-584, 584-585, 585-586, 586-587, 587 60, 562, 582-585, 588-591, 603-604, 611, 613-616, 659, 661, 699-700, 702, 705-707, 724-725, 770-771, 812-816, 838-842, 845-852, 856, 883-885, 889, 893-897, 936, 940-941, 945, 948, 950, 955, 959-961, 965-968, 972-973, 999, 1007, 1016-1017, 1019, 1021-1022, 1036, 1040-1045, 1 047, 1170, 1172-1173, 1211, 1216, 1222, 1235, 1240-1242, 1244-1249, 1251-1252, 1254-1259, 1268, 1316, 1318-1322, 1328-1329, 1373-1375, 1379-1383, 1386-1387, 1407-1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1541, 1565-1566, 1579, 1581-1588 9, 1591, 1600-1607, 1610, 1625, 1627-1629, 1631-1639, 1643, 1650-1660, 1663-1665, 1668-1675, 1713-1714, 1716-1722, 1724, 1727-1731, 1741, 1745-1747, 1751-1755, 1799-1801, 1859-1866, 1868-1869, 1894-1896, 1905-1908, 1954, 1964-1966, 1969, 2066-2070, 2075-2079, 2108,It consists of any one of the nucleic acid base sequences 2138, 2143-2147, 2157-2160, 2193-2194, 2299-2300, 2312-2313, 2385, 2388, 2390-2395, 2416-2418, 2460, 2462, and 2463. In one embodiment, the oligonucleotide is selected from the group consisting of SEQ ID NOs: 20, 22, 25-29, 31-32, 81-82, 115, 130, 132-134, 144, 145, 147, 168, 210, 212-215, 290-293, 295-296, 299-305, 309, 351-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 459-460, 479, 482-493, 497-498, 500-501, 503-506, 508-51 2, 543-550, 552-560, 562, 582-585, 589-591, 603-604, 611, 613-616, 659, 661, 699-700, 702, 705-707, 724, 770-771, 812-816, 838-842, 845-852, 856, 883-885, 889, 893-897, 936, 940-941, 945, 948, 950, 955, 959-961, 965-968, 972-973, 1041-1045, 1047, 1170, 1 172, 1216, 1222, 1235, 1241-1242, 1244-1249, 1251-1252, 1254-1259, 1268, 1316, 1318-1319, 1321-1322, 1328, 1373, 1379-1383, 1386-1387, 1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1541, 1565-1566, 1579, 1581-1582, 1584- 1589, 1591, 1601-1607, 1610, 1625, 1627-1629, 1631-1638, 1650-1655, 1659, 1665, 1668-1675, 1713-1714, 1716-1722, 1727-1731, 1745, 1747, 1751-1755, 1799-1800, 1859, 1861-1862, 1865-1866, 1868-1869, 1895-1896, 1905-1908, 1954, 1964-1966, 2066-2070,It consists of any one of the nucleic acid base sequences 2075-2079, 2108, 2138, 2144-2146, 2158-2160, 2193-2194, 2299, 2300, 2313, 2385, 2388, 2390-2392, 2394-2395, 2418, 2460, 2462, and 2463. In one embodiment, the oligonucleotide is selected from the group consisting of SEQ ID NOs: 20, 25-29, 32, 81-82, 130, 133-134, 144-145, 147, 210, 212-213, 215, 290-293, 295-296, 299-304, 309, 351, 352-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 460, 479, 482-486, 488-492, 497-498, 500-501, 503-504, 505-506, 507-508, 509-510, 511-512, 513-514, 514-515, 515-516, 516-517, 517-518, 518-519, 520-521, 521-522, 522-523, 523-524, 524-525, 525-526, 526-527, 527-528, 528-529, 530-531, 532-533, 534-535, 535-536, 537-538, 538-539, 540-541, 542-543, 544, 545-546, 546-547, 547 6, 508-512, 544-550, 553-558, 560, 582-585, 589, 603-604, 611, 613-616, 659, 661, 699-700, 702, 705-707, 770-771, 812-816, 838-842, 845-851, 856, 883, 885, 889, 893, 895-897, 936, 940, 945, 961, 965-968, 972-973, 1041-1045, 1047, 1170, 1172, 12 16, 1222, 1235, 1244, 1246-1249, 1251-1252, 1254-1255, 1257-1259, 1268, 1319, 1321-1322, 1380-1381, 1386-1387, 1408, 1433-1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1540, 1565-1566, 1579, 1581-1582, 1584-1589, 1591, 1 601~1604, 1606~1607, 1610, 1625, 1627~1629, 1631~1638, 1651~1654, 1668, 1670~1674, 1714, 1717~1722, 1727~1731, 1745, 1751~1755, 1799, 1861, 1869, 1908, 1964, 1966, 2066~2069, 2075~2076, 2078~2079, 2108, 2144~2145, 2158~2160, 2193, 2385,2390 and 2460. In one aspect, the oligonucleotide consists of any one of the nucleic acid base sequences of SEQ ID NOs: 145, 147, 210, 352, 365, 366, 407, 408, 439-442, 444, 492, 500, 504, 511, 512, 544-547, 582, 604, 616, 699, 700, 702, 705-707, 839-842, 848, 1042-1045, 1172, 1255, 1454-1457, 1477-1499, 1538, 1539, 1581, 1582, 1606, 1607, 1610 and 1631-1633. In one aspect, the oligonucleotide consists of any one of the nucleic acid base sequences of SEQ ID NOs: 407, 408, 441, 442, 444, 545, 582, 616, 705-707, 841, 1043, 1044, 1252, 1255, 1268, 1321, 1451, 1454-1460, 1497-1499, 1538, 1539, 1581, 1582, 1587, 1601, 1602, 1606, 1607, 1610, 1631-1633, 1719, 1721, 1730, 1731, 1861 and 2068. In one embodiment, the oligonucleotide consists of any one of the nucleic acid base sequences of SEQ ID NOs: 479, 482 to 491, 770, 771, 973, 998 to 1000, 1007, 1008, 1040 to 1043, 1387, 1454, 1456, 1459 to 1461, 1538, 1539, 1606, 1607, 1610, 1643 to 1665, 1668 to 1675, and 1862 to 1869.

[0118] In one embodiment, the oligonucleotide exhibits at least 50% mRNA inhibition at 20 nM compared to control cells as determined using a cellular assay. In one embodiment, the oligonucleotide exhibits at least 60% mRNA inhibition at an oligonucleotide concentration of 20 nM compared to control cells as determined using a cellular assay. In one embodiment, the oligonucleotide exhibits at least 70% mRNA inhibition at an oligonucleotide concentration of 20 nM compared to control cells as determined using a cellular assay. In one embodiment, the oligonucleotide exhibits at least 85% mRNA inhibition at an oligonucleotide concentration of 20 nM compared to control cells as determined using a cellular assay. In one embodiment, the oligonucleotide exhibits at least 50% mRNA inhibition at 2 nM compared to control cells as determined using a cellular assay. In one embodiment, the oligonucleotide exhibits at least 60% mRNA inhibition at an oligonucleotide concentration of 2 nM compared to control cells as determined using a cellular assay. In one embodiment, the oligonucleotide exhibits at least 70% mRNA inhibition at an oligonucleotide concentration of 2 nM when compared to control cells as determined using a cell assay. In one embodiment, the oligonucleotide exhibits at least 85% mRNA inhibition at an oligonucleotide concentration of 2 nM when compared to control cells as determined using a cell assay.

[0119] The cellular assay can include transfecting mammalian cells, such as HEK293, NIH3T3, or HeLa cells, with a desired concentration of oligonucleotide (e.g., 2 nM or 20 nM) using Lipofectamine 2000 (Invitrogen) and comparing the MSH3 mRNA levels of the transfected cells with those of control cells. The control cells can be transfected with an oligonucleotide not specific for MSH3 or can be mock-transfected. The mRNA levels can be determined using RT-qPCR, and the MSH3 mRNA levels can be normalized to GAPDH mRNA levels. The percent inhibition can be calculated as the percentage of the MSH3 mRNA concentration compared to that of the control cells.

[0120] In some embodiments, the oligonucleotide, or contiguous nucleotide region thereof, has a gapmer design or structure, also referred to herein simply as a "gapmer." In a gapmer structure, the oligonucleotide comprises at least three distinct structural regions in a "5->3" orientation: a 5' flanking sequence (also known as a 5' wing), a DNA core sequence (also known as a gap), and a 3' flanking sequence (also known as a 3' wing). In this design, the 5' and 3' flanking sequences comprise at least one alternative nucleoside adjacent to the DNA core sequence, and in some embodiments, may comprise a contiguous stretch of 2 to 7 alternative nucleosides, or a contiguous stretch of alternative and DNA nucleosides (a mixed flanking sequence comprising both alternative and DNA nucleosides).

[0121] The length of the 5' flanking sequence region can be at least two nucleosides long (e.g., at least 2, at least 3, at least 4, at least 5, or more nucleosides long). The length of the 3' flanking sequence region can be at least two nucleosides long (e.g., at least 2, at least 3, at least 4, at least 5, or more nucleosides long). The 5' and 3' flanking sequences can be symmetric or asymmetric with respect to the number of nucleosides they contain. In some embodiments, the DNA core sequence comprises about 10 nucleosides flanked by 5' and 3' flanking sequences each comprising about 5 nucleosides, also referred to as a 5-10-5 gapmer.

[0122] As a result, the nucleosides of the 5' and 3' flanking sequences flanking the DNA core sequence are alternate nucleosides, e.g., alternate 2' nucleosides. The DNA core sequence comprises a contiguous stretch of nucleotides capable of recruiting RNase H when the oligonucleotide is duplexed with an MSH3 target nucleic acid. In some embodiments, the DNA core sequence comprises a contiguous stretch of 5-16 DNA nucleosides. In other embodiments, the DNA core sequence comprises a region of at least 10 contiguous nucleobases having at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) complementarity to the MSH3 gene. In some embodiments, gapmers comprise a region complementary to at least 17 contiguous nucleotides, 19-23 contiguous nucleotides, or 19 contiguous nucleotides of the MSH3 gene. Gapmers are complementary to the MSH3 target nucleic acid and thus can be contiguous nucleoside regions of oligonucleotides.

[0123] The 5' and 3' flanking sequences adjacent to the 5' and 3' ends of the DNA core sequence may contain one or more affinity-enhancing alternative nucleosides. In some embodiments, the 5' and / or 3' flanking sequences contain at least one 2'-O-methoxyethyl (MOE) nucleoside. In some embodiments, the 5' and / or 3' flanking sequences contain at least two MOE nucleosides. In some embodiments, the 5' flanking sequence contains at least one MOE nucleoside. In some embodiments, both the 5' and 3' flanking sequences contain one MOE nucleoside. In some embodiments, all nucleosides in the flanking sequences are MOE nucleosides. In other embodiments, the flanking sequences can include both MOE nucleosides and other nucleosides, e.g., DNA nucleosides and / or non-MOE surrogate nucleosides, e.g., bicyclic nucleosides (BNAs) (e.g., LNA nucleosides or cET nucleosides) or other 2'-substituted nucleosides (mixed flanking sequences). In this case, the DNA core sequence is defined as a contiguous sequence of at least five RNase H-recruiting nucleosides (e.g., 5-16 DNA nucleosides) flanked on the 5' and 3' ends by affinity-enhancing surrogate nucleosides, e.g., MOE nucleosides.

[0124] In other embodiments, the 5' and / or 3' flanking sequences comprise at least one BNA (e.g., at least one LNA nucleoside or cET nucleoside). In some embodiments, the 5' and / or 3' flanking sequences comprise at least two bicyclic nucleosides. In some embodiments, the 5' flanking sequence comprises at least one BNA. In some embodiments, both the 5' and 3' flanking sequences comprise BNAs. In some embodiments, all nucleosides in the flanking sequences are BNAs. In other embodiments, the flanking sequences can comprise both BNAs and other nucleosides, e.g., DNA nucleosides and / or non-BNA alternative nucleosides, e.g., 2'-substituted nucleosides (mixed flanking sequences). In this case, the DNA core sequence is defined as a contiguous sequence of at least five RNase H-recruiting nucleosides (e.g., 5-16 DNA nucleosides) flanked at the 5' and 3' ends by affinity-enhancing surrogate nucleosides, e.g., BNAs, e.g., LNAs, e.g., beta-D-oxy-LNAs.

[0125] The 5' flanking sequence attached to the 5' end of the DNA core sequence comprises, contains, or consists of at least one alternative sugar moiety (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 or more alternative sugar moieties). In some embodiments, the flanking sequence comprises or consists of 1 to 7 alternative nucleobases, e.g., 2 to 6 alternative nucleobases, e.g., 2 to 5 alternative nucleobases, e.g., 2 to 4 alternative nucleobases, e.g., 1 to 3 alternative nucleobases, e.g., 1, 2, 3, or 4 alternative nucleobases. In some embodiments, the flanking sequence comprises or consists of at least one alternative internucleoside linkage (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 or more alternative internucleoside linkages).

[0126] The 3' flanking sequence attached to the 3' end of the DNA core sequence comprises, contains, or consists of at least one alternative sugar moiety (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 or more alternative sugar moieties). In some embodiments, the flanking sequence comprises or consists of 1 to 7 alternative nucleobases, e.g., 2 to 6 alternative nucleobases, e.g., 2 to 5 alternative nucleobases, e.g., 2 to 4 alternative nucleobases, e.g., 1 to 3 alternative nucleobases, e.g., 1, 2, 3, or 4 alternative nucleobases. In some embodiments, the flanking sequence comprises or consists of at least one alternative internucleoside linkage (e.g., at least 3, at least 4, at least 5, at least 6, at least 7 or more alternative internucleoside linkages).

[0127] In some embodiments, one or more or all of the alternative sugar moieties in the flanking sequences are 2' alternative sugar moieties.

[0128] In further embodiments, one or more of the 2' alternative sugar moieties in the wing region are selected from a 2'-O-alkyl-sugar moiety, a 2'-O-methyl-sugar moiety, a 2'-amino-sugar moiety, a 2'-fluoro-sugar moiety, a 2'-alkoxy-sugar moiety, an MOE sugar moiety, an LNA sugar moiety, an arabinonucleic acid (ANA) sugar moiety, and a 2'-fluoro-ANA sugar moiety.

[0129] In one embodiment, all alternative nucleosides in the flanking sequence are bicyclic nucleosides. In a further embodiment, the bicyclic nucleosides in the flanking sequence are independently selected from the group consisting of oxy-LNA, thio-LNA, amino-LNA, cET and / or ENA, in either the beta-D or alpha-L configuration or a combination thereof.

[0130] In some embodiments, one or more alternative internucleoside linkages in a flanking sequence are phosphorothioate internucleoside linkages. In some embodiments, the phosphorothioate linkages are stereochemically pure phosphorothioate linkages. In some embodiments, the phosphorothioate linkages are Sp phosphorothioate linkages. In other embodiments, the phosphorothioate linkages are Rp phosphorothioate linkages. In some embodiments, the alternative internucleoside linkages are 2'-alkoxy internucleoside linkages. In other embodiments, the alternative internucleoside linkages are alkylphosphate internucleoside linkages.

[0131] The DNA core sequence can comprise, contain, or consist of at least 5-16 consecutive DNA nucleosides capable of recruiting RNase H. In some embodiments, all of the nucleosides in the DNA core sequence are DNA units. In further embodiments, the DNA core region can consist of a mixture of DNA and other nucleosides capable of mediating RNase H cleavage. In some embodiments, at least 50% of the nucleosides in the DNA core sequence are DNA, e.g., at least 60%, at least 70%, or at least 80%, or at least 90% are DNA. In some embodiments, all of the nucleosides in the DNA core sequence are RNA units.

[0132] The oligonucleotide comprises a contiguous region complementary to the target nucleic acid. In some embodiments, the oligonucleotide may further comprise additional linked nucleosides located 5' and / or 3' to either the 5' and 3' flanking sequences. These additional linked nucleosides may be attached to the 5' end of the 5' flanking sequence or the 3' end of the 3' flanking sequence, respectively. In some embodiments, the additional nucleosides may form part of the contiguous sequence complementary to the target nucleic acid, or in other embodiments, may be non-complementary to the target nucleic acid.

[0133] The inclusion of additional nucleosides in either or both of the 5' and 3' flanking sequences can independently include 1, 2, 3, 4, or 5 additional nucleotides, which can be complementary or non-complementary to the target nucleic acid. In this embodiment, the oligonucleotide can, in some embodiments, include a contiguous sequence in which the additional nucleotides can modulate the target adjacent to the 5' and / or 3' end. Such additional nucleosides can function as nuclease-sensitive biocleavable linkers and can therefore be used to attach functional groups, such as conjugate moieties, to the oligonucleotide. In some embodiments, the additional 5' and / or 3' terminal nucleosides are linked by phosphodiester linkages and can be DNA or RNA. In another embodiment, the additional 5' and / or 3' terminal nucleosides are alternative nucleosides that can be included, for example, to enhance nuclease stability or for ease of synthesis.

[0134] In other embodiments, the oligonucleotide utilizes an "altimer" design and contains alternating 2'-fluoro-ANA and DNA regions that alternate every 3 nucleosides. Altimer oligonucleotides are discussed in more detail in Min, et al., Bioorganic & Medicinal Chemistry Letters, 2002, 12(18): 2651-2654 and Kalota, et al., Nuc. Acid Res. 2006, 34(2): 451-61 (hereby incorporated by reference).

[0135] In other embodiments, the oligonucleotide utilizes a "hemimer" design, comprising a single 2'-modified flanking sequence adjacent to a DNA core sequence (either 5' or 3' to the DNA core sequence). Hemimer oligonucleotides are discussed in more detail in Geary et al., 2001, J. Pharm. Exp. Therap., 296: 898-904, which is hereby incorporated by reference.

[0136] In some embodiments, the oligonucleotide has a nucleic acid sequence that has at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 6-2545. In some embodiments, the oligonucleotide has a nucleic acid sequence that has at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 6-2545.

[0137] Although the sequences in SEQ ID NOs: 6-2545 are set forth as unmodified and / or unconjugated sequences, it will be understood that the nucleosides of the oligonucleotides, e.g., the oligonucleotides, may comprise any one of the sequences set forth in any one of SEQ ID NOs: 6-2545, which are alternative nucleosides and / or conjugated as described in detail below.

[0138] Those skilled in the art will appreciate that oligonucleotides having structures between about 18 and 20 base pairs can be particularly effective in inducing RNase H-mediated degradation. However, it will be appreciated that shorter or longer oligonucleotides may also be effective. In the above embodiments, due to the nature of the oligonucleotide sequences provided herein, the oligonucleotides described herein may contain shorter or longer oligonucleotide sequences. It can be reasonably expected that shorter oligonucleotides minus only a few linked nucleosides on one or both ends may be similarly effective compared to the above-described oligonucleotides. Thus, oligonucleotides having a sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous linked nucleosides derived from one of the sequences provided herein, but differing in their ability to inhibit MSH3 gene expression by no more than about 5, 10, 15, 20, 25, or 30% from oligonucleotides containing the entire sequence, are contemplated as being within the scope of the present invention.

[0139] The oligonucleotides described herein can function through nuclease-mediated degradation of target nucleic acids, where the oligonucleotide is capable of recruiting a nuclease, e.g., an endonuclease-like endoribonuclease (RNase) (e.g., RNase H). Examples of oligonucleotide designs that operate via a nuclease-mediated mechanism are oligonucleotides that typically include a region of at least five or six DNA nucleosides, flanked on one or both sides by affinity-enhancing alternative nucleosides, e.g., gapmers, headmers, and tailmers.

[0140] The RNase H activity of an oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO01 / 23613 provides an in vitro method for determining RNase H activity that can be used to determine the ability to recruit RNase H. Typically, when a complementary target nucleic acid sequence is provided, an oligonucleotide is considered to be capable of recruiting RNase H if it has an initial rate, measured in pmol / l / min, that is at least 5%, e.g., at least 10%, or greater than 20%, of the initial rate determined when using an oligonucleotide having the same base sequence as the modified oligonucleotide being tested but containing only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide, and the methodology provided in Examples 91-95 of WO01 / 23613 (hereby incorporated by reference).

[0141] Additionally, the oligonucleotides described herein identify a site or sites in an MSH3 transcript that are susceptible to RNase H-mediated cleavage. As used herein, an oligonucleotide is said to target a specific site within an RNA transcript if the oligonucleotide promotes cleavage of the transcript anywhere within that specific site. Such oligonucleotides generally contain at least about 5-10 contiguous linked nucleosides from one of the sequences provided herein, coupled to an additional linked nucleoside sequence taken from a region contiguous with the selected sequence in the MSH3 gene.

[0142] Inhibitory oligonucleotides can be designed by methods well known in the art. Target sequences are generally about 10 to 30 linked nucleosides in length, although there is wide variation in the suitability of particular sequences within this range to direct cleavage of any given target RNA. Oligonucleotides with sufficient homology to provide the necessary sequence specificity to uniquely degrade any RNA can be designed using programs known in the art.

[0143] Systematic testing of several kinds of inhibitory oligonucleotide sequences can be carried out according to the teachings provided herein to optimize them.The considerations when designing interference oligonucleotide include but are not limited to biophysical, thermodynamic and structural considerations, base preference at specific positions, and homology.The preparation and use of inhibitory therapeutic agents based on non-coding oligonucleotides are also known in the art.

[0144] Although the various software packages and guidelines provided herein provide guidance for identifying optimal target sequences for any given gene target, an empirical approach can be taken in which a "window" or "mask" of a given size (for example, 21 nucleotides) is literally or figuratively (including, for example, in silico) present on the target RNA sequence to identify sequences within a size range that can function as target sequences. The next potential target sequence can be identified by gradually moving the sequence "window" one nucleotide upstream or downstream of the initial target sequence position until a complete set of possible sequences for any given target size is identified. This process, coupled with the systematic synthesis and testing of identified sequences (using assays described herein or known in the art) to identify optimally functioning sequences, can identify RNA sequences that mediate the best inhibition of target gene expression when targeted with an oligonucleotide drug. Thus, while the sequences identified herein represent effective target sequences, it is contemplated that further optimization of inhibitory efficiency may be achieved by progressively "walking the window" one nucleotide upstream or downstream of a given sequence to identify sequences with equal or better inhibitory characteristics.

[0145] Furthermore, it is contemplated that for any sequence identified herein, further optimization can be achieved by systematically adding or removing linked nucleosides to generate longer or shorter sequences, and testing the generated sequences by walking from this point up or down the target RNA in longer or shorter size windows.In addition, coupling this approach to generate new candidate targets with testing the effectiveness of oligonucleotides based on target sequences in inhibition assays known in the art and / or described herein can result in further improvements in the efficiency of inhibition.

[0146] Furthermore, such optimized sequences can be adjusted, for example, by introducing alternative nucleosides, sugar moieties, and / or internucleoside linkages described herein or known in the art, including alternative nucleosides, sugar moieties, and / or internucleoside linkages known in the art and / or discussed herein, to further optimize the molecule as an expression inhibitor (e.g., increase serum stability or circulatory half-life, increase thermostability, enhance transmembrane delivery, target to a specific location or cell type, increase interaction with silencing pathway enzymes, increase release from endosomes). The oligonucleotide agents described herein can contain one or more mismatches with the target sequence. In one embodiment, the oligonucleotides described herein contain three or fewer mismatches. When an oligonucleotide contains mismatches with a target sequence, in some embodiments, the region of mismatch is not located in the center of the region of complementarity. If an oligonucleotide contains mismatches with a target sequence, in some embodiments, the mismatches should be limited to within the last five nucleotides from either the 5' or 3' end of the region of complementarity. For example, for a 30-linked nucleoside oligonucleotide drug, the contiguous nucleobase region complementary to a region of the MSH3 gene generally does not contain any mismatches within the central 5-10 linked nucleosides. Methods described herein or known in the art can be used to determine whether an oligonucleotide containing mismatches with a target sequence is effective in inhibiting MSH3 gene expression. Consideration of the effectiveness of oligonucleotides with mismatches in inhibiting MSH3 gene expression is particularly important when a particular region of complementarity in the MSH3 gene is known to have polymorphic sequence variants within the population.

[0147] The construction of vector for expressing polynucleotide can be achieved by using conventional techniques that do not require detailed explanation for those skilled in the art.For the production of efficient expression vector, it is necessary to have a regulatory sequence that controls the expression of polynucleotide.These regulatory sequences include promoter and enhancer sequences, and these regulatory sequences are affected by specific cellular factors that interact with these sequences, and are well known in the art.

[0148] A. Alternative Oligonucleosides In one embodiment, one or more of the linked nucleosides or internucleoside linkages of the oligonucleotide are naturally occurring, and do not include, for example, chemical modifications and / or conjugations known in the art and described herein.In another embodiment, one or more of the linked nucleosides or internucleoside linkages of the oligonucleotide are 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 reduce immunogenicity.For example, the oligonucleotide can include nucleotides (e.g., adenine, thymidine, guanosine, cytidine, uridine, or inosine) that are naturally found in DNA or RNA, or can include alternative nucleosides or internucleoside linkages that have one or more chemical modifications to one or more components of the nucleotide (e.g., nucleobase, sugar, or phospho-linker portion). The oligonucleotides may be linked to each other via naturally occurring phosphodiester bonds or may contain alternative linkages (e.g., covalently linked via phosphorothioate (e.g., Sp phosphorothioate or Rp phosphorothioate), 3'-methylene phosphonate, 5'-methylene phosphonate, 3'-phosphoamidate, 2'-5' phosphodiester, guanidinium, S-methylthiourea, 2'-alkoxy, alkyl phosphate, or peptide bonds).

[0149] In some embodiments, substantially all of the nucleosides or internucleoside linkages of an oligonucleotide are alternative nucleosides. In other embodiments, all of the nucleosides or internucleoside linkages of an oligonucleotide are alternative nucleosides. An oligonucleotide in which "substantially all of the nucleosides are alternative nucleosides" may be mostly, but not completely, modified and may contain no more than 5, 4, 3, 2, or 1 naturally occurring nucleoside. In yet other embodiments, an oligonucleotide may contain no more than 5, 4, 3, 2, or 1 alternative nucleoside.

[0150] 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, SL et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated by reference. Alternative nucleotides and nucleosides include those with terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, reverse linkage) or 3'-terminal modifications (conjugation, DNA nucleotides, reverse linkage, etc.); base modifications, such as replacement with a stabilizing base, a destabilizing base, or a base that base pairs with an extended repertoire of partners, removal of a base (abasic nucleotide), or a conjugated base; sugar modifications (e.g., at the 2' or 4' position) or sugar replacement; and / or backbone modifications, including modification or replacement of a phosphodiester linkage. The nucleobase may 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 oligonucleotide compounds useful in the embodiments described herein include, but are not limited to, alternative nucleosides that contain modified backbones or do not contain natural internucleoside linkages.Nucleotides and nucleosides with 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 referred to in the art, alternative RNAs that do not have a phosphorus atom in their internucleoside backbone can be considered to be oligonucleosides.In some embodiments, oligonucleotides have a phosphorus atom in their internucleoside backbone.

[0151] 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'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boronophosphates with the normal 3'-5' linkage, their 2'-5' linked analogs, and those with opposite polarity, in which 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.

[0152] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include 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 ; Same No. 5,571,799; Same No. 5,587,361; Same No. 5,625,050; Same No. 6,028,188; Same No. 6,124,445; Same No. 6,160,109; Same No. 6,169 , No. 6,172,209; No. 6,239,265; No. 6,277,603; No. 6,326,199; No. 6,346,614; No. 6,444,423; No. 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. Reissue Patent No. 39464.

[0153] Alternative internucleoside linkages that do not contain a phosphorus atom have backbones formed by short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH constituent moieties.

[0154] Representative United States patents that teach the preparation of the above oligonucleosides include U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; No. 7; No. 5,470,967; No. 5,489,677; No. 5,541,307; No. 5,561,225; No. 5,596,086; No. 5,602,240; No. 5,608,046; No. 5,610,289; No. 5,618,704; No. 5,623,070; No. 5,663,312; No. 5,633,360; No. 5,677,437; and No. 5,677,439.

[0155] In other embodiments, suitable oligonucleotides 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 appropriate nucleic acid target compounds. One such oligomeric compound mimic that has been shown to have excellent hybridization properties is called peptide nucleic acid (PNA). In PNA compounds, the sugar of the nucleoside is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the 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 by reference herein. Further PNA compounds suitable for use in oligonucleotides are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0156] Some embodiments include oligonucleotides having phosphorothioate backbones, as well as oligonucleotides having heteroatom backbones, particularly -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- (also known as methylene(methylimino) or MMI backbones), -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- (wherein the native phosphodiester backbone is designated as -OPO-CH2-) of the above-referenced U.S. Patent No. 5,489,677, and the amide backbones of the above-referenced U.S. Patent No. 5,602,240. In some embodiments, the oligonucleotides featured herein have the morpholino backbone structure of the above-referenced U.S. Patent No. 5,034,506. In other embodiments, the oligonucleotides described herein include phosphorodiamidate morpholino oligomers (PMOs), in which the deoxyribose moieties are replaced by morpholine rings and the charged phosphodiester intersubunit linkages are replaced by uncharged phosphorodiamidate linkages, as described in Summerton, et al., Antisense Nucleic Acid Drug Dev. 1997, 7:63-70.

[0157] Alternative nucleosides and nucleotides can include one or more substituted sugar moieties. Oligonucleotides, such as those 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, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl and alkynyl. Exemplary suitable modifications include -O[(CH) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n -NH2, -O(CH2)n CH3, -O(CH2) n -ONH2 and -O(CH2) n -ON[(CH2) n CH3]2, where n and m are from 1 to about 10. In other embodiments, the oligonucleotide comprises, at the 2' position, one of the following: C1 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CHCHOCH, 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 oligonucleotides compared to unmodified oligonucleotides, including, but not limited to, increased nuclease resistance, improved pharmacokinetic properties, reduced non-specific protein binding, reduced toxicity, reduced immunostimulatory properties, and enhanced target affinity.

[0158] Another exemplary alternative includes 2'-dimethylaminooxyethoxy, i.e., the -O(CH)ON(CH), also known as 2'-DMAOE, as described in the Examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-(CH)-O-(CH)-N(CH). Further exemplary alternatives include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers within these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0159] Other alternatives include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the nucleosides and nucleotides of oligonucleotides, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides, and the 5' position of 5' terminal nucleotide. Oligonucleotides can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; and 5,700,920, certain of which are commonly owned with the present application. The entire contents of each of the foregoing are hereby incorporated by reference.

[0160] Oligonucleotides may contain nucleobase (often simply referred to in the art as "base") substitutions (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-thiouridine, pseudouridine, 1-methyl-pseudouridine, deoxyuridine, 5-hydroxyuridine, 5-hydroxy- Hydroxybutyl-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, Included are 5-propynyl uridine and cytidine, 6-azolidine, cytidine and thymine, 4-thiouridine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and 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.Additional 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, those 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, ST and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of oligonucleotides. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST, and Lebleu, B., Eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are an exemplary base substitution, even more particularly when combined with a 2'-O-methoxyethyl sugar modification.

[0161] Representative United States patents that teach the preparation of certain of the above-described alternative nucleobases, as well as other alternative nucleobases, include the above-mentioned 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; and 5,552,540, the entire contents of each of which are hereby incorporated by reference herein. ;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;and7,495,088.

[0162] In other embodiments, 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.

[0163] An oligonucleotide may contain one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a two-atom bridge. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety containing a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In some embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, an oligonucleotide may contain one or more locked nucleosides. A locked nucleoside is a nucleoside having a modified ribose moiety that contains an extra bridge connecting the 2' and 4' carbons of the ribose moiety. In other words, a locked nucleoside is a nucleoside containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in a structural conformation at the 3' end. The addition of a locked nucleoside to an oligonucleotide has been shown to increase the stability of the oligonucleotide in serum and reduce off-target effects (Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in polynucleotides include, but are not limited to, nucleosides that comprise a bridge between the 4'-ribosyl ring atom and the 2'-ribosyl ring atom. In some embodiments, polynucleotide agents comprise one or more bicyclic nucleosides that comprise a 4'-to-2' bridge.Examples of such 4' to 2' bridged bicyclic nucleosides include 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(CHOCH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH 3) (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-ON(CH3)2-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2', where R is H, C1-C. 12 alkyl or 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 by reference herein.

[0164] Additional representative U.S. patents and published U.S. patent applications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; and 7,053,202, the entire contents of each of which are hereby incorporated by reference herein. No. 7; No. 7,034,133; No. 7,084,125; No. 7,399,845; No. 7,427,672; No. 7,569,686; No. 7,741,457; No. 8,022,193; No. 8,030,467; No. 8,278,425; No. 8,278,426; No. 8,278,283; U.S. Patent Application Publication No. 2008 / 0039618; and No. 2009 / 0012281.

[0165] For example, any of the bicyclic nucleosides described above can be prepared with one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0166] Oligonucleotides can be modified to contain one or more constrained ethyl nucleosides. As used herein, a "constrained ethyl nucleoside" or "cEt" is a locked nucleoside containing a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge. In one embodiment, the constrained ethyl nucleoside is in the S conformation, referred to herein as an "S-cEt."

[0167] An oligonucleotide may contain one or more "conformationally restricted nucleosides" ("CRNs"). CRNs are nucleoside analogs with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. CRNs lock the ribose ring into a stable conformation, increasing hybridization affinity to mRNA. The linker is long enough to position the oxygen in an optimal position for stability and affinity, resulting in less ribose ring puckering.

[0168] Representative publications that teach the preparation of certain of the above-mentioned CRNs include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383; and PCT Application Publication No. WO2013 / 036868, the entire contents of each of which are hereby incorporated by reference herein.

[0169] In some embodiments, an oligonucleotide comprises one or more monomers that are UNA (unlocked nucleoside) nucleosides. UNA is an unlocked acyclic nucleoside, in which one of the sugar bonds is removed to form an unlocked "sugar" residue. In one example, UNA also encompasses a monomer in which the C1'-C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) is removed. In another example, the C2'-C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar is removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are hereby incorporated by reference).

[0170] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Application Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of each of which are hereby incorporated by reference herein.

[0171] The ribose molecule can be modified with a cyclopropane ring to produce tricyclodeoxynucleic acid (tricycloDNA). The ribose moiety can be replaced with another sugar, such as 1,5-anhydrohexitol, threose to produce threose nucleosides (TNAs), or arabinose to produce arabinonucleosides. The ribose molecule can be replaced with a non-sugar, such as cyclohexene to produce cyclohexene nucleosides, or glycol to produce glycol nucleosides.

[0172] Potentially stabilizing modifications to the termini of the 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 the like. Disclosure of this modification can be found in PCT Application Publication No. WO2011 / 005861.

[0173] Other alternative chemistries for oligonucleotides include 5' phosphates or 5' phosphate mimics, such as 5' terminal phosphates or phosphate mimics of oligonucleotides. Suitable phosphate mimics are disclosed, for example, in U.S. Patent Application Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0174] Exemplary oligonucleotides include nucleosides with alternative sugar moieties and may include DNA or RNA nucleosides. In some embodiments, oligonucleotides include nucleosides with alternative sugar moieties and DNA nucleosides. The incorporation of alternative nucleosides into oligonucleotides can enhance the affinity of the oligonucleotide for target nucleic acids. In this case, the alternative nucleosides can be referred to as affinity-enhancing alternative nucleotides.

[0175] In some embodiments, the oligonucleotide comprises at least one alternative nucleoside, e.g., 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 embodiments, the oligonucleotide comprises 1 to 10 alternative nucleosides, e.g., 2 to 9 alternative nucleosides, e.g., 3 to 8 alternative nucleosides, e.g., 4 to 7 alternative nucleosides, e.g., 6 or 7 alternative nucleosides. In certain embodiments, the oligonucleotide can comprise alternatives independently selected from these three types of alternatives (alternative sugar moieties, alternative nucleobases, and alternative internucleoside linkages), or combinations thereof. In one embodiment, the oligonucleotide comprises one or more nucleosides comprising an alternative sugar moiety, e.g., a nucleoside with a 2' sugar alternative. In some embodiments, the oligonucleotide comprises one or more nucleosides with a 2' sugar alternative 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, arabinonucleic acid (ANA), 2'-fluoro-ANA, and BNA (e.g., LNA) nucleosides. In some embodiments, one or more alternative nucleosides are BNA.

[0176] In some embodiments, at least one of the alternative nucleosides is a BNA (e.g., an LNA), e.g., at least two, e.g., at least three, at least four, at least five, at least six, at least seven, or at least eight of the alternative nucleosides are BNAs. In still further embodiments, all of the alternative nucleosides are BNAs.

[0177] In further embodiments, the oligonucleotide comprises at least one alternative internucleoside linkage. In some embodiments, the internucleoside linkage in the consecutive nucleotide sequence is a phosphorothioate or boronophosphate internucleoside linkage. In some embodiments, all internucleoside linkages in the consecutive sequence of the oligonucleotide are phosphorothioate linkages. In some embodiments, the phosphorothioate linkage is a stereochemically pure phosphorothioate linkage. In some embodiments, the phosphorothioate linkage is an Sp phosphorothioate linkage. In other embodiments, the phosphorothioate linkage is an Rp phosphorothioate linkage.

[0178] In some embodiments, the oligonucleotide comprises at least one alternative nucleoside that is 2'-MOE-RNA, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-MOE-RNA nucleoside units. In some embodiments, the 2'-MOE-RNA nucleoside units are connected by phosphorothioate linkages. In some embodiments, at least one of the alternative nucleosides is 2'-fluoro DNA, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-fluoro-DNA nucleoside units. In some embodiments, the oligonucleotide comprises at least one BNA unit and at least one 2'-substituted modified nucleoside. In some embodiments, the oligonucleotide comprises both a 2'-sugar-modified nucleoside and a DNA unit. In some embodiments, the oligonucleotide, or a contiguous nucleotide region thereof, is a gapmer oligonucleotide.

[0179] B. Ligand-Conjugated OligonucleotidesThe oligonucleotides may be chemically linked to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include lipid moieties, e.g., cholesterol moieties (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), thioethers, e.g., beryl-S-tritylthiol (Manoharan et al., (1992) Ann. NY Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20:533-538), aliphatic chains such as 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), phospholipids such as 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), polyamine or polyethylene glycol chains (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), palmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0180] In one aspect, the ligand alters the distribution, targeting, or lifetime of the incorporated oligonucleotide agent. In some aspects, the ligand provides enhanced affinity for a selected target, e.g., a molecule, a cell or cell type, a compartment, e.g., a cell or organ compartment, a tissue, an organ, or a region of the body, e.g., compared to the species in the absence of such ligand.

[0181] Ligands can include naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolized) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0182] The ligand can include a targeting group, such as, for example, a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid, or protein, e.g., an antibody that binds to a specific cell type, such as a kidney cell. The targeting group can be thyrotropin, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

[0183] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid 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 enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0184] Ligands can be proteins, e.g., glycoproteins or peptides, e.g., molecules with specific affinity for a co-ligand, or antibodies, e.g., antibodies that bind to specific cell types such as hepatocytes. Ligands can include hormones and hormone receptors. These can include non-peptidic species, e.g., lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose.

[0185] The ligand can be a substance, such as a drug, that can increase the uptake of an oligonucleotide agent into a cell, for example, by disrupting the cytoskeleton of the cell, for example, by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell. The drug can be, for example, a taxane, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0186] In some embodiments, the ligands attached to the oligonucleotides described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipid-soluble drugs, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides containing multiple phosphorothioate linkages in the backbone, e.g., oligonucleotides of about 5, 10, 15, or 20 bases, are also suitable as ligands (e.g., as PK-modulating ligands). Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0187] Ligand-conjugated oligonucleotides can be synthesized by using oligonucleotides bearing pendant reactive functionality, such as those derived from the attachment of a linking molecule to the oligonucleotide (described below). This reactive oligonucleotide can be reacted directly with commercially available ligands, synthesized ligands bearing any of a variety of protecting groups, or ligands with a linking moiety attached to it.

[0188] The oligonucleotides used in the conjugates can be easily and routinely produced through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art can also or alternatively be used. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0189] For ligand-conjugated oligonucleotides, e.g., sequence-specific linked nucleosides bearing ligand molecules, oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already bearing a linking moiety, ligand-nucleotide or nucleoside-conjugate precursors already bearing a ligand molecule, or non-nucleoside ligand-bearing building blocks.

[0190] When using a conjugate precursor that already possesses a linking moiety, synthesis of the sequence-specific linked nucleoside is typically completed, and then the ligand molecule is reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotide or linked nucleoside is synthesized by automated synthesizer using phosphoramidites derived from the ligand-nucleoside conjugate, in addition to standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0191] i. Lipid conjugates In one embodiment, ligand or conjugate is lipid or lipid-based molecule.This lipid or lipid-based molecule can be bound to serum protein, for example, human serum albumin (HSA).HSA binding ligand allows conjugate to be distributed to target tissue, for example, non-renal target tissue of the body.Lipid or lipid-based ligand can (a) increase the resistance of conjugate to degradation, (b) increase targeting or transport into target cell or cell membrane, and / or (c) can be used to adjust the binding to serum protein, for example, HSA.

[0192] In another embodiment, the ligand is a moiety, e.g., a vitamin, that is taken up by target cells, e.g., proliferating cells. Exemplary vitamins include vitamins A, E, and K.

[0193] ii. Cell permeabilizing agents In another embodiment, the ligand is a cell-penetrating agent, e.g., a helical cell-penetrating agent. In one embodiment, the agent is amphipathic. An exemplary agent is a peptide, e.g., tat or antennopedia. If the agent is a peptide, it can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudo-peptide linkages, and the use of D-amino acids. In one embodiment, the helical agent is an alpha-helical agent, which can have a lipophilic phase and a lipophobic phase.

[0194] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. The attachment of peptides and peptidomimetics to oligonucleotide drugs can affect the pharmacokinetic distribution of the oligonucleotide, for example, by enhancing cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0195] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can contain a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP. An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP) can be a targeting moiety. The peptide moiety can be a "delivery" peptide, capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, 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. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). For cell targeting purposes, an example of a peptide or peptidomimetic tethered to an oligonucleotide drug via an incorporated monomer unit is an arginine-glycine-aspartic acid (RGD) peptide or RGD mimetic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, for example, to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

[0196] The RGD peptide for use in compositions and methods can be linear or cyclic, and can be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissue(s).RGD-containing peptides and peptidomimetics can include D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties can be used to target integrin ligands.Some conjugates of this ligand target PECAM-1 or VEGF.

[0197] The cell-penetrating peptide can penetrate cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). The cell-penetrating peptide can include a nuclear localization signal (NLS). For example, the cell-penetrating peptide can be a bisected amphipathic peptide, such as MPG 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).

[0198] iii. Carbohydrate conjugates In some embodiments of the compositions and methods described herein, the oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated oligonucleotides are advantageous for the compositions described herein suitable for in vivo delivery of nucleic acids and for in vivo therapeutic use. As used herein, "carbohydrate" refers to a compound that is a carbohydrate (which can be linear, branched, or cyclic) that is itself composed of one or more monosaccharide units with at least six carbon atoms, each of which is bound to an oxygen, nitrogen, or sulfur atom; or a compound that has as its part a carbohydrate moiety (which can be linear, branched, or cyclic) that is composed of one or more monosaccharide units, each of which has at least six carbon atoms, each of which is bound to an oxygen, nitrogen, or sulfur atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include C5 and higher (e.g., C5, C6, C7, or C8) sugars; disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0199] In one aspect, the carbohydrate conjugates for use in the compositions and methods described herein are monosaccharides.

[0200] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, for example, but not limited to, a PK modulator and / or a cell-penetrating peptide.

[0201] Additional carbohydrate conjugates (and linkers) suitable for use include those described in PCT Application Publication Nos. WO2014 / 179620 and WO2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0202] iv. Linker In some aspects, the conjugates or ligands described herein can be attached to the oligonucleotide using a variety of linkers, which can be cleavable or non-cleavable.

[0203] The linker is typically a direct bond or an atom, such as oxygen or sulfur, a unit, such as NR 8, C(O), C(O)NH, SO, SO2, SO2NH, or, for example, 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, alkenylheteroarylalkynyl alkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, wherein one or more methylene is selected from the group consisting of O, S, S(O), SO, N(R 8 ), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; 8is hydrogen, acyl, aliphatic, or substituted aliphatic. In one aspect, the linker is between about 1 to 24, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18, 7 to 17, 8 to 17, 6 to 16, 7 to 17, 8 to 16 atoms, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, or 24 atoms.

[0204] A cleavable linking group is one that is sufficiently stable outside a cell but is cleaved upon entry into a target cell to release the two moieties held together by the linker. In some embodiments, the cleavable linking group is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more, or at least about 100-fold faster in the target cell or under a first reference condition (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (which may, for example, be selected to mimic or represent conditions found in blood or serum).

[0205] Cleavable linking groups are sensitive to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are more prevalent or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include: oxidizing or reducing enzymes or reducing agents present in cells that can degrade redox-cleavable linking groups by reduction, such as mercaptans, which are selective for specific substrates or do not have substrate specificity; esterases; endosomes, or agents that can create an acidic environment, such as those that produce a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which can be substrate specific) and phosphatases.

[0206] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH, approximately 5.0. Some linkers have cleavable linking groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell or into a desired compartment of the cell.

[0207] Linker can contain cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can depend on the cell to be targeted.For example, liver targeting ligand can be linked to cationic lipid through linker that contains ester group.Liver cell is rich in esterase, therefore, linker is more efficiently cleaved in liver cell than in cell type that is not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testicular cells.

[0208] Linkers containing peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synovial cells.

[0209] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or when contacted with other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between at least two conditions, where at least one condition is selected to demonstrate cleavage in target cells, and another condition is selected to demonstrate cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be carried out in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to perform initial evaluation in a cell-free or culture condition and confirm by further evaluation in whole animals. In some embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0210] a. Redox-cleavable linking group In one embodiment, the cleavable linking group is a redox-cleavable linking group that is cleaved upon reduction or oxidation.An example of a reductively cleavable linking group is a disulfide linking group (-SS-).To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group", or whether it is suitable for use with, for example, a specific oligonucleotide moiety and a specific targeting agent, the method described herein can be used.For example, the candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agent, using a reagent known in the art that mimics the cleavage rate observed in cells, for example, target cells.The candidate can be evaluated under conditions selected to mimic blood or serum conditions.In one embodiment, the candidate compound is cleaved at most about 10% in blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media compared to conditions selected to mimic extracellular media.

[0211] b. Phosphate-based cleavable linker In another embodiment, the cleavable linker comprises a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by an agent that decomposes or hydrolyzes the phosphate group. An example of an agent that cleaves a phosphate group in a cell is an enzyme such as a phosphatase in the cell. An example of a phosphate-based linking group is -OP(O)(OR k )-O-, -OP(S)(OR k )-O-, -OP(S)(SR k )-O-, -SP(O)(OR k )-O-, -OP(O)(OR k )-S-, -SP(O)(OR k )-S-, -OP(S)(ORk )-S-, -SP(S)(OR k )-O-, -OP(O)(R k )-O-, -OP(S)(R k )-O-, -SP(O)(R k )-O-, -SP(S)(R k )-O-, -SP(O)(R k )-S-, -OP(S)(R k )-S-. These candidates can be evaluated using methods similar to those described above.

[0212] c. acid-cleavable linking group In another embodiment, the 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 embodiments, the acid-cleavable linking group is cleaved in an acidic environment having 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 an agent such as an enzyme that can act as a general acid. Within cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linking group. 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 embodiment, the carbon is linked to the oxygen of an ester (alkoxy group), an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0213] d. Ester-based linking groups In another embodiment, the cleavable linker comprises an ester-based cleavable linking group. Ester-based cleavable linking groups are 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. Cleavable ester linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0214] e. Peptide-based cleavage groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linker. Peptide-based cleavable linkers are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linkers are peptide bonds formed between amino acids to give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to give rise to peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give rise to peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable linkers have the general formula -NHCHR A C(O)NHCHR B C(O)—, where R A and R B are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0215] In one embodiment, oligonucleotide is conjugated to carbohydrate via linker.Linker includes bivalent and trivalent branched-chain linker group.Linker for oligonucleotide carbohydrate conjugate includes but is not limited to those described in formula 24-35 of PCT application publication number WO2018 / 195165.

[0216] Representative United States patents that teach the preparation of oligonucleotide conjugates include 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;No. 5,414,077;No. 5,486,603;No. 5,512,439;No. 5,578,718; Same No. 5,608,046; Same No. 4,587,044; Same No. 4,605,735; Same No. 4,667,025; Same No. 4,76 No. 2,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335 ; Same No. 4,904,582; Same No. 4,958,013; Same No. 5,082,830; Same No. 5,112,963; Same No. 5,21 No. 4,136; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,245,022 ; Same No. 5,254,469; Same No. 5,258,506; Same No. 5,262,536; Same No. 5,272,250; Same No. 5,2 No. 92,873; No. 5,317,098; No. 5,371,241, No. 5,391,723; No. 5,416,203 No. 5,451,463; No. 5,510,475; No. 5,512,667; No. 5,514,785; No. 5,5 These include, but are not limited to, Nos. 6,552; 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; and 8,106,022.

[0217] Not all positions in a given compound need be uniformly modified; in fact, more than one of the above modifications can be incorporated into a single compound, or even a single nucleoside within an oligonucleotide.Chimera oligonucleotide compounds are also contemplated.Chimeric oligonucleotides typically contain at least one region in which RNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to target nucleic acids.An additional region of the oligonucleotide can serve as a substrate for enzymes capable of cleaving RNA:DNA.For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex.Therefore, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression.As a result, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used compared to phosphorothioate deoxynucleotides hybridized to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, optionally, associated nucleic acid hybridization techniques known in the art.

[0218] In certain cases, the nucleotides of the oligonucleotide may be modified with non-ligand groups. Some non-ligand molecules have been conjugated to oligonucleotides to enhance the activity, cellular distribution or cellular uptake of the oligonucleotide, and procedures for carrying out such conjugation are available in the scientific literature. Such non-ligand moieties include 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), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), and the like. 20:533), aliphatic chains such as dodecanediol 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), phospholipids such as 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), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such oligonucleotide conjugates are listed above. A typical conjugation protocol involves the synthesis of an oligonucleotide bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule to be conjugated using an appropriate coupling or activating reagent. The conjugation reaction can be carried out with the oligonucleotide still attached to the solid support, or after cleavage of the oligonucleotide in solution phase. Purification of the oligonucleotide conjugate by HPLC typically yields the pure conjugate.

[0219] IV. Medicinal Uses The oligonucleotide compositions described herein are useful in the methods described herein and, without being bound by theory, are believed to exert their desired effects through their ability to modulate the level, state and / or activity of the MSH3-containing MutSβ heterodimer, for example, by inhibiting the activity or levels of MSH3 protein in cells in a mammal.

[0220] One embodiment relates to a method for treating a DNA mismatch repair-associated disorder, such as a trinucleotide repeat expansion disorder, in a subject in need of such treatment. Another embodiment involves reducing the level of MSH3 in cells of a subject identified as having a trinucleotide repeat expansion disorder. Yet another embodiment involves a method for inhibiting expression of MSH3 in cells in a subject. A further embodiment involves a method for reducing trinucleotide repeat expansion in cells. These methods include contacting the cells with an amount of an oligonucleotide effective to inhibit expression of MSH3 in the cells, thereby inhibiting expression of MSH3 in the cells.

[0221] Based on the above method, oligonucleotides or compositions comprising such oligonucleotides are contemplated for use in therapy, or as medicines, or for treating DNA mismatch repair-related disorders, such as repeat expansion disorders, in a subject in need of such treatment, or for reducing the level of MSH3 in cells of a subject identified as having a trinucleotide repeat expansion disorder, or for inhibiting the expression of MSH3 in cells in a subject, or for reducing trinucleotide repeat expansion in cells. These uses include contacting cells with an amount of oligonucleotide effective to inhibit the expression of MSH3 in the cells, thereby inhibiting the expression of MSH3 in the cells. The embodiments described below in relation to the methods described herein are also applicable to these further embodiments.

[0222] Contacting cells with oligonucleotides can be carried out in vitro or in vivo. Contacting cells with oligonucleotides in vivo includes contacting cells or a group of cells in a subject, for example, a human subject, with oligonucleotides. A combination of in vitro and in vivo cell contact methods is also possible. Contacting cells can be direct or indirect, as discussed above. Furthermore, contacting cells can be achieved through a targeting ligand, including any ligand described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, such as a GalNAc3 ligand, or any other ligand that directs oligonucleotides to the desired site. The cell can include cells of the central nervous system or muscle cells.

[0223] Inhibiting expression of the MSH3 gene includes any level of inhibition of the MSH3 gene, e.g., at least partial suppression of expression of the MSH3 gene, e.g., at least about 20% inhibition. In some embodiments, the inhibition is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% inhibition.

[0224] Expression of the MSH3 gene can be evaluated based on the level of any variable associated with MSH3 gene expression, for example, MSH3 mRNA level or MSH3 protein level.

[0225] Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level, which can be any type of control level available in the art, such as a baseline level before dosing, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (e.g., a buffer-only control or an inactive agent control).

[0226] In some embodiments, surrogate markers may be used to detect inhibition of MSH3. For example, effective treatment of a trinucleotide repeat expansion disorder with an agent that reduces MSH3 expression may be understood to demonstrate a clinically relevant reduction in MSH3, as demonstrated by accepted diagnostic and monitoring criteria.

[0227] In some embodiments of the method, expression of the MSH3 gene is inhibited 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 embodiments, the method includes clinically relevant inhibition of MSH3 expression, as demonstrated, for example, by a clinically relevant outcome following treatment of the subject with an agent that reduces expression of MSH3.

[0228] Inhibition of expression of the MSH3 gene may be manifested by a reduction in the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which the MSH3 gene is transcribed that has been treated (e.g., by contacting the cells with an oligonucleotide or by administering an oligonucleotide to a subject in which the cells are or were present) such that expression of the MSH3 gene is inhibited compared to a second cell or group of cells that are substantially identical to the first cell or group of cells but have not been so treated (control cells that have not been treated with the oligonucleotide or with an oligonucleotide targeted to the gene of interest). The degree of inhibition may be expressed in terms of:

number

[0229] In other embodiments, inhibition of MSH3 gene expression can be assessed with respect to a parameter functionally associated with MSH3 gene expression, such as a reduction in MSH3 protein expression or the MSH3 signaling pathway. MSH3 gene silencing can be determined by any assay known in the art in any cell expressing endogenous MSH3 or heterologous MSH3 from an expression construct.

[0230] Inhibition of MSH3 protein expression can be manifested by a reduction in the level of MSH3 protein 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 assessment of mRNA suppression, inhibition 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.

[0231] A control cell or group of cells that can be used to assess inhibition of MSH3 gene expression includes a cell or group of cells that has not yet been contacted with an oligonucleotide. For example, a control cell or group of cells can be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an oligonucleotide.

[0232] The level of MSH3 mRNA expressed by a cell or group of cells can be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of MSH3 expression in a sample is determined by detecting a transcribed polynucleotide of the MSH3 gene or a portion thereof, e.g., mRNA. RNA can be extracted from cells using RNA extraction techniques, including, for example, acid phenol / guanidine thiocyanate extraction (RNAzol B; Biogenesis), the RNEASY™ RNA preparation kit (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 the methods described in PCT Application Publication WO 2012 / 177906, the entire contents of which are hereby incorporated by reference. In some embodiments, the level of MSH3 expression is determined using a nucleic acid probe. The term "probe" as used herein refers to any molecule capable of selectively binding to a specific MSH3 sequence, such as an mRNA or polypeptide. Probes can be synthesized by those skilled in the art or derived from appropriate biological preparations. Probes can be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0233] The isolated mRNA can be used in hybridization or amplification assays, including, but not limited to, Southern or Northern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to MSH3 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the 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 embodiment, 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. Those skilled in the art can easily adapt known mRNA detection methods for use in determining MSH3 mRNA levels.

[0234] Alternative methods for determining the level of expression of MSH3 in a sample include, for example, RT-PCR (Mullis, 1987, experimental aspects shown in 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), transcription amplification systems (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 detection of the amplified molecules using techniques well known to those of skill in the art, involve the processes of nucleic acid amplification of mRNA in a sample and / or reverse transcriptase (to prepare cDNA). These detection schemes are particularly useful for the detection of nucleic acid molecules when such molecules are present in very low numbers. In some embodiments, the level of expression of MSH3 is determined by quantitative fluorogenic RT-PCR (i.e., the TAQMAN™ System) or the DUAL-GLO® Luciferase assay.

[0235] The expression level of MSH3 mRNA can be monitored using membrane blots (e.g., as used in hybridization analyses, such as Northern, Southern, dot, etc.), or microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acids). See U.S. Patent Nos. 5,770,722; 5,874,219; 5,744,305; 5,677,195; and 5,445,934, which are incorporated herein by reference. Determining the expression level of MSH3 can involve using a nucleic acid probe in solution.

[0236] In some embodiments, the level of mRNA expression is assessed using a branched-chain DNA (bDNA) assay or real-time PCR (qPCR). The use of this PCR method is described and exemplified in the examples provided herein. Such methods can be used to detect MSH3 nucleic acids.

[0237] The level of MSH3 protein expression can be determined using any method known in the art for measuring protein levels, including, for example, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reaction, absorption spectroscopy, colorimetric assay, spectrophotometric assay, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, etc. Such assays can be used to detect proteins indicating the presence or replication of MSH3 protein.

[0238] In some embodiments of the method described herein, oligonucleotide is administered to the subject so that the oligonucleotide is delivered to the specific site in the subject.The inhibition of MSH3 expression can be evaluated by measuring the level or change in level of MSH3 mRNA or MSH3 protein in the sample from the specific site in the subject.In some embodiments, the method comprises the clinically relevant inhibition of MSH3 expression, for example, as demonstrated by clinically relevant outcome after the treatment of the subject with the agent that reduces MSH3 expression.

[0239] In other embodiments, the oligonucleotide is administered in an amount and for a time period effective to produce one (or more, e.g., two or more, three or more, four or more) of the following: (a) a reduction in the number of repeats; (b) a reduction in the level of polyglutamine; (c) a reduction in cell death (e.g., CNS cell death and / or muscle cell death); (d) a delay in the onset of the disorder; (e) an increase in survival of the subject; and (f) an increase in progression-free survival of the subject.

[0240] Treating trinucleotide repeat expansion disorders can result in an increase in the average survival time of individuals or a group of subjects treated with the oligonucleotides described herein compared to a group of untreated subjects.For example, the survival time of an individual or the average survival time of a group is increased by more than 30 days (more than 60 days, 90 days, or 120 days).The increase in the average survival time of an individual or a group can be measured by any reproducible means.The increase in the survival time of an individual can be measured, for example, by calculating the length of survival time for an individual after the start of treatment with a compound described herein.The increase in the average survival time of a group can be measured, for example, by calculating the average length of survival time for an individual after the start of treatment with a compound described herein.The increase in the survival time of an individual can be measured, for example, by calculating the length of survival time for an individual after the start of treatment with a compound described herein or a pharmaceutically acceptable salt of the compound.The increase in the survival time of an individual can be measured, for example, by calculating the length of survival time for an individual after the completion of the first round of treatment with a compound described herein or a pharmaceutically acceptable salt of the compound. An increase in the average survival time of a population can be measured, for example, by calculating for the population the average length of survival time after completion of a first round of treatment with a compound described herein or a pharmaceutically acceptable salt of the compound.

[0241] Treating trinucleotide repeat expansion disorder can cause the mortality rate of the treated subject population to decrease compared with the untreated population.For example, mortality rate is reduced by more than 2% (for example, more than 5%, 10% or 25%).The mortality rate of the treated subject population can be measured by any reproducible means, for example, by calculating the average number of disease-related deaths per unit time for the population after the start of treatment with the compound described herein or a pharmaceutically acceptable salt of the compound.The mortality rate of the population can be measured by, for example, calculating the average number of disease-related deaths per unit time for the population after the completion of the first round of treatment with the compound described herein or a pharmaceutically acceptable salt of the compound.

[0242] A. Delivery of Anti-MSH3 Agents The delivery of oligonucleotide to cells, for example, to cells in a subject, for example, a human subject, for example, a subject that requires delivery of oligonucleotide, for example, a subject that has a trinucleotide repeat expansion disorder, can be achieved by several different methods.For example, delivery can be carried out by contacting cells with oligonucleotide either in vitro or in vivo.In vivo delivery can be carried out directly by administering a composition comprising oligonucleotide to a subject.These alternative methods will be further discussed below.

[0243] Generally, any method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with oligonucleotides (see, for example, Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144 and WO94 / 02595, the entire contents of which are incorporated herein by reference). For in vivo delivery, factors to consider when delivering oligonucleotide molecules include, for example, the biological stability of the delivered molecule, the prevention of non-specific effects, and the accumulation of the delivered molecule in the target tissue. The non-specific effects of oligonucleotides can be minimized by local administration, for example, by direct injection or implantation into tissue, or by topical administration of a preparation. Local administration to the treatment site maximizes the local concentration of the drug, limits the exposure of the drug to systemic tissues that may otherwise be harmed by the drug or degrade the drug, and allows a lower total dose of oligonucleotide to be administered.

[0244] To administer oligonucleotides systemically for disease treatment, the oligonucleotides may contain alternative nucleobases, alternative sugar moieties, and / or alternative internucleoside linkages, or alternatively, may be delivered using a drug delivery system; both methods act to prevent rapid in vivo degradation of the oligonucleotide by endonucleases and exonucleases. Modification of the oligonucleotide or pharmaceutical carrier may enable targeting of the oligonucleotide composition to the target tissue and avoid undesirable off-target effects. Oligonucleotide molecules may be modified by chemical conjugation to lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation. In alternative embodiments, oligonucleotides may be delivered using a drug delivery system, such as nanoparticles, lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems promote binding of the oligonucleotide molecules (which are negatively charged) and also enhance interactions with the negatively charged cell membrane, allowing efficient uptake of the oligonucleotide by cells. Cationic lipids, dendrimers or polymers can be bound to oligonucleotides or can be induced to form vesicles or micelles that encase oligonucleotides.The formation of vesicles or micelles further prevents the degradation of oligonucleotides when administered systemically.Generally, any method of nucleic acid delivery known in the art can be adapted for the delivery of the oligonucleotides described herein.The methods for making and administering cationic oligonucleotide complexes are well within the skill of those skilled in the art (see, for example, 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 oligonucleotides 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. Aug 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 embodiments, the oligonucleotides are complexed with cyclodextrins for systemic administration. Methods and pharmaceutical compositions for the administration of oligonucleotides and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereby incorporated by reference in its entirety. In some embodiments, the oligonucleotides described herein are delivered by polyplex or lipoplex nanoparticles.Methods and pharmaceutical compositions for administration of oligonucleotides and polyplex 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 hereby incorporated by reference in their entireties.

[0245] i. Membranous molecular assembly delivery method Oligonucleotides can be delivered using various membrane-based molecular assembly delivery methods, including polymeric biodegradable microparticle or microcapsule delivery devices known in the art. For example, colloidal dispersion systems can be used for targeted delivery of the oligonucleotide agents described herein. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Liposomes are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. Large unilamellar vesicles (LUVs), ranging in size from 0.2 to 4.0 μm, have been shown to be capable of encapsulating a substantial percentage of an aqueous buffer containing large macromolecules. Liposomes are useful for the translocation and delivery of active ingredients to the site of action. Because liposome membranes are structurally similar to biological membranes, when liposomes are applied to tissues, the liposome bilayer fuses with the cell membrane bilayer. As liposomes and cells integrate, the aqueous contents containing oligonucleotides are delivered into the cells, where the oligonucleotides can specifically bind to target RNA and mediate RNase H-mediated gene silencing.In some cases, liposomes can also be specifically targeted, for example, to direct oligonucleotides to specific cell types.The composition of liposomes is usually a combination of phospholipids, usually combined 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.

[0246] Liposomes containing oligonucleotides can be prepared by various methods. In one example, the lipid components of the liposome are dissolved in a detergent to form micelles with the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The oligonucleotide preparation is then added to the micelles containing the lipid components. The cationic groups on the lipids interact with the oligonucleotides and condense around them to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposome preparation of oligonucleotides.

[0247] If necessary, a carrier compound that aids in coagulation can be added during the coagulation reaction, for example, by controlled addition. For example, the carrier compound can be a polymer other than nucleic acid (e.g., spermine or spermidine). The pH can be adjusted to favor coagulation.

[0248] Methods for producing stable polynucleotide delivery vehicles that incorporate polynucleotide / cationic lipid complexes as structural components of the delivery vehicle are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation is described in Feigner, PL et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; US Pat. No. 4,897,355; US Pat. 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 suitable size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, for example, Mayer et al., (1986) Biochim. Biophys. Acta 858:161). When consistently small (50-200 nm) and relatively uniform aggregates are desired, microfluidization can be used (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169). These methods are easily adapted to package oligonucleotide preparations into liposomes.

[0249] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complex binds to the negatively charged cell surface and is internalized in endosomes. Due to the acidic pH within the endosome, the liposomes rupture, releasing their contents into the cell cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).

[0250] pH-sensitive or negatively charged liposomes encapsulate nucleic acids rather than complexing with them. Because both nucleic acids and lipids are similarly charged, repulsion occurs rather than complex formation. Nevertheless, some nucleic acids are encapsulated in the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding thymidine kinase genes to cell monolayers in culture. The expression of exogenous genes was detected in target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).

[0251] One major type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholine. Neutral liposome compositions can be formed, for example, from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0252] Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO94 / 00569; WO93 / 24640; WO91 / 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.

[0253] Nonionic liposome systems, especially those containing nonionic surfactants and cholesterol, have also been tested to determine their usefulness in delivering drugs to the skin.Nonionic liposome formulations containing NOVASOME™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and NOVASOME™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine A into the dermis of mouse skin.The results showed that such nonionic liposome systems were effective in promoting the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) STP Pharma. Sci., 4(6):466).

[0254] The liposomes may be sterically stabilized liposomes containing one or more specialized lipids that result in enhanced circulation lifetime compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome is (A) one or more glycolipids, e.g., monosialoganglioside G M1or (B) derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without wishing to be bound by any particular theory, it is believed 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 is due to 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).

[0255] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., (1987), 507:64) reported the use of monosialoganglioside G M1 reported the ability of galactocerebroside sulfate and phosphatidylinositol to improve the blood half-life of liposomes. These findings were explained by Gabizon et al. (Proc. Natl. Acad. Sci. USA, (1988), 85:6949). U.S. Patent No. 4,837,028 and WO 88 / 04924, both to Allen et al., reported the ability of (1) sphingomyelin and (2) ganglioside G M1 or galactocerebroside sulfate esters. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97 / 13499 (Lim et al.).

[0256] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell membrane. Non-cationic liposomes cannot fuse efficiently with the plasma membrane, but they can be taken up by macrophages in vivo and used to deliver oligonucleotides to macrophages.

[0257] Additional advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs; and liposomes can protect oligonucleotides encapsulated in their internal compartments from metabolism and degradation (Rosoff, "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 aqueous volume of the liposomes.

[0258] The 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 spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with negatively charged lipids in the plasma membrane of tissue culture cells, resulting in delivery of oligonucleotides (see, e.g., Feigner, PL 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).

[0259] DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP), can be used in combination with phospholipids 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, which comprises positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. When sufficiently positively charged liposomes are used, the net charge on the resulting complex is also positive. The positively charged complexes prepared in this way spontaneously bind 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.

[0260] Other reported cationic lipid compounds include compounds conjugated to various moieties, including, for example, carboxyspermine, including compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (TRANSFECTAM™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES"), conjugated to one of two types of lipids (see, e.g., U.S. Pat. No. 5,171,678).

[0261] Another cationic lipid conjugate includes lipid derivatization with cholesterol ("DC-Chol") formulated into liposomes in combination with DOPE (see Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, produced 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 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 delivery of oligonucleotides are described in WO98 / 39359 and WO96 / 37194.

[0262] Liposomal formulations are particularly suitable for topical administration, and liposomes offer several advantages over other formulations. Such advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug in the desired target, and the ability to administer oligonucleotides to the skin. In some implementations, liposomes are used to deliver oligonucleotides to epithelial cells and also to enhance penetration of oligonucleotides into dermal tissue, e.g., the skin. For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been reported (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, RJ 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, RM and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, CY and Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

[0263] Nonionic liposome systems, especially those containing nonionic surfactants and cholesterol, have also been tested to determine their usefulness in delivering drugs to the skin.Nonionic liposome formulations containing NOVASOME I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and NOVASOME II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) have been used to deliver drugs into the dermis of mouse skin.Such formulations containing oligonucleotides are useful for treating dermatological disorders.

[0264] Liposome targeting can also be based on, for example, organ specificity, cell specificity and organelle specificity, and is known in the art.In the case of liposome targeting delivery system, lipid group can be incorporated into the lipid bilayer of liposome to maintain targeting ligand in stable association with the liposome bilayer.Various linking groups can be used to connect lipid chains to targeting ligand.Additional methods are known in the art, and for example, the linking groups are described in US Patent Application Publication No. 20060058255, which is hereby incorporated by reference.

[0265] Liposomes containing oligonucleotides can be made highly deformable. Such deformability can allow liposomes to penetrate through pores smaller than the average radius of the liposomes. For example, transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates as drug delivery vehicles. Transfersomes can be described as lipid droplets that are so highly deformable that they can easily penetrate through pores smaller than the droplets. Transfersomes can be made by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Transfersomes containing oligonucleotides can be delivered subcutaneously, for example, by infection to deliver oligonucleotides to keratinocytes in the skin. To cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter of less than 50 nm, under the influence of an appropriate transdermal gradient. Furthermore, due to their lipid properties, these transfersomes can be self-optimizing (e.g., adapting to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and are often self-filling. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0266] Other suitable formulations are described in U.S. Provisional Patent Application No. 61 / 018,616, filed January 2, 2008; U.S. Provisional Patent Application No. 61 / 018,611, filed January 2, 2008; U.S. Provisional Patent Application No. 61 / 039,748, filed March 26, 2008; U.S. Provisional Patent Application No. 61 / 047,087, filed April 22, 2008, and U.S. Provisional Patent Application No. 61 / 051,528, filed May 8, 2008. PCT Application No. PCT / US2007 / 080331, filed October 3, 2007, also describes suitable formulations. Surfactants find wide application in formulations, such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the properties of many different types of surfactants, both natural and synthetic, is by using hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing different surfactants used in formulations (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0267] If a 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. Their HLB values ​​generally range from 2 to approximately 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. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0268] When surfactant molecule carries negative charge when dissolved or dispersed in water, this surfactant is classified as anionic.Anionic surfactants include carboxylate, such as soap, acyl lactylate, acyl amide of amino acid, sulfuric acid ester, such as alkyl sulfate and ethoxylated alkyl sulfate, sulfonate, such as alkyl benzene sulfonate, acyl isethionate, acyl taurate and sulfosuccinate, and phosphate.The most important members of the class of anionic surfactants are alkyl sulfate and soap.

[0269] If the surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

[0270] 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 alkyl amides, N-alkyl betaines, and phosphatides.

[0271] The use of surfactants in drug products, formulations and emulsions has been reviewed (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0272] The oligonucleotide for use in the method can be provided as a micelle formulation.Micelle is a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure, so that all hydrophobic parts of the molecule are facing inward, while the hydrophilic parts remain in contact with the surrounding aqueous phase.When the environment is hydrophobic, the opposite arrangement exists.

[0273] ii. Lipid nanoparticle-based delivery methods Oligonucleotides can be fully encapsulated in lipid formulations, such as lipid nanoparticles (LNPs) or other nucleic acid-lipid particles. LNPs exhibit extended circulatory life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the administration site), making them extremely useful for systemic application. Examples of LNPs include "pSPLPs," which contain encapsulated coagulant-nucleic acid complexes, as described in PCT Application Publication No. WO 00 / 03683. The particles typically have an average 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, and most typically about 70 nm to about 90 nm, and are substantially nontoxic. Furthermore, when present in nucleic acid-lipid particles, nucleic acids are resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Patent Application Publication No. 2010 / 0324120; and PCT Application Publication No. WO 96 / 40964.

[0274] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to oligonucleotide ratio) is in the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above-listed ranges are also contemplated.

[0275] 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-dilinoleyloxy-N,N-dimethylaminopropane (DLen ... Dicarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 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-propanediol(propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogs, (3aR, 5s, 6aS )-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-ylethylazanediyedidodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid may comprise, for example, about 20 mol% to about 50 mol%, or about 40 mol% of the total lipid present in the particle.

[0276] Ionizable / non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine. The lipids may be anionic or neutral, including, but not limited to, 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. The non-cationic lipid, when cholesterol is included, may comprise about 5 mol% to about 90 mol%, about 10 mol%, or about 60 mol% of the total lipid present in the particle.

[0277] The conjugated lipid that inhibits particle aggregation can be, for example, a polyethylene glycol (PEG)-lipid, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. PEG-DAA conjugates can be, for example, PEG-dilauryloxypropyl (C 12 ), PEG-dimyristyloxypropyl (C 14 ), PEG-dipalmityloxypropyl (C 16 ) or PEG-distearyloxypropyl (C 18The conjugated lipid that prevents particle aggregation can be, for example, 0 mol % to about 20 mol %, or about 2 mol % of the total lipid present in the particle.

[0278] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, for example, from about 10 mol % to about 60 mol %, or about 50 mol % of the total lipid present in the particle.

[0279] B. Combination Therapy Oligonucleotide can be used alone or in combination with at least one additional therapeutic agent, for example, other drugs for treating trinucleotide repeat expansion disorder or its associated symptoms, or in combination with other types of treatment for treating trinucleotide repeat expansion disorder.In combined treatment, the dosage of one or more therapeutic compounds can be reduced from the standard dosage when administered alone.For example, dosage can be empirically determined from drug combinations and permutations, or can be estimated by isobolographic analysis (for example, Black et al., Neurology 65:S3-S6 (2005)).In this case, the dosage of the compound when combined should provide therapeutic effect.

[0280] In some embodiments, the oligonucleotide agents described herein can be used in combination with at least one additional therapeutic agent to treat trinucleotide repeat expansion disorders associated with genes having trinucleotide repeats (e.g., any of the trinucleotide repeat expansion disorders and related genes having nucleotide repeats listed in Table 1). In some embodiments, at least one of the additional therapeutic agents can be an oligonucleotide (e.g., ASO) that hybridizes with the mRNA of a gene associated with a trinucleotide repeat expansion disorder (e.g., any of the genes listed in Table 1). In some embodiments, the trinucleotide repeat expansion disorder is Huntington's disease (HD). In some embodiments, the gene associated with a trinucleotide repeat expansion disorder is huntingtin (HTT). Several allelic variants of the huntingtin gene are associated with the cause of Huntington's disease. In some cases, these variants are identified based on having unique HD-associated single nucleotide polymorphisms (SNPs). In some embodiments, the oligonucleotide hybridizes to a huntingtin gene mRNA containing any of the HD-associated SNPs known in the art (e.g., any of the HD-associated SNPs described in Skotte et al., PLoS One 2014, 9(9): e107434; Carroll et al., Mol. Ther. 2011, 19(12): 2178-85; Warby et al., Am. J. Hum. Gen. 2009, 84(3): 351-66, which are hereby incorporated by reference). In some embodiments, the additional therapeutic oligonucleotide hybridizes to a huntingtin gene mRNA lacking any HD-associated SNPs. In some embodiments, the additional therapeutic oligonucleotide hybridizes to a huntingtin gene mRNA having any of the SNPs selected from the group consisting of rs362307 and rs365331. In some embodiments, the additional therapeutic oligonucleotide may be a modified oligonucleotide (eg, an oligonucleotide containing any of the modifications described herein).In some embodiments, the modified oligonucleotide that is an additional therapeutic agent comprises one or more phosphorothioate internucleoside linkages. In some embodiments, the modified oligonucleotide comprises one or more 2'-MOE moieties. In some embodiments, the oligonucleotide that is an additional therapeutic agent that hybridizes to the mRNA of the huntingtin gene has a sequence selected from SEQ ID NOS: 6-285 of U.S. Patent No. 9,006,198; SEQ ID NOS: 6-8 of U.S. Patent Application Publication No. 2017 / 0044539; SEQ ID NOS: 1-1565 of U.S. Patent Application Publication No. 2018 / 0216108; and SEQ ID NOS: 1-2432 of PCT Application Publication WO2017 / 192679, which sequences are hereby incorporated by reference.

[0281] In some embodiments, at least one of the additional therapeutic agents is a chemotherapeutic agent (eg, a cytotoxic agent or other chemical compound useful in the treatment of a trinucleotide repeat expansion disorder).

[0282] In some embodiments, at least one of the additional therapeutic agents can be a therapeutic agent that is a non-drug treatment, for example, at least one of the additional therapeutic agents is physical therapy.

[0283] In any of the combination embodiments described herein, two or more therapeutic agents are administered simultaneously or sequentially in any order. For example, a first therapeutic agent can be administered immediately before or after one or more of the additional therapeutic agents, or 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, up to 14 hours, up to 16 hours, up to 17 hours, up to 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 to 7, 1 to 14, 1 to 21, or 1 to 30 days before or after one or more of the additional therapeutic agents.

[0284] V. Pharmaceutical Compositions The oligonucleotides described herein are formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for in vivo administration.

[0285] The compounds described herein can be used in the form of free base, salt, solvate, or prodrug. All forms are within the scope of the methods described herein. According to the methods described herein, the described oligonucleotides or their salts, solvates, or prodrugs can be administered to patients in various forms depending on the selected administration route, as will be understood by those skilled in the art. The compounds described herein can be administered, for example, orally, parenterally, intrathecally, intracerebroventricularly, intraparenchymal, buccal, sublingually, nasally, rectally, via patch, pump, or transdermal administration, and by appropriately formulated pharmaceutical compositions. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, intranasal, intrapulmonary, intrathecally, intracerebroventricularly, intraparenchymal, rectal, and topical administration. Parenteral administration can be by continuous infusion over a selected period of time.

[0286] The compounds described herein can be orally administered, for example, with an inert diluent or absorbable edible carrier, or enclosed in hard or soft shell gelatin capsules, or compressed into tablets, or directly incorporated with food or diet.For oral therapeutic administration, the compounds described herein can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups and wafers.The compounds described herein can be administered parenterally.Solutions of the compounds described herein can be prepared in water appropriately mixed with a surfactant such as hydroxypropyl cellulose.Dispersions can be prepared in glycerol, liquid polyethylene glycol, DMSO, and their mixtures, with or without alcohol, and in oils.Under normal conditions of storage and use, these preparations can contain preservatives to prevent microbial growth. Conventional procedures and ingredients for the selection and preparation of appropriate formulations are described, for example, in Remington's Pharmaceutical Sciences (2012, 22nd ed.) and The United States Pharmacopeia: The National Formulary (USP 41 NF 36), published in 2018. 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 fluid to the extent that it can be easily administered via syringe. Compositions for nasal administration can be conveniently formulated as aerosols, drops, gels, and powders. Aerosol formulations typically contain a solution or fine suspension of the active agent in a physiologically acceptable aqueous or non-aqueous solvent, usually presented in single- or multi-dose sterile form in a sealed container, which may take the form of a cartridge or refill for use with an atomization device. Alternatively, the sealed container may be a unit-dispensing device, such as a single-dose nasal inhaler or aerosol dispenser fitted with a metering valve, intended for disposal after use.When the dosage form comprises an aerosol dispenser, it contains a propellant that can be a compressed gas, such as compressed air, or an organic propellant, such as fluorochlorohydrocarbon.The aerosol dosage form can take the form of a pump-atomizer.Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, in which the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, gelatin and glycerin.Compositions for rectal administration are conveniently in the form of suppositories, containing conventional suppository bases such as cocoa butter.

[0287] The compounds described herein, as noted herein, can be administered to animals, e.g., humans, alone or in combination with pharmaceutically acceptable carriers, the proportions being determined by the solubility and chemical properties of the compound, the chosen route of administration, and standard pharmaceutical practice.

[0288] VI. Dosage The dosage of the compositions described herein (for example, compositions comprising oligonucleotides) can vary depending on many factors, such as the pharmacodynamic properties of the compound; the mode of administration; the age, health condition and weight of recipient; the nature and severity of symptoms; the frequency of treatment, and if present, the type of concurrent treatment; and the clearance rate of the compound in the treated animal.The compositions described herein can be initially administered at an appropriate dosage, which can be adjusted as necessary depending on clinical response.In some embodiments, the dosage of the compositions (for example, compositions comprising oligonucleotides) is a preventively effective amount or a therapeutically effective amount.

[0289] VII. Kit Kits are contemplated that include (a) a pharmaceutical composition comprising an oligonucleotide agent that reduces the level and / or activity of MSH3 in a cell or subject as described herein, and (b) a package insert with instructions for practicing any of the methods described herein. In some embodiments, the kit includes (a) a pharmaceutical composition comprising an oligonucleotide agent that reduces the level and / or activity of MSH3 in a cell or subject as described herein, (b) an additional therapeutic agent, and (c) a package insert with instructions for practicing any of the methods described herein. [Example]

[0290] Example 1 Design and Selection of Antisense Oligonucleotides Target transcript identification and selection: Target transcript selection and off-target scoring (below) utilized NCBI RefSeq sequences downloaded from NCBI on November 21, 2018. The experimentally validated "NM" transcript model was used, except for cynomolgus monkey, which only had an "XM" predicted model for the majority of genes. The longest human, mouse, rat, and cynomolgus monkey MSH3 transcripts containing fully mapped internal exons were selected (SEQ ID NOs: 1, 3, 4, and 5 for human, mouse, rat, and cynomolgus monkey, respectively; SEQ ID NO: 2 is the protein sequence).

[0291] Selection of 20-mer oligonucleotide sequences: For each transcript, all antisense 20-mer subsequences were generated. Candidate antisense oligonucleotides ("ASOs") were selected that met the following thermodynamic and physical characteristics, as determined by the inventors: a predicted melting temperature ("T") of the ASO:target duplex between 30 and 65°C; m ”), the predicted melting temperature of the hairpin (“T ヘアピン ”) <35°C, the predicted melting temperature for homopolymer formation (“T ホモ") <25°C, 20-60% GC content, no G homopolymers longer than 4, and no A, T, or C homopolymers longer than 6. These selected or "preferred" oligonucleotides were further evaluated for specificity (off-target scoring, below).

[0292] Off-target scoring: The specificity of preferred ASOs was assessed via alignment to all unspliced ​​RefSeq transcripts ("NM" model for human, mouse, and rat; "NM" and "XM" models for cynomolgus monkey) using the FASTA algorithm with an E-value cutoff of 1000. The number of mismatches (by species) between each ASO and each transcript was tallied. The "off-target score" for each ASO in each species was calculated as the minimum number of mismatches to any transcript other than the transcript encoded by the MSH3 gene.

[0293] Selection of ASOs for Screening: A set of 480 favorable ASOs was selected for screening according to both specificity and ASO:mRNA (target) hybridization energy maximization information as follows: All candidate ASOs were analyzed for the delta G (ΔG) of hybridization with the predicted target mRNA secondary structure according to Xu and Mathews (Methods Mol Biol. 1490:15-34 (2016)). 全体 Next, two subsets of ASOs were selected: first, 69 ASOs that matched human, cynomolgus monkey, and mouse target transcripts had an off-target score of at least 1 in the three species and a negative ΔG overall Second, 411 ASOs that matched human and cynomolgus monkey target transcripts had an off-target score of at least 2 in both species, and ΔG 全体 was below -9.5°C.

[0294] The sequence of each ASO, its location in the human transcript, its conservation in other species, and its species-specific off-target score are given in Table 2. Where "NC" is indicated, the ASO did not match the MSH3 gene in that species and therefore did not generate an off-target score.

[0295] ASOs were synthesized as 5-10-5 "flanking sequence-DNA core sequence-flanking sequence" antisense oligonucleotides with ribonucleotides at positions 1-5 and 16-20 and deoxyribonucleotides at positions 6-15, with the following general structure: 5'-Nm s Nm s Nm s Nm s Nm s N s N s N s N s N s N s N s N s N s N s Nm s Nm s Nm s Nm s Nm-3' During the ceremony, Nm: 2'-MOE residues (including 5-methyl-2'-MOE-C and 5-methyl-2'-MOE-U) N: DNA / RNA residue · s : Phosphorothioate (the backbone is fully phosphorothioate modified) All C's in the DNA core (positions 6-15) are 5'-methyl-2'-MOE-dC All "T"s at positions 1-5 or 16-20 are 5'-methyl-2'-MOE-U. Desalted oligonucleotides were used for primary screening at 2 nM and 20 nM. For detailed characterization of a subset of oligonucleotides, the oligonucleotides were further purified by HPLC.

[0296]

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

Table 2-29

Table 2-30

Table 2-31

Table 2-32

Table 2-33

Table 2-34

Table 2-35

Table 2-36

Table 2-37

Table 2-38

Table 2-39

Table 2-40

Table 2-41

Table 2-42

Table 2-43

Table 2-44

Table 2-45

Table 2-46

Table 2-47

Table 2-48

Table 2-49

Table 2-50

Table 2-51

Table 2-52

Table 2-53

Table 2-54

Table 2-55

Table 2-56

Table 2-57

Table 2-58

Table 2-59

Table 2-60

Table 2-61

Table 2-62

Table 2-63

Table 2-64

Table 2-65

Table 2-66

Table 2-67

Table 2-68

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

[0297] Example 3 In vitro screening for reduced elongation DNA triplet repeat expansion can be reproduced in vitro using patient-derived cell lines and DNA-damaging agents. Human fibroblasts from Huntington's disease (GM04281, GM04687, and GM04212), Friedreich's ataxia patients (GM03816 and GM02153), or myotonic dystrophy 1 (GM04602, GM03987, and GM03989) were purchased from Coriell Cell Repositories and maintained in culture medium according to 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, fibroblasts are treated with an oxidizing agent, such as hydrogen peroxide (H2O2), potassium chromate (K2CrO4) or potassium bromate (KBrO3), for up to 2 hours (Kovtum et al., ibid.). The cells are washed, the medium is replaced, and the cells are allowed to recover for 3 days. The treatment is repeated up to two more times, after which the cells are harvested and DNA is isolated. The CAG repeat length is determined using the method described below.

[0298] DNA triplet repeat expansion can be reproduced in vitro using a patient-derived cell line. Induced pluripotent stem cells (iPSCs) derived from Huntington's patient-derived human fibroblasts (CS09iHD-109n1) were purchased from the Cedars-Sinai RMI Induced Pluripotent Stem Cell Core and maintained according to the manufacturer's recommendations (https: / / www.cedars-sinai.org / content / dam / cedars-sinai / research / documents / biomanufacturing / recommended-guidelines-for-handling-ipscsv1.pdf). CAG repeats from an iPSC line with 109 CAGs show an increase in CAG repeat size over time in dividing iPS cells, with an average expansion of four CAG repeats over 70 days (Gold et al., 2019 Human Molecular Genetics Feb 15; 28(4): 650-661).

[0299] CS09iHD-109n1 iPSCs were treated with either LNP-formulated siRNA or ASO for sustained knockdown of target mRNA, and CAG repeat expansion was determined by DNA fragment analysis as described below. siRNA or ASO was added to cells at varying concentrations every 3–15 days, and mRNA knockdown was determined by RT-qPCR using standard molecular biology techniques. DNA and mRNA were isolated from cells at t = 0, 14, 28, 42, 56, and 80 days according to standard techniques. Lines indicate linear regression best fits. Differences in expansion between treatment and control were compared at each time point according to a linear repeated measures model with Tukey's post hoc test.

[0300] Example 4 Genomic DNA extraction and quantification of CAG repeat length by Small Pool-PCR (sp-PCR) analysis

[0301] Genomic DNA was purified using standard proteinase K digestion and extracted using DNAzol (Invitrogen) according to the manufacturer's instructions. CAG repeat length was determined by small-pool PCR analysis as previously described (Mario Gomes-Pereira and Darren Monckton, 2017, Front Cell Neuro 11:153). Briefly, DNA was digested with HindIII and diluted to a final concentration between 1 and 6 pg / μl, and approximately 10 pg was used in the subsequent PCR reaction. Primers flanking exon 1 of human HTT were used to amplify the CAG allele, and PCR products were resolved by electrophoresis. Subsequently, Southern blot hybridization was performed, and the CAG allele was observed by autoradiography or visualized by ethidium bromide staining. CAG length can be measured directly by sequencing on a MiSeQ or appropriate machine. Changes in CAG repeat number in the various treatment groups compared to controls are calculated using simple descriptive statistics (e.g., mean ± standard deviation).

[0302] Genomic DNA extraction and quantification of CAG repeat length by DNA fragment analysis Genomic DNA is purified using the DNAeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions. DNA is quantified using the Qubit dsDNA assay (ThemoScientific), and CAG repeat length is determined by fragment analysis using Laragen (Culver City, CA).

[0303] Example 5 Mouse studies Natural history study in HD mouse model: The R6 / 2 strain of HD mice is transgenic for the 5' end of the human HD gene (HTT), which carries an approximately 120-CAG repeat expansion. HTT is ubiquitously expressed. Transgenic mice exhibit a progressive neurological phenotype mimicking many of the pathological features of HD, including chorea-like movements, involuntary stereotypic movements, tremors and epileptiform seizures, and non-motor components including abnormal vocalizations. They urinate frequently and exhibit weight and muscle mass loss throughout the course of the disease. Neurologically, these mice develop neuronal intranuclear inclusions (NIIs) containing both huntingtin and ubiquitin proteins. These previously unknown NIIs were subsequently identified in HD patients. The onset of HD symptoms in R6 / 2 mice has been reported to occur between 9 and 11 weeks of age (Mangiarini et al., 1996 Cell 87: 493-506).

[0304] Somatic expansion was reported in the striatum, cortex, and liver of R6 / 2 mice. Somatic instability increased with increasing constitutive length (Larson et al., Neurobiology of Disease 76 (2015) 98-111). A natural history study was conducted in R6 / 2 mice carrying 120 CAG repeats. Their genotypes and CAG expansion lengths were determined. R6 / 2 mice (four male and four female mice per age group) were sacrificed at 4, 8, 12, and 16 weeks of age. The striatum, cerebellum, cortex, liver, kidney, heart, spleen, lung, duodenum, colon, quadriceps muscle, CSF, and plasma were collected and flash-frozen in liquid nitrogen. Genomic DNA was extracted, CAG repeat length was measured, and instability index was calculated from the striatum, cerebellum, cortex, liver, and kidney according to Lee et al. BMC Systems Biology 2010, 4:29. At 12 and 16 weeks of age, the striatum showed a significant increase in somatic elongation as measured by the instability index ( **** p<0.0001, one-way ANOVA) (Fig. 1). In the R6 / 2 mouse cerebellum, no changes in somatic outgrowth were observed across all ages (Fig. 2).

[0305] Mouse models that recapitulate many of the features of trinucleotide repeat expansion disorders, including HD, FA, and DM1, are readily available from commercial suppliers 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 performed in accordance with local IACUC guidelines. Included below are three examples of different affected mouse models and how they can be used to investigate the utility of pharmacological interventions targeting MSH3 for somatic expansion.

[0306] In the Huntington's disease research, several transgenic and knock-in mouse models have been generated to investigate the underlying pathological mechanisms involved in the disease. For example, the R6 / 2 transgenic mouse contains a 1.9-kb transgene of human HTT containing 144 copies of CAG repeats (Mangiarini et al., 1996 Cell 87:493-506), while the HdhQ111 model was generated by replacing mouse HTT exon 1 with human exon 1 containing 111 copies of CAG repeats (Wheeler et al., 2000 Hum Mol Genet 9:503-513). Both the R6 / 2 and HdhQ111 models recapitulate many of the features of human HD, including motor and behavioral dysfunction, neuronal loss, and CAG repeat expansions 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).

[0307] R6 / 2 mice are genotyped using DNA from tail snips at weaning to determine CAG repeat size. Mice are randomized into groups (n=12 / group) at 4 weeks of age at weaning and administered monthly (weeks 4 and 8) ICV injections of either PBS (control) or up to 500 μg of oligos targeting MSH3. A series of oligos targeting different regions of MSH3 can be tested to identify the most effective oligo sequence in vivo. At 12 weeks of age, mice are euthanized and tissues are extracted for analysis. The list of tissues includes, but is not limited to, the striatum, cortex, cerebellum, and liver. Genomic DNA is extracted and CAG repeat length is measured as described below. CSF and plasma are collected for biomarker analysis. Additional suitable mouse models of HD can be considered.

[0308] In Friedreich's ataxia, the YG8 FRDA transgenic mouse model is commonly used to understand the pathology (Al-Mahdawi 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 a null FRDA mouse background. The YG8 model demonstrates somatic expansion of a GAA triplet repeat expansion in neural tissue, accompanied by only mild motor deficits. YG8 FRDA mice were genotyped using DNA derived from tail snips at weaning, and CAG repeat size was determined using a method. To determine whether MSH3 plays a role in the somatic expansion of the disease allele, hemizygous YG8 FRDA animals were intravenously administered the oligos targeted to knockdown MSH3 identified above.

[0309] Approximately 2 months later, animals are euthanized and tissues are collected for molecular analysis.Suitable tissues are heart, quadriceps, dorsal root ganglion (DRG), cerebellum, kidney and liver.Genomic DNA is extracted and the length of CAG repeat is measured as described above in Example 4.

[0310] In myotonic dystrophy, the DM300-328 transgenic mouse model is suitable for investigating the pathology behind DM1. This mouse model contains a large human genome sequence (approximately 45 kb) containing more than 300 CTG repeats, and exhibits both somatic elongation 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 from tail fragments at weaning time to determine CAG repeat size. To determine whether MSH3 plays a role in the somatic expansion of disease alleles in myotonic dystrophy, DM300-328 transgenic animals were administered an ASO targeting MSH3 knockdown via subcutaneous (sc), intraperitoneal (ip), or intravenous tail injection (iv). Mice were administered the ASO up to twice weekly for up to 8 weeks of treatment. Animals were euthanized at multiple time points, and tissues were collected for molecular analysis. Appropriate tissues included quadriceps, heart, diaphragm, cortex, cerebellum, sperm, kidney, and liver. Genomic DNA was extracted, and CAG repeat length was measured and compared with parallel controls.

[0311] The HdhQ111 mouse model for Huntington's disease is a heterozygous knock-in system in which most of exon 1 and part of intron 1 on one allele of the huntingtin gene (i.e., the HTT or Huntington's disease gene) are replaced with human DNA containing approximately 111 CAG repeats. In this example, an ASO that knocks down MSH3 activity or levels is administered. After the treatment period, brain tissue from treated or untreated mice is isolated (e.g., striatal tissue) and analyzed using qRT-PCR as previously described to determine MSH3 RNA levels. Huntingtin gene repeat analysis is performed using mouse tissue (e.g., striatal tissue) after the treatment period using a human-specific PCR assay that amplifies the HTT CAG repeat from the knock-in allele but not the mouse sequence (i.e., the wild-type allele). In this protocol, the forward primer is fluorescently labeled (e.g., with 6-FAM as previously described in Pinto RM, 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 the product can be resolved using an analyzer with comparison to an internal size standard to generate a CAG repeat size distribution trace. Repeat size is determined from the peak with the greatest intensity from control tissue (e.g., mouse tail tissue) and from affected tissue (e.g., brain striatum tissue or brain cortex tissue). Immunohistochemistry is performed using a polyclonal anti-huntingtin antibody (e.g., EM48) on paraffin-embedded or otherwise prepared brain tissue sections and can be quantified using a standardized staining index to capture both nuclear staining intensity and the number of stained nuclei.The reduction in repeat size in affected tissues indicates that agents that reduce the levels and / or activity of MSH3 may reduce the repeats responsible for the toxic and / or defective gene product in Huntington's disease.

[0312] Other Aspects All publications, patents, and patent applications mentioned herein are incorporated 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. In the event that a term in this application is found to be defined differently in a document incorporated by reference herein, the definition provided herein serves as the definition of that term.

[0313] While the invention has been described in connection with specific embodiments thereof, it will be understood that the invention is capable of further modifications, and this application...

Claims

1. A single-stranded oligonucleotide 20 nucleosides in length, comprising a region of at least 10 contiguous nucleic acid bases having at least 95% complementarity to the MSH3 gene; The oligonucleotide is (a) a DNA core sequence comprising linked deoxyribonucleosides; (b) a 5' flanking sequence comprising linked nucleosides; and (c) a 3' flanking sequence containing linked nucleosides Including, the DNA core comprises a region of at least 10 contiguous nucleobases located between the 5' flanking sequence and the 3' flanking sequence; the 5' flanking sequence and the 3' flanking sequence each comprise at least two linked nucleosides; and at least one nucleoside in each flanking sequence comprises an alternative nucleoside; The oligonucleotides are selected from the group consisting of SEQ ID NOs: 20, 22-29, 31-32, 77-78, 81-82, 115, 117, 130, 132-134, 144-145, 147, 167-168, 210, 212-215, 290-293, 295-296, 299-305, 309, 351-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 459-460, 479, 482-493, 497-498, 500-501, 503-512, 543-550, 552-560, 562, 582-585, 590-591, 592-593, 594-595, 596-600, 598-601, 599-602, 599-603, 599-604, 599-605, 599-606, 599-607, 599-608, 591-609, 592-609, 593-601, 594-602, 595-603, 596-604, 597-605, 598-606, 599-607, 599-608, 599-609, 591-601, 592-603, 5 88-591, 603-604, 611, 613-616, 659, 661, 699-700, 702, 705-707, 724-725, 770-771, 812-816, 838-842, 845-852, 856, 883-885, 889, 893-897, 936, 94 0 to 941, 945, 948, 950, 955, 959 to 961, 965 to 968, 972 to 973, 999, 1007, 1016 to 1017, 1019, 1021 to 1022, 1036, 1040 to 1045, 1047, 1170, 1172 to 1173, 1211, 1216, 1222, 1235, 1240-1242, 1244-1249, 1251-1252, 1254-1259, 1268, 1316, 1318-1322, 1328-1329, 1373-1375, 1379-1383, 1386-1387, 1407-1408, 1433- 1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1541, 1565-1566, 1579, 1581-1589, 1591, 1600-1607, 1610, 1625, 1627-1629, 1631- 1639, 1643, 1650-1660, 1663-1665, 1668-1675, 1713-1714, 1716-1722, 1724, 1727-1731, 1741, 1745-1747, 1751-1755, 1799-1801, 1859-1866, 1868- 1869, 1894-1896, 1905-1908, 1954, 1964-1966, 1969, 2066-2070, 2075-2079, 2108, 2138, 2143-2147, 2157-2160, 2193-2194, 2299-2300, 2312-2313,An oligonucleotide comprising any one of the nucleic acid base sequences 2385, 2388, 2390-2395, 2416-2418, 2460, 2462, or 2463.

2. 1. A composition for inhibiting expression of the human MSH3 gene in a cell, said composition comprising a single-stranded oligonucleotide 20 nucleosides in length, said oligonucleotide comprising a region of at least 10 contiguous nucleobases having at least 95% complementarity to the MSH3 gene; The oligonucleotide is (a) a DNA core sequence comprising linked deoxyribonucleosides; (b) a 5' flanking sequence comprising linked nucleosides; and (c) a 3' flanking sequence containing linked nucleosides Including, the DNA core comprises a region of at least 10 contiguous nucleobases located between the 5' flanking sequence and the 3' flanking sequence; the 5' flanking sequence and the 3' flanking sequence each comprise at least two linked nucleosides; and at least one nucleoside in each flanking sequence comprises an alternative nucleoside; The oligonucleotides are selected from the group consisting of SEQ ID NOs: 20, 22-29, 31-32, 77-78, 81-82, 115, 117, 130, 132-134, 144-145, 147, 167-168, 210, 212-215, 290-293, 295-296, 299-305, 309, 351-359, 361-362, 365-366, 368, 407-409, 432, 437-442, 444, 459-460, 479, 482-493, 497-498, 500-501, 503-512, 543-550, 552-560, 562, 582-585, 590-591, 592-593, 594-595, 596-600, 598-601, 599-602, 599-603, 599-604, 599-605, 599-606, 599-607, 599-608, 591-609, 592-609, 593-601, 594-602, 595-603, 596-604, 597-605, 598-606, 599-607, 599-608, 599-609, 591-601, 592-603, 5 88-591, 603-604, 611, 613-616, 659, 661, 699-700, 702, 705-707, 724-725, 770-771, 812-816, 838-842, 845-852, 856, 883-885, 889, 893-897, 936, 94 0 to 941, 945, 948, 950, 955, 959 to 961, 965 to 968, 972 to 973, 999, 1007, 1016 to 1017, 1019, 1021 to 1022, 1036, 1040 to 1045, 1047, 1170, 1172 to 1173, 1211, 1216, 1222, 1235, 1240-1242, 1244-1249, 1251-1252, 1254-1259, 1268, 1316, 1318-1322, 1328-1329, 1373-1375, 1379-1383, 1386-1387, 1407-1408, 1433- 1435, 1450-1451, 1454-1461, 1476-1477, 1496-1499, 1532, 1538-1541, 1565-1566, 1579, 1581-1589, 1591, 1600-1607, 1610, 1625, 1627-1629, 1631- 1639, 1643, 1650-1660, 1663-1665, 1668-1675, 1713-1714, 1716-1722, 1724, 1727-1731, 1741, 1745-1747, 1751-1755, 1799-1801, 1859-1866, 1868- 1869, 1894-1896, 1905-1908, 1954, 1964-1966, 1969, 2066-2070, 2075-2079, 2108, 2138, 2143-2147, 2157-2160, 2193-2194, 2299-2300, 2312-2313,A composition comprising any one of the nucleic acid base sequences: 2385, 2388, 2390-2395, 2416-2418, 2460, 2462, or 2463.

3. 3. The oligonucleotide of claim 1 or the composition of claim 2, wherein the oligonucleotide comprises at least one alternative internucleoside linkage.

4. 4. The oligonucleotide or composition of claim 3, wherein said at least one alternative internucleoside linkage is a phosphorothioate internucleoside linkage.

5. 4. The oligonucleotide or composition of claim 3, wherein said at least one alternative internucleoside linkage is a 2'-alkoxy internucleoside linkage.

6. 4. The oligonucleotide or composition of claim 3, wherein said at least one alternative internucleoside linkage is an alkylphosphate internucleoside linkage.

7. 3. The oligonucleotide of claim 1 or the composition of claim 2, wherein the oligonucleotide comprises at least one alternative nucleobase.

8. The oligonucleotide or composition of claim 7, wherein the alternative nucleobase is 5'-methylcytosine, pseudouridine or 5-methoxyuridine.

9. 3. The oligonucleotide of claim 1 or the composition of claim 2, wherein the oligonucleotide comprises at least one alternative sugar moiety.

10. 10. The oligonucleotide or composition of claim 9, wherein the alternative sugar moiety is 2'-OMe or a bicyclic nucleic acid.

11. The oligonucleotide of claim 1 or the composition of claim 2, wherein the oligonucleotide further comprises a ligand conjugated to the 5' or 3' end of the oligonucleotide via a monovalent or branched divalent or trivalent linker.

12. 3. The oligonucleotide of claim 1 or the composition of claim 2, wherein the oligonucleotide comprises a region complementary to at least 17 consecutive nucleotides of the MSH3 gene.

13. 3. The oligonucleotide of claim 1 or the composition of claim 2, wherein the oligonucleotide comprises a region complementary to at least 19 consecutive nucleotides of the MSH3 gene.

14. 3. The oligonucleotide of claim 1 or the composition of claim 2, wherein the oligonucleotide comprises a region complementary to 19 consecutive nucleotides of the MSH3 gene.

15. 3. The oligonucleotide of claim 1 or the composition of claim 2, wherein the oligonucleotide comprises a region complementary to 20 consecutive nucleotides of the MSH3 gene.

16. A pharmaceutical composition comprising one or more of the oligonucleotides of claim 1 or the composition of claim 2, and a pharmaceutically acceptable carrier or excipient.

17. A composition comprising one or more of the oligonucleotides of claim 1 or the composition of claim 2, and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome.

18. 18. A composition comprising one or more of the oligonucleotides of claim 1, the composition of claim 2, the pharmaceutical composition of claim 16, or the composition of claim 17 for use in a method of inhibiting transcription of MSH3 in a cell, said method comprising contacting said cell with one or more of the oligonucleotides of claim 1, the composition of claim 2, the pharmaceutical composition of claim 16, or the composition of claim 17 for a time sufficient to result in degradation of mRNA transcripts of the MSH3 gene, thereby inhibiting expression of the MSH3 gene in said cell.

19. 18. A composition comprising one or more of the oligonucleotides of claim 1, the composition of claim 2, the pharmaceutical composition of claim 16, or the composition of claim 17 for treating, preventing, or delaying the progression of a trinucleotide repeat expansion disorder in a subject in need thereof.

20. 18. A composition comprising one or more of the oligonucleotides of claim 1, the composition of claim 2, the pharmaceutical composition of claim 16, or the composition of claim 17, for use in 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, said method comprising contacting said cell with one or more of the oligonucleotides of claim 1, the composition of claim 2, the pharmaceutical composition of claim 16, or the composition of claim 17.

21. 19. A composition comprising one or more of the oligonucleotides of claim 1, the composition of claim 2, the pharmaceutical composition of claim 16, or the composition of claim 17, for use in a method for inhibiting expression of the MSH3 gene in a cell, the method comprising contacting the cell with one or more of the oligonucleotides of claim 1, the composition of claim 2, the pharmaceutical composition of claim 16, or the composition of claim 17, and maintaining the cell for a time sufficient to obtain degradation of mRNA transcripts of the MSH3 gene, thereby inhibiting expression of the MSH3 gene in the cell.

22. 19. A composition comprising one or more of the oligonucleotides of claim 1, the composition of claim 2, the pharmaceutical composition of claim 16, or the composition of claim 17, for use in a method for reducing trinucleotide repeat expansions in a cell, the method comprising contacting the cell with one or more of the oligonucleotides of claim 1, the composition of claim 2, the pharmaceutical composition of claim 16, or the composition of claim 17.

23. The composition of claim 21 or 22, wherein the cell is in a subject.

24. 24. The composition of any one of claims 19, 20, and 23, wherein the subject is a human.

25. 22. The composition of any one of claims 18, 19, and 21, wherein the cell is a cell of the central nervous system or a muscle cell.

26. 26. The composition of any one of claims 19, 20, and 23-25, wherein the subject is identified as having a trinucleotide repeat expansion disorder.

27. The composition of any one of claims 19, 20, and 22 to 26, wherein the trinucleotide repeat expansion disorder is a polyglutamine disease.

28. 28. The composition of claim 27, wherein the polyglutamine disease is selected from the group consisting of dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-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.

29. The composition of any one of claims 19 to 26, wherein the trinucleotide repeat expansion disorder is a non-polyglutamine disease.

30. 30. The composition of claim 29, 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.

31. A composition comprising one or more oligonucleotides according to claim 1, the composition according to claim 2, the pharmaceutical composition according to claim 16 or the composition according to claim 17 for use in the prevention or treatment of a trinucleotide repeat expansion disorder.

32. 32. The composition of claim 31, wherein the trinucleotide repeat expansion disorder is selected from the group consisting of dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-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.

33. 33. The composition of claim 31 or 32, wherein the trinucleotide repeat expansion disorder is Huntington's disease.

34. 33. The composition of claim 31 or 32, wherein the trinucleotide repeat expansion disorder is Friedreich's ataxia.

35. 33. The composition of claim 31 or 32, wherein the trinucleotide repeat expansion disorder is myotonic dystrophy type 1.

36. The composition according to any one of claims 31 to 35, characterized in that the composition is administered intrathecally.

37. The composition according to any one of claims 31 to 35, wherein the composition is administered intracerebroventricularly.

38. The composition according to any one of claims 31 to 35, characterized in that the composition is administered intramuscularly.

39. 18. A composition comprising one or more of the oligonucleotides of claim 1, the composition of claim 2, the pharmaceutical composition of claim 16 or the composition of claim 17 for treating, preventing or delaying the progression of a disorder in a subject in need thereof, wherein the subject is suffering from a trinucleotide repeat expansion disorder.

40. 40. The composition of claim 39, wherein the composition is administered in combination with an additional therapeutic agent.

41. 41. The composition of claim 40, wherein the additional therapeutic agent is another oligonucleotide that hybridizes to mRNA encoding the huntingtin gene.

42. 18. A composition comprising one or more of the oligonucleotides of claim 1, the composition of claim 2, the pharmaceutical composition of claim 16, or the composition of claim 17, for preventing or delaying the progression of a trinucleotide repeat expansion disorder in a subject.

43. 43. The composition of claim 42, wherein the trinucleotide repeat expansion disorder is selected from the group consisting of dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-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.

44. 44. The composition of claim 42 or 43, wherein the trinucleotide repeat expansion disorder is Huntington's disease.

45. 44. The composition of claim 42 or 43, wherein the trinucleotide repeat expansion disorder is Friedreich's ataxia.

46. 41. The composition of claim 39 or 40, wherein the trinucleotide repeat expansion disorder is myotonic dystrophy type 1.

47. 41. The composition of claim 39 or 40, wherein the composition is administered in combination with an additional therapeutic agent.

48. 48. The composition of claim 47, wherein the additional therapeutic agent is an oligonucleotide that hybridizes to mRNA encoding the huntingtin gene.

49. 49. The composition of any one of claims 42-48, wherein the progression of the trinucleotide repeat expansion disorder is delayed from at least 120 days to at least 10 years or longer when compared to expected progression.

50. 18. A composition comprising one or more oligonucleotides according to claim 1, the composition according to claim 2, the pharmaceutical composition according to claim 16 or the composition according to claim 17 for use in preventing or delaying the progression of a trinucleotide repeat expansion disorder in a subject.

51. 51. The composition of claim 50, wherein the trinucleotide repeat expansion disorder is selected from the group consisting of dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-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.

52. 52. The composition of claim 50 or 51, wherein the trinucleotide repeat expansion disorder is Huntington's disease.

53. 52. The composition of claim 50 or 51, wherein the trinucleotide repeat expansion disorder is Friedreich's ataxia.

54. 52. The composition of claim 50 or 51, wherein the trinucleotide repeat expansion disorder is myotonic dystrophy type 1.

55. 55. The composition of any one of claims 50 to 54, wherein the progression of the trinucleotide repeat expansion disorder is delayed from at least 120 days to at least 10 years or longer when compared to expected progression.

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