Methods for the treatment of 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 a sequence listing The entire contents of a 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 into this specification in their entirety.

Background Art

[0002] Background Triplet repeat expansion disorders are genetic disorders caused by triplet repeat expansions. Triplet repeat expansion is a type of genetic mutation in which a nucleotide repeat in a particular gene or intron exceeds the normal stable threshold for that gene. Triplet repeats can give rise to truncated or toxic gene products, can impair RNA transcription, and / or can cause toxic effects by forming toxic mRNA transcripts.

[0003] Triplet 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 give rise to a series of glutamine residues known as polyglutamine tracts, type 2 disorders are caused by generally small heterogeneous expansions, and type 3 disorders such as fragile X syndrome are characterized by large repeat expansions generally located outside the protein-coding region of the gene. Triplet repeat expansion disorders are characterized by a wide variety of symptoms such as the progressive degeneration of nerve cells common to type 1 disorders.

[0004] Subjects having trinucleotide repeat expansion disorders or considered to be at risk of developing trinucleotide repeat expansion disorders have constitutive nucleotide expansions in genes related to the diseases (i.e., trinucleotide repeat expansions are present in the genes during embryogenesis). The constitutive trinucleotide repeat expansions can undergo expansion after embryogenesis (i.e., somatic trinucleotide repeat expansions). Both constitutive trinucleotide repeat expansions and somatic trinucleotide repeat expansions can be related to the presence of the disease, the age of onset of the disease, and / or the rate of progression of the disease. Summary of the Invention Means for Solving the Problems

[0005] Gist of the Invention The present disclosure features, for example, useful compositions and methods for treating trinucleotide repeat expansion disorders in subjects in need of treatment of trinucleotide repeat expansion disorders. In some embodiments, the compositions and methods described herein are useful in the treatment of disorders related to MSH3 activity.

[0006] Oligonucleotide Some aspects of the present disclosure relate to single-stranded oligonucleotides having a length of 10 to 30 linked nucleosides and comprising a region of at least 10 consecutive nucleobases having at least 80% complementarity to the MSH3 gene. In some aspects, the present disclosure provides a single-stranded oligonucleotide having a length of 10 to 30 linked nucleosides, wherein the oligonucleotide comprises: (a) a DNA core sequence comprising linked deoxyribonucleosides; (b) a 5' adjacent sequence comprising linked nucleosides; and (c) a 3' adjacent sequence comprising linked nucleosides; the DNA core comprises a region of at least 10 consecutive nucleobases having at least 80% complementarity to the MSH3 gene and is positioned between the 5' adjacent sequence and the 3' adjacent sequence; the 5' adjacent sequence and the 3' adjacent sequence each comprise at least 2 linked nucleosides; and at least one nucleoside of each adjacent sequence comprises an alternative nucleoside.

[0007] In some aspects, the present disclosure relates to a single-stranded oligonucleotide having a length of 10 to 30 linked nucleosides for inhibiting the expression of the human MSH3 gene in a cell and comprising a region of at least 10 consecutive nucleobases having at least 80% complementarity to the MSH3 gene. In some aspects, the present disclosure provides a single-stranded oligonucleotide having a length of 10 to 30 linked nucleosides for inhibiting the expression of the human MSH3 gene in a cell, wherein the oligonucleotide comprises: (a) a DNA core comprising linked deoxyribonucleosides; (b) a 5' adjacent sequence comprising linked nucleosides; and (c) a 3' adjacent sequence comprising linked nucleosides; the DNA core comprises a region of at least 10 consecutive nucleobases having at least 80% complementarity to the MSH3 gene and is positioned between the 5' adjacent sequence and the 3' adjacent sequence; the 5' adjacent sequence and the 3' adjacent sequence each comprise at least 2 linked nucleosides; and at least one nucleoside of each adjacent 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 complementary to the MSH3 gene corresponding to the sequence of reference mRNA NM_002439.4 at one or more positions among positions 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, 2262-2293, 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 or 4281-4319 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 positions among positions 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, 2304-2329, 2371-2410, 2433-2458, 2494-2521, 2539-2647, 2679-2713, 2727-2753, 2767-2920, 2933-3000, 3046-3072, 3132-3245, 3266-3303, 3397-3484, 3528-3575, 3591-3617, 3753-3792, 3901-3936, 4076-4101 or 4281-4319 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 positions among positions 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, 2265 - 2293, 2378 - 2410, 2433 - 2458, 2494 - 2521, 2539 - 2647, 2679 - 2712, 2727 - 2753, 2767 - 2919, 2934 - 3000, 3046 - 3071, 3144 - 3183, 3220 - 3245, 3397 - 3484, 3534 - 3575, 3591 - 3616, 3901 - 3931 or 4281 - 4306 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 positions among 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 positions among 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 one aspect, 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 positions among 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.

[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 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 to 560, 562, 582 to 585, 588 to 591, 603 to 604, 611, 613 to 616, 659, 661, 699 to 700, 702, 705 to 707, 724 to 725, 770 to 771, 812 to 816, 838 to 842, 845 to 852, 856, 883 to 885, 889, 893 to 897, 936, 940 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 to 1242, 1244 to 1249, 1251 to 1252, 1254 to 1259, 1268, 1316, 1318 to 1322, 1328 to 1329, 1373 to 1375, 1379 to 1383, 1386 to 1387, 1407 to 1408, 1433 to 1435, 1450 to 1451, 1454 to 1461, 1476 to 1477, 1496 to 1499, 1532, 1538 to 1541, 1565 to 1566, 1579, 1581 to 1589, 1591, 1600 to 1607, 1610, 1625, 1627 to 1629, 1631 to 1639, 1643, 1650 to 1660, 1663 to 1665, 1668 to 1675, 1713 to 1714, 1716 to 1722, 1724, 1727 to 1731, 1741, 1745 to 1747, 1751 to 1755, 1799 to 1801, 1859 to 1866, 1868 to 1869, 1894 to 1896, 1905 to 1908, 1954, 1964 to 1966, 1969, 2066 to 2070, 2075 to 2079, 2108,It contains any one of the nucleotide sequences of 2138, 2143 to 2147, 2157 to 2160, 2193 to 2194, 2299 to 2300, 2312 to 2313, 2385, 2388, 2390 to 2395, 2416 to 2418, 2460, 2462 or 2463. In some embodiments, the oligonucleotide has the sequences of SEQ ID NO: 20, 22, 25 to 29, 31 to 32, 81 to 82, 115, 130, 132 to 134, 144, 145, 147, 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 506, 508 to 512, 543 to 550, 552 to 560, 562, 582 to 585, 589 to 591, 603 to 604, 611, 613 to 616, 659, 661, 699 to 700, 702, 705 to 707, 724, 770 to 771, 812 to 816, 838 to 842, 845 to 852, 856, 883 to 885, 889, 893 to 897, 936, 940 to 941, 945, 948, 950, 955, 959 to 961, 965 to 968, 972 to 973, 1041 to 1045, 1047, 1170, 1172, 1216, 1222, 1235, 1241 to 1242, 1244 to 1249, 1251 to 1252, 1254 to 1259, 1268, 1316, 1318 to 1319, 1321 to 1322, 1328, 1373, 1379 to 1383, 1386 to 1387, 1408, 1433 to 1435, 1450 to 1451, 1454 to 1461, 1476 to 1477, 1496 to 1499, 1532, 1538 to 1541, 1565 to 1566, 1579, 1581 to 1582, 1584 to 1589, 1591, 1601 to 1607, 1610, 1625, 1627 to 1629, 1631 to 1638, 1650 to 1655, 1659, 1665, 1668 to 1675, 1713 to 1714, 1716 to 1722, 1727 to 1731, 1745, 1747, 1751 to 1755, 1799 to 1800, 1859, 1861 to 1862, 1865 to 1866, 1868 to 1869, 1895 to 1896, 1905 to 1908, 1954, 1694 to 1966, 2066 to 2070,It contains any one of the nucleotide sequences of 2075~2079, 2108, 2138, 2144~2146, 2158~2160, 2193~2194, 2299, 2300, 2313, 2385, 2388, 2390~2392, 2394~2395, 2418, 2460 or 2462~2463. In some embodiments, the oligonucleotide has the sequences of SEQ ID NO: 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, 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, 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, 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,It contains any one of the nucleic acid base sequences of 2390 or 2460. In some embodiments, the oligonucleotide contains any one of the nucleic acid base sequences of SEQ ID NO: 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 contains any one of the nucleic acid base sequences of SEQ ID NO: 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 contains any one of the nucleic acid base sequences of SEQ ID NO: 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 nucleobase sequence of the oligonucleotide consists of any one of SEQ ID NOs: 6 to 2545. In some embodiments, the oligonucleotide is SEQ ID NO: 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 to 560, 562, 582 to 585, 588 to 591, 603 to 604, 611, 613 to 616, 659, 661, 699 to 700, 702, 705 to 707, 724 to 725, 770 to 771, 812 to 816, 838 to 842, 845 to 852, 856, 883 to 885, 889, 893 to 897, 936, 940 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 to 1242, 1244 to 1249, 1251 to 1252, 1254 to 1259, 1268, 1316, 1318 to 1322, 1328 to 1329, 1373 to 1375, 1379 to 1383, 1386 to 1387, 1407 to 1408, 1433 to 1435, 1450 to 1451, 1454 to 1461, 1476 to 1477, 1496 to 1499, 1532, 1538 to 1541, 1565 to 1566, 1579, 1581 to 1589, 1591, 1600 to 1607, 1610, 1625, 1627 to 1629, 1631 to 1639, 1643, 1650 to 1660, 1663 to 1665, 1668 to 1675, 1713 to 1714, 1716 to 1722, 1724, 1727 to 1731, 1741, 1745 to 1747, 1751 to 1755, 1799 to 1801, 1859 to 1866, 1868 to 1869, 1894 to 1896, 1905 to 1908, 1954, 1964 to 1966, 1969, 2066 to 2070, 2075 to 2079, 2108,It consists of any one of the nucleotide sequences of 2138, 2143 to 2147, 2157 to 2160, 2193 to 2194, 2299 to 2300, 2312 to 2313, 2385, 2388, 2390 to 2395, 2416 to 2418, 2460, 2462 or 2463. In some embodiments, the oligonucleotide has the sequences of SEQ ID NO: 20, 22, 25 to 29, 31 to 32, 81 to 82, 115, 130, 132 to 134, 144, 145, 147, 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 506, 508 to 512, 543 to 550, 552 to 560, 562, 582 to 585, 589 to 591, 603 to 604, 611, 613 to 616, 659, 661, 699 to 700, 702, 705 to 707, 724, 770 to 771, 812 to 816, 838 to 842, 845 to 852, 856, 883 to 885, 889, 893 to 897, 936, 940 to 941, 945, 948, 950, 955, 959 to 961, 965 to 968, 972 to 973, 1041 to 1045, 1047, 1170, 1172, 1216, 1222, 1235, 1241 to 1242, 1244 to 1249, 1251 to 1252, 1254 to 1259, 1268, 1316, 1318 to 1319, 1321 to 1322, 1328, 1373, 1379 to 1383, 1386 to 1387, 1408, 1433 to 1435, 1450 to 1451, 1454 to 1461, 1476 to 1477, 1496 to 1499, 1532, 1538 to 1541, 1565 to 1566, 1579, 1581 to 1582, 1584 to 1589, 1591, 1601 to 1607, 1610, 1625, 1627 to 1629, 1631 to 1638, 1650 to 1655, 1659, 1665, 1668 to 1675, 1713 to 1714, 1716 to 1722, 1727 to 1731, 1745, 1747, 1751 to 1755, 1799 to 1800, 1859, 1861 to 1862, 1865 to 1866, 1868 to 1869, 1895 to 1896, 1905 to 1908, 1954, 1694 to 1966, 2066 to 2070,It consists of any one of the nucleotide sequences of 2075 - 2079, 2108, 2138, 2144 - 2146, 2158 - 2160, 2193 - 2194, 2299, 2300, 2313, 2385, 2388, 2390 - 2392, 2394 - 2395, 2418, 2460 or 2462 - 2463. In some embodiments, the oligonucleotide has the sequences of SEQ ID NO: 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, 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, 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, 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,It consists of any one of the nucleotide sequences of 2390 or 2460. In some embodiments, the oligonucleotide consists of any one of the nucleotide sequences of SEQ ID NO: 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 any one of the nucleotide sequences of SEQ ID NO: 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 nucleotide sequences of SEQ ID NO: 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.,

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

[0013] The cell assay can include the steps of transfecting mammalian cells, such as HEK293, NIH3T3, or HeLa, with the oligonucleotide using Lipofectamine 2000 (Invitrogen), and measuring the mRNA level as 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, the at least one alternative internucleoside linkage is a phosphorothioate internucleoside linkage. In some embodiments, the at least one alternative internucleoside linkage is a 2'-alkoxy internucleoside linkage. In some embodiments, the at least one alternative internucleoside linkage is an alkyl phosphate internucleoside linkage.

[0015] In some embodiments, the oligonucleotide comprises at least one alternative nucleobase. In 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. In 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 the oligonucleotide via a monovalent or branched divalent or trivalent linker.

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

[0019] Pharmaceutical Composition and Method of Treatment Using the Same In some embodiments, the present application relates to a pharmaceutical composition comprising one or more of the oligonucleotides described herein and a pharmaceutically acceptable carrier or excipient.

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

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

[0022] In some embodiments, 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, the method comprising contacting the cell with one or more of the oligonucleotides described herein, a pharmaceutical composition comprising one or more of the oligonucleotides described herein, or a composition comprising one or more of the oligonucleotides described herein and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles or liposomes for a time sufficient to effect degradation of the mRNA transcript of the MSH3 gene, thereby inhibiting the expression of the MSH3 gene in the cell.

[0023] In one aspect, the present application relates to a method of reducing the level and / or activity of MSH3 in cells of a subject identified as having a trinucleotide repeat expansion disorder, the method comprising contacting the cells with one or more of the oligonucleotides described herein, one or more pharmaceutical compositions of the oligonucleotides described herein, or a composition of one or more of the oligonucleotides described herein and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles or liposomes for a time sufficient to obtain degradation of the mRNA transcript of the MSH3 gene, thereby inhibiting the expression of the MSH3 gene in the cells.

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

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

[0026] In some aspects, the cell is in a subject. In some aspects, the subject is human. In some aspects, the cell is a central nervous system cell or a muscle cell.

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

[0028] In some aspects, the trinucleotide repeat expansion disorder is a non-polyglutamine disease. In some aspects, the non-polyglutamine disease is selected from the group consisting of fragile X syndrome, fragile X-associated tremor / ataxia syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy type 1, spinocerebellar ataxia type 8, spinocerebellar ataxia type 12, oculopharyngeal muscular dystrophy, fragile X-associated premature ovarian insufficiency, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy. In some aspects, the trinucleotide repeat expansion disorder is Friedreich's ataxia. In some aspects, the trinucleotide repeat expansion disorder is myotonic dystrophy type 1.

[0029] In some embodiments, the present application relates to one or more of the oligonucleotides described herein, one or more pharmaceutical compositions of one or more of the oligonucleotides described herein, or a composition 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 embodiments, one or more of the oligonucleotides described herein, one or more pharmaceutical compositions of one or more of the oligonucleotides described herein, or a composition 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, one or more pharmaceutical compositions of one or more of the oligonucleotides described herein, or a composition of one or more of the oligonucleotides described herein and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles or liposomes are administered intracerebroventricularly.

[0031] In some embodiments, one or more of the oligonucleotides described herein, one or more pharmaceutical compositions of one or more of the oligonucleotides described herein, or a composition of one or more of the oligonucleotides described herein and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles or liposomes are 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 has a trinucleotide repeat expansion disorder, and administering to the subject one or more of the oligonucleotides described herein, one or more pharmaceutical compositions of the oligonucleotides described herein, or a composition of one or more of the oligonucleotides described herein and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles or liposomes. 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 the mRNA encoding the huntingtin gene.

[0033] In some aspects, the method of treating, preventing or delaying the progression of a disorder in a subject delays the progression of the trinucleotide repeat expansion disorder 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 longer when compared to the predicted progression.

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

[0035] Definitions For the sake of simplicity, the meanings of some terms and phrases used in the specification, examples, and the appended claims are provided below. Unless otherwise indicated or implicitly indicated by the context, the following terms and phrases include the meanings provided below. Since the scope of the technology is limited only by the claims, the definitions are provided to assist in describing a particular embodiment 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 obvious discrepancy between the usage of a term in the art and the definition provided herein, the definition provided within this specification shall prevail.

[0036] In this application, unless otherwise apparent from the context: (i) the term "a" can be understood to mean "at least one"; (ii) the term "or" can be understood to mean "and / or"; (iii) the terms "including" and "comprising", whether presented alone or in conjunction with one or more additional components or steps, can be understood to encompass the recited components or steps.

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

[0038] The term "at least" before a number or series of numbers is understood to include the number adjacent to the term "at least", as well as all subsequent numbers and integers that may be logically included, as apparent from the context. For example, the number of nucleotides in a nucleic acid molecule should be an integer. For example, "at least 18 nucleotides of a 21 - nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" is present before a series of numbers or a range, "at least" is understood to modify each of the numbers in that series or range. "At least" is also not limited to integers (e.g., "at least" 5% includes 5.0%, 5.1%, 5.18% without considering significant digits).

[0039] As used herein, "less than" or "fewer than" is understood logically from the context to mean the value adjacent to the phrase and all values and integers from the theoretical lower limit or zero up to that value. For example, an oligonucleotide having "fewer than 3 mismatches with a target sequence" has 3, 2, 1, or 0 mismatches with the target sequence. When "less than" is present before a series of numbers or a range, "less than" is understood to modify each of the numbers in that series or range.

[0040] As used herein, the term "administer" refers to the administration of a composition (e.g., a compound or preparation containing a compound described herein) 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 defines the dosage and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, the delivery of two or more agents is simultaneous or concurrent, and these agents may be co-formulated. In some embodiments, two or more agents are not co-formulated and are administered in a sequential manner as part of the 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 effects of the two treatments can be partially additive, fully additive, or greater than additive (e.g., synergistic). The sequential or substantially concurrent administration of each therapeutic agent can be effected by any suitable route, including but not limited to the oral, intravenous, intramuscular, and direct absorption through mucosal tissues. The therapeutic agents can be administered by the same route or different routes. For example, one therapeutic agent in the combination can be administered by intravenous injection, while a further therapeutic agent in the combination can be administered orally.

[0042] As used herein, the term "MSH3" refers to MutS homolog 3, a DNA mismatch repair protein, having an amino acid sequence from any vertebrate or mammalian source including, but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey and guinea pig, unless otherwise specified. This term also refers to fragments and variants of native MSH3 that maintain at least one in vivo or in vitro activity of native MSH3. This 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 shown in NCBI reference NM_002439.4 or SEQ ID NO: 1. The term "MSH3" also refers to 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 shown in NCBI reference number NP_002430.3 or SEQ ID NO: 2, which is a natural variant of the wild-type MSH3 protein. The nucleic acid sequence of an exemplary Mus musculus (mouse) MSH3 gene is shown in NCBI reference number NM_010829.2 or SEQ ID NO: 3. The nucleic acid sequence of an exemplary Rattus norvegicus (rat) MSH3 gene is shown in NCBI reference number 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 naturally occurring DNA sequence variants of the MSH3 gene, e.g., specific polypeptides expressed in cells by single nucleotide polymorphisms in the MSH3 gene. A number of SNPs within the MSH3 gene have been identified and can be found, for example, in NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov / snp). Non-limiting examples of SNPs within the MSH3 gene can be found at NCBI dbSNP accession 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, rs1428030, rs1478834, rs1650694, rs1650737, rs1677626, rs1677658, rs1805355, rs2897298, rs3045983, rs3797897, rs4703819, 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 the mRNA that is the product of RNA processing of the primary transcript. In one aspect, the target portion of the sequence is at least long enough to function as a substrate for oligonucleotide-directed (e.g., antisense oligonucleotide (ASO)-directed) cleavage at or near that portion of the nucleotide sequence of the mRNA molecule formed during transcription of the MSH3 gene. The target sequence can be, for example, about 9 to 36 nucleotides in length, such as about 15 to 30 nucleotides in length. For example, the target sequence can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, or about 15 to 30 nucleotides, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 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 nucleotides in length. Intermediate ranges and lengths between the ranges and lengths listed above are also contemplated.

[0045] "G", "C", "A", "T", and "U" each generally denote naturally occurring nucleotides that contain guanine, cytosine, adenine, thymidine, and uracil, respectively, as bases. However, it is understood that the term "nucleotide" can refer to alternative nucleotides, or surrogate replacement moieties, as further detailed below. One of ordinary skill in the art is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of oligonucleotides containing nucleotides with such replacement moieties. For example, without limitation, a nucleotide that contains inosine as its base can base pair with a nucleotide that contains adenine, cytosine, or uracil. Thus, a nucleotide that contains uracil, guanine, or adenine can be replaced, for example, by a nucleotide that contains inosine in the nucleotide sequence of an oligonucleotide. In another example, adenine and cytosine at any position in an oligonucleotide can be replaced by guanine and uracil, respectively, to form G-U wobble base pairs with a target mRNA. Sequences that contain such replacement moieties are suitable for the compositions and methods featured herein.

[0046] The terms "nucleobase" and "base" include the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides that form hydrogen bonds in nucleic acid hybridization. The term "nucleobase" includes 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 the monomeric unit of an oligonucleotide or polynucleotide that has a nucleobase and a sugar moiety. Nucleosides can include naturally occurring nucleosides such as those described herein as well as nucleosides that are alternative nucleosides. 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 that has an alternative sugar or an alternative nucleobase, such as those described herein.

[0049] In some embodiments, the nucleobase moiety is modified by changing a purine or pyrimidine to an "alternative nucleobase" selected from modified purines or pyrimidines such as substituted purines or substituted pyrimidines such as isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazolo-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] The nucleobase moiety can be represented by a letter code for each corresponding nucleobase, e.g., A, T, G, C or U, where each letter can include alternative nucleobases of equivalent function. In some embodiments, for example, for gapmers, 5-methylcytosine LNA nucleosides can be used.

[0051] "Sugar" or "sugar moiety" includes naturally occurring sugars having a furanose ring. Sugar also includes "alternative sugars" defined as structures capable of replacing 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 an open system. Such structures include simple changes compared to the natural furanose ring, such as 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 by another ring system, such as a morpholino or hexitol ring system. Sugar moieties useful in the preparation of 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., when the ribose ring is replaced by 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 having an alternative sugar moiety, the heterocyclic nucleic acid base is generally maintained to allow hybridization.

[0052] "Nucleotide," as used herein, refers to the monomeric units of an oligonucleotide or polynucleotide, including nucleosides and internucleoside linkages. Internucleoside linkages can include phosphate linkages. Similarly, "linked nucleosides" can be linked by phosphate linkages. Many "alternative internucleoside linkages" are known in the art, including, but not limited to, phosphate, phosphorothioate, and boronophosphate linkages. Alternative nucleosides include bicyclic nucleosides (BNA) (e.g., locked nucleic acid (LNA) and constrained ethyl (cEt) nucleosides), peptide nucleosides (PNA), phosphotriesters, phosphorothionates, phosphoramidates, and other variants of the native nucleoside phosphate backbone, including those described herein.

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

[0054] As used herein, the terms "oligonucleotide" and "polynucleotide" are defined as molecules containing two or more covalently linked nucleosides, as generally understood by those skilled in the art. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are generally prepared in the laboratory by solid-phase chemical synthesis followed by purification. When referring to the sequence of an oligonucleotide, reference is made to the sequence or order of the nucleobase portions of the covalently linked nucleotides or nucleosides, or modifications thereof. Oligonucleotides can be artificial. For example, oligonucleotides can be chemically synthesized, purified or isolated. Oligonucleotides are also intended to include: (i) compounds in which one or more furanose moieties are replaced by any cyclic or acyclic structure that can be used as a point of attachment by a furanose derivative or by a covalent bond 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 a suitable linking moiety as in the case of formacetal or riboacetal linkages; and / or (iii) compounds in which one or more linked furanose-phosphodiester linkage moieties are replaced by any cyclic or acyclic structure that can be used as a point of attachment by a covalent bond for the base moiety. Oligonucleotides can contain one or more alternative nucleosides or nucleotides (including, for example, those described herein). It is also understood that oligonucleotides can include compositions that lack a sugar moiety or nucleobase but are still capable of forming a pairing or hybridizing with a target sequence.

[0055] "Oligonucleotide" refers to short polynucleotides (e.g., those 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 of which is composed of at least one monomer unit, i.e., a nucleotide or nucleoside in the case of an oligonucleotide. Chimeric oligonucleotides also include "gapmers".

[0057] The oligonucleotide can be of any length that allows for the specific degradation of a desired target RNA via the RNase H-mediated pathway, and can range from about 10 to 30 nucleoside lengths, for example, from about 15 to 30 nucleoside lengths or from about 18 to 20 nucleoside lengths, such as about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleoside lengths, such as from about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 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 nucleoside lengths. Intermediate ranges and lengths within the ranges and lengths listed above are also contemplated.

[0058] As used herein, the term "oligonucleotide comprising a nucleic acid sequence" refers to an oligonucleotide that contains a strand of nucleotides or nucleosides described by the sequence referred to using standard nucleotide nomenclature.

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

[0060] As used herein, the term "gapmer" refers to an oligonucleotide comprising a region (wing or flanking sequence) containing one or more affinity-enhancing alternative nucleosides flanking on the 5' and 3' sides a region (gap or DNA core) of an RNase H recruiting oligonucleotide. Various gapmer designs are described herein. A headmer and a tailmer are oligonucleotides capable of recruiting RNase H, where one of the flanks is missing, i.e., only one end of the oligonucleotide contains an affinity-enhancing alternative nucleoside. For a headmer, the 3' flanking sequence is missing (i.e., the 5' flanking sequence contains an affinity-enhancing alternative nucleoside), and for a tailmer, the 5' flanking sequence is missing (i.e., the 3' flanking sequence contains an affinity-enhancing alternative nucleoside). A "mixed flanking sequence gapmer" refers to a gapmer where the flanking sequence contains at least one alternative nucleoside, e.g., at least one DNA nucleoside or at least one 2'-substituted alternative nucleoside, e.g., 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), etc., 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') containing an alternative nucleoside and the other flanking sequence (3' or 5', respectively) contains a 2'-substituted alternative nucleoside(s).

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

[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 with an oligonucleotide or polynucleotide containing the second nucleotide sequence and form a double-stranded structure, as understood by one of ordinary skill in the art. Such conditions can be, for example, stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12 to 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 appropriate conditions that may be encountered inside an organism, can be used. One of ordinary skill in the art can determine the set of conditions most appropriate for testing the complementarity of two sequences according to the ultimate application of the hybridized nucleotides or nucleosides.

[0063] A "complementary" array, as used herein, can include, or can be entirely formed from, non-Watson-Crick base pairs and / or base pairs formed from non-natural and alternative nucleotides or nucleosides, so long as the above requirements are met with respect to their ability to hybridize. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobbles or Hoogstein base pairing. The complementary arrays between an oligonucleotide and a target sequence described herein include the base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide or nucleoside sequence to an oligonucleotide or polynucleotide comprising a second nucleotide or nucleoside sequence, over the entire length of one or both of the nucleotide or nucleoside sequences. Such arrays can be referred to herein as "perfectly complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences can be perfectly complementary or can form one or more, but generally 5, 4, 3, or 2 or fewer, mismatched base pairs upon hybridization, 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, for duplexes up to 30 base pairs in length. "Substantially complementary" can refer to a polynucleotide that is substantially complementary to a contiguous portion of a target mRNA (e.g., the mRNA encoding MSH3). For example, a polynucleotide is complementary to at least a portion of MSH3 mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding MSH3.

[0064] As used herein, the term "complementary region" 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 that interferes with the expression of an endogenous gene (e.g., MSH3). If the complementary region is not completely complementary to the target sequence, the mismatch can be in the internal or terminal region of the molecule. Generally, the most tolerated mismatches are within 5, 4, 3, or 2 nucleotides of the terminal region, e.g., the 5' end and / or the 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 expression of MSH3 or inhibits the expression of MSH3 in a cell or subject. The phrase "inhibits the expression of MSH3," as used herein, includes inhibition of the expression of any MSH3 gene (e.g., the mouse MSH3 gene, the rat MSH3 gene, the monkey MSH3 gene, or the human MSH3 gene, etc.) as well as variants or mutants of the MSH3 gene that encode the MSH3 protein. Thus, the MSH3 gene can be a wild-type MSH3 gene, a mutant MSH3 gene, or a transgenic MSH3 gene in the context of a genetically engineered cell, group of cells, or organism.

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

[0067] "Reducing the level of MSH3" means, for example, decreasing the level of MSH3 in a cell or subject 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 the MutSβ heterodimer containing MSH3" means altering the level of an activity associated with the MutSβ heterodimer or an associated downstream effect. The level of activity of the 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., ASO). The term "inhibit", as used herein, is used interchangeably with "reduce", "silence", "down-regulate", "suppress" and other similar terms and includes any level of inhibition.

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

[0071] Contacting cells in vitro can be carried out, for example, by incubating the cells with an oligonucleotide. Contacting cells in vivo can be carried out, for example, by injecting the oligonucleotide into or near the tissue in which the cells are located, or by injecting the oligonucleotide drug into another region, such as the bloodstream or subcutaneous space, such that the drug subsequently reaches the tissue in which the cells to be contacted are located. For example, the oligonucleotide can comprise and / or be coupled to a ligand, such as GalNAc3, that directs the oligonucleotide to the site of interest, such as the liver. Combinations of in vitro and in vivo methods of contact are also possible. For example, cells can be contacted with an oligonucleotide in vitro and subsequently transplanted into a subject.

[0072] In one aspect, contacting a cell with an oligonucleotide includes "introducing" or "delivering the oligonucleotide into the cell" by promoting or effecting uptake or absorption into the cell. Absorption or uptake of the ASO can occur via a passive diffusion process or an active cellular process, or by an adjunct or device. Introducing 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 systemically administered. In vitro introduction into cells includes methods known in the art such as electroporation and lipofection. Further approaches are described below in this specification and / or are known in the art.

[0073] As used herein, "lipid nanoparticle" or "LNP" is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, e.g., a nucleic acid molecule such as an oligonucleotide. LNP refers to stable nucleic acid-lipid particles. LNPs typically comprise a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particles (e.g., a PEG-lipid conjugate). LNPs are described, for example, in U.S. Patent Nos. 6,858,225; 6,815,432; 8,158,601; and 8,058,069, the entire contents of each 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, e.g., one bilayer or multiple bilayers. Liposomes include unilamellar and multilamellar vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains an oligonucleotide composition. The lipophilic material separates the aqueous interior from an aqueous exterior that may, in some instances, contain an oligonucleotide composition but typically does not. Liposomes also include "sterically stabilized" liposomes, which term, as used herein, refers to liposomes containing one or more special lipids that, when incorporated into a liposome, result in an enhanced circulation lifetime compared to liposomes lacking such special lipids.

[0075] "Micelle" is defined herein as a particular type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all hydrophobic portions of the molecule face inward while keeping the hydrophilic portion in contact with the surrounding aqueous phase. When the environment is hydrophobic, the reverse arrangement exists.

[0076] As used herein, the term "antisense" refers to a nucleic acid comprising an oligonucleotide or polynucleotide that is sufficiently complementary to all or a portion of a gene, primary transcript, or processed mRNA such as to interfere with the expression of an endogenous gene (e.g., MSH3). A "complementary" polynucleotide is one that is capable of base pairing according to the standard Watson-Crick complementarity rules. Specifically, a purine base pairs with a pyrimidine to form combinations of guanine paired with cytosine (G:C), and in the case of DNA, adenine paired with thymine (A:T) or in the case of RNA, adenine paired with uracil (A:U). 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 the level and / or activity of MSH3 (e.g., in a cell or subject) as described herein refer to an amount sufficient to produce a beneficial or desired result, including clinical results, when administered to a subject, including a human, and thus, the "effective amount" or its synonyms depend on the context in which it is applied. For example, in the context of treating a trinucleotide repeat expansion disorder, this is an amount of an agent that reduces the level and / or activity of MSH3 sufficient to achieve a treatment response as compared to the response obtained without administration of an agent that reduces the level and / or activity of MSH3. The amount of a given agent that reduces the level and / or activity of MSH3 as described herein corresponding to such an amount will vary depending on various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, and / or weight), or the host being treated, etc., but can nevertheless be routinely determined by one of ordinary 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 as compared to a control. As defined herein, a therapeutically effective amount of an agent that reduces the level and / or activity of MSH3 of the present disclosure can be readily determined by one of ordinary skill in the art by conventional methods known in the art. The dosage regimen can be adjusted to provide the optimal therapeutic response.

[0078] "Preventive effective amount", as used herein, is intended to include an amount of an oligonucleotide that, when administered to a subject having a trinucleotide repeat expansion disorder or a subject predisposed to having a trinucleotide repeat expansion disorder, is sufficient to prevent or alleviate a disease or one or more symptoms of the disease. Alleviating a disease includes slowing the progression of the disease or reducing the severity of a disease that develops later. The "preventive effective amount" can vary depending on the oligonucleotide, how the agent is administered, the degree of risk of the disease, and medical history, age, weight, family history, genetic constitution, the type of prior or concomitant treatment if any, and other individual characteristics of the patient being treated. The preventive effective amount can refer to, for example, an amount of an agent that reduces the level and / or activity of MSH3 (e.g., in a cell or subject) as described herein, or when administered to a subject including a human, when compared to the predicted onset, delays the onset of one or more of the trinucleotide repeat disorders described herein by at least 120 days, for example, by 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 longer.

[0079] A "therapeutically effective amount" or "preventive effective amount" also includes an amount of an oligonucleotide (administered either as 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 "complementary region" 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 that interferes with the expression of an endogenous gene (e.g., MSH3). If the complementary region is not completely complementary to the target sequence, the mismatch can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are within 5, 4, 3, or 2 nucleotides of the terminal regions, e.g., the 5' end 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 that reduces the level and / or activity of MSH3 (e.g., in a cell or subject) as described herein, or an amount sufficient to reduce trinucleotide repeat expansion of a particular gene (e.g., a gene associated with a trinucleotide repeat expansion disorder as described herein) when administered to a subject, including a human.

[0082] As used herein, the term "subject identified as having a trinucleotide repeat expansion disorder" refers to the identification of a subject having, or suspected of having, a trinucleotide repeat expansion disorder, or a molecular or pathological condition, disease, or state associated therewith, such as the identification of a trinucleotide repeat expansion disorder or its symptoms, or a subject who 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 in a subject that exceed the normal stable threshold for that gene or intron. Nucleotide repeats are common in the human genome and are usually not associated with disease. However, in some cases, the number of repeats can expand beyond the stable threshold and cause disease, and the severity of the 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" means detecting a protein, or mRNA encoding a protein, by methods known in the art, either directly or indirectly. "Determining directly" 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 defined herein). "Determining indirectly" refers to receiving a physical entity or value from another entity or source (e.g., a third-party laboratory that has directly obtained the physical entity or value). Methods for measuring protein levels generally include, but are not limited to, assays based on the properties of the protein, including 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, as well as enzyme activity, or interaction with other protein partners. Methods for measuring mRNA levels 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 the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps (DNA core sequences) as necessary to achieve the maximum percent sequence identity. Alignments 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 the art, for example, using publicly available computer software such as BLAST, BLAST-2 or Megalign software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared. For example, percent sequence identity values can be generated using the sequence comparison computer program BLAST. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to (or, this can be expressed as a given nucleic acid or amino acid sequence A having a particular percent sequence identity to) a given nucleic acid or amino acid sequence B is calculated as follows: 100 × (fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in the alignment of the programs 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, the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0086] "Level" means the level or activity of a protein, or of an mRNA encoding a protein (e.g., MSH3), which is compared to a reference as needed. The reference can be any useful reference as defined herein. A "decreased level" or "increased level" of a protein means a decrease or increase in the protein level compared to the reference (e.g., a decrease or increase of 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; a decrease or increase greater than about 10%, about 15%, about 20%, about 50%, about 75%, about 100% or about 200% compared to the reference; 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 greater than or equal to 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 greater). The level of a protein 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 a sample.

[0087] As used herein, the term "pharmaceutical composition" refers to a composition comprising a compound described herein formulated with a pharmaceutically acceptable excipient, which can be manufactured or sold under approval by a government regulatory agency as part of a therapeutic regimen for the treatment of diseases in mammals. The pharmaceutical composition can be formulated, for example, for oral administration in unit dosage form (e.g., tablets, capsules, caplets, gelcaps or syrups); for topical administration (e.g., as creams, gels, lotions or ointments); for intravenous administration (e.g., as a sterile solution without particulate plugs and in a solvent system suitable for intravenous use); for intrathecal injection; for intracerebroventricular injection; for parenchymal injection; or in any other pharmaceutically acceptable formulation.

[0088] "Pharmaceutically acceptable excipient", as used herein, refers to any component other than the compounds described herein that has the property of being substantially non-toxic and non-inflammatory in a patient (e.g., a vehicle capable of suspending or dissolving an active compound). Excipients can include, for example, anti-adhesives, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavorants, 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, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0089] As used herein, the term "pharmaceutically acceptable salt" means any pharmaceutically acceptable salt of any of the compounds described herein. For example, pharmaceutically acceptable salts of any of the compounds described herein are within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, and have a reasonable benefit / risk ratio commensurate with the condition being treated. 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. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base moiety with a suitable organic acid.

[0090] The compounds described herein may have ionizable groups so as to be prepared as pharmaceutically acceptable salts. These salts can be acid addition salts involving inorganic or organic acids, or the salts can be prepared from inorganic or organic bases in the case of the compounds described herein in acidic form. Frequently, the compounds are prepared or used as pharmaceutically acceptable salts that are prepared as addition products of pharmaceutically acceptable acids or bases. Methods for appropriate pharmaceutically acceptable acids and bases and for the preparation of appropriate salts are well known in the art. The salts can be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, 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 metal salts or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, and magnesium, as well as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine, including non-toxic ammonium, quaternary ammonium, and amine cations.

[0091] "Reference" means any useful reference used to compare protein or mRNA levels or activities. 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 prior sample taken from the same subject; a sample from a normal healthy subject, e.g., normal cells or normal tissue; a sample from a subject without a disease (e.g., cells or tissue); a sample from a subject diagnosed with a disease but not yet treated with the compounds described herein; a sample from a subject treated with the compounds described herein; or a sample of a purified protein at a known normal concentration (e.g., any of those described herein). A "reference standard or level" means a value or number derived from a reference sample. A "normal control value" is a predetermined value indicating a non-diseased 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 ("less than or equal to X") or a low threshold ("greater than or equal to 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. A normal reference standard or level can be a normal subject without a disease or disorder (e.g., a trinucleotide repeat expansion disorder); a value or number derived from a subject treated with the compounds 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, gender, disease stage and general health. A standard curve of the level of a purified protein within a normal reference range, e.g., any of those described herein, can be used as a reference.

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

[0093] As used herein, the terms "treat", "treated", and "treating" mean both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or blunt (reduce) an undesired physiological state, disorder, or disease, or to obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include alleviation of symptoms; reduction in the degree of a state, disorder, or disease; a stabilized (i.e., not worsening) state of a state, disorder, or disease; delay in onset, or slowing of progression of a state, disorder, or disease; remission, or amelioration (partial or complete), whether detectable or undetectable, of a state, disorder, or disease state; remission of at least one measurable physical parameter not necessarily distinguishable by the patient; or enhancement or improvement of a state, disorder, or disease, but are not limited thereto. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes extending survival as compared to expected survival in the absence of treatment.

[0094] As used herein, the terms "variant" and "derivative" are used interchangeably and refer to naturally occurring, synthetic, and semi-synthetic analogs of a compound, peptide, protein, or other substance described herein. A variant or derivative of a compound, peptide, protein, or other substance described herein can retain or improve the biological activity of the original material.

[0095] The details of one or more aspects are set forth in the following description. 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 having a linked nucleoside length of 10 to 30, comprising a region of at least 10 consecutive nucleobases having at least 80% complementarity to the MSH3 gene. (Item 2) The oligonucleotide is (a) A DNA core sequence comprising linked deoxyribonucleosides; (b) A 5' adjacent sequence comprising linked nucleosides; and (c) A 3' adjacent sequence comprising linked nucleosides and the DNA core comprises a region of at least 10 consecutive nucleobases having at least 80% complementarity to the MSH3 gene, positioned between the 5' adjacent sequence and the 3' adjacent sequence; the 5' adjacent sequence and the 3' adjacent sequence each comprise at least 2 linked nucleosides; and at least one nucleoside of each adjacent sequence comprises an alternative nucleoside. The oligonucleotide according to Item 1. (Item 3) A single-stranded oligonucleotide having a linked nucleoside length of 10 to 30 for inhibiting the expression of the human MSH3 gene in a cell, 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 comprising linked deoxyribonucleosides; (b) A 5' adjacent sequence comprising linked nucleosides; and (c) A 3' adjacent sequence comprising linked nucleosides and the DNA core comprises a region of at least 10 consecutive nucleobases having at least 80% complementarity to the MSH3 gene, positioned between the 5' adjacent sequence and the 3' adjacent sequence; the 5' adjacent sequence and the 3' adjacent sequence each comprise at least 2 linked nucleosides; and at least one nucleoside of each adjacent sequence comprises an alternative nucleoside. The oligonucleotide according to Item 3. (Item 5) The oligonucleotide according to any one of Items 1 to 4, wherein the region of at least 10 nucleobases has at least 90% complementarity to the MSH3 gene. (Item 6) The oligonucleotide according to any one of Items 1 to 5, wherein the region of at least 10 nucleobases has at least 95% complementarity to the MSH3 gene. (Item 7) The oligonucleotide according to any one of items 1 to 6, wherein said 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 positions among positions 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, 2304 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, 3132 - 3245, 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 of the MSH3 gene. (Item 8) The oligonucleotide according to any one of items 1 to 6, wherein said 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 positions among positions 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, 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 of the MSH3 gene. (Item ⑨) The oligonucleotide according to any one of items 1 to 6, wherein said 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 positions among positions 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, 2265 - 2293, 2378 - 2410, 2433 - 2458, 2494 - 2521, 2539 - 2647, 2679 - 2712, 2727 - 2753, 2767 - 2919, 2934 - 3000, 3046 - 3071, 3144 - 3183, 3220 - 3245, 3397 - 3484, 3534 - 3575, 3591 - 3616, 3901 - 3931 or 4281 - 4306 of said MSH3 gene. (Item 10) The oligonucleotide according to any one of items 1 to 6, wherein said 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 positions among 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 said MSH3 gene. (Item 11) The oligonucleotide according to any one of items 1 to 6, wherein said 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 positions among 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 said MSH3 gene. (Item 12) The oligonucleotide according to any one of items 1 to 6, wherein said 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 positions among positions 984 - 1021, 1467 - 1493, 1722 - 1747, 1767 - 1802, 1833 - 1861, 2385 - 2410, 2554 - 2581, 2816 - 2845, 2861 - 2920 or 3151 - 3183 of said MSH3 gene. (Item 13) The oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleobase sequences of SEQ ID NOs: 6 - 2545. (Item 14) Array numbers 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 - 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, 2385, 2388The oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleobase sequences of 2390~2395, 2416~2418, 2460, 2462 or 2463. (Item 15) comprising any one of the nucleotide sequences 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, 508-512, 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-?61, 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, 2075-2079, 2108, 2138, 2144-2146, 2158-2160, 2193-2194, 2299, 2300, 2313, 2385, 2388, 2390-2392, 2394-2395, 2418, 2460 or 2462-2463The oligonucleotide according to any one of items 1 to 6., (Item 16) The oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleotide sequences of SEQ ID NO: 20, 25 to 29, 32, 81 to 82, 130, 133 to 134, 144 to 145, 147, 210, 212 to 213, 215, 290 to 293, 295 to 296, 299 to 304, 309, 351, 352 to 359, 361 to 362, 365 to 366, 368, 407 to 409, 432, 437 to 442, 444, 460, 479, 482 to 486, 488 to 492, 497 to 498, 500 to 501, 503 to 506, 508 to 512, 544 to 550, 553 to 558, 560, 582 to 585, 589, 603 to 604, 611, 613 to 616, 659, 661, 699 to 700, 702, 705 to 707, 770 to 771, 812 to 816, 838 to 842, 845 to 851, 856, 883, 885, 889, 893, 895 to 897, 936, 940, 945, 961, 965 to 968, 972 to 973, 1041 to 1045, 1047, 1170, 1172, 1216, 1222, 1235, 1244, 1246 to 1249, 1251 to 1252, 1254 to 1255, 1257 to 1259, 1268, 1319, 1321 to 1322, 1380 to 1381, 1386 to 1387, 1408, 1433 to 1435, 1450 to 1451, 1454 to 1461, 1476 to 1477, 1496 to 1499, 1532, 1538 to 1540, 1565 to 1566, 1579, 1581 to 1582, 1584 to 1589, 1591, 1601 to 1604, 1606 to 1607, 1610, 1625, 1627 to 1629, 1631 to 1638, 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) An oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleic acid base sequences of SEQ ID NO: 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. (Item 18) An oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleic acid base sequences of SEQ ID NO: 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. (Item 19) An oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleic acid base sequences of SEQ ID NO: 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. (Item 20) An 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 NO: 6-2545. (Item 21) Array numbers 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 - 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, 2385, 2388The oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleotide sequences of 2390 to 2395, 2416 to 2418, 2460, 2462 or 2463. (Item 22) Comprising any one of the nucleotide sequences of SEQ ID NO: 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, 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, 2075 - 2079, 2108, 2138, 2144 - 2146, 2158 - 2160, 2193 - 2194, 2299, 2300, 2313, 2385, 2388, 2390 - 2392, 2394 - 2395, 2418, 2460 or 2462 - 2463The oligonucleotide according to any one of items 1 to 6. (Item 23) The oligonucleotide according to any one of items 1 to 6, which consists of any one of the nucleotide sequences of SEQ ID NO: 20, 25 to 29, 32, 81 to 82, 130, 133 to 134, 144 to 145, 147, 210, 212 to 213, 215, 290 to 293, 295 to 296, 299 to 304, 309, 351, 352 to 359, 361 to 362, 365 to 366, 368, 407 to 409, 432, 437 to 442, 444, 460, 479, 482 to 486, 488 to 492, 497 to 498, 500 to 501, 503 to 506, 508 to 512, 544 to 550, 553 to 558, 560, 582 to 585, 589, 603 to 604, 611, 613 to 616, 659, 661, 699 to 700, 702, 705 to 707, 770 to 771, 812 to 816, 838 to 842, 845 to 851, 856, 883, 885, 889, 893, 895 to 897, 936, 940, 945, 961, 965 to 968, 972 to 973, 1041 to 1045, 1047, 1170, 1172, 1216, 1222, 1235, 1244, 1246 to 1249, 1251 to 1252, 1254 to 1255, 1257 to 1259, 1268, 1319, 1321 to 1322, 1380 to 1381, 1386 to 1387, 1408, 1433 to 1435, 1450 to 1451, 1454 to 1461, 1476 to 1477, 1496 to 1499, 1532, 1538 to 1540, 1565 to 1566, 1579, 1581 to 1582, 1584 to 1589, 1591, 1601 to 1604, 1606 to 1607, 1610, 1625, 1627 to 1629, 1631 to 1638, 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 24) An oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleotide sequences of SEQ ID NO: 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 or 1631 to 1633. (Item 25) An oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleotide sequences of SEQ ID NO: 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 26) An oligonucleotide according to any one of items 1 to 6, comprising any one of the nucleotide sequences of SEQ ID NO: 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. (Item 27) An oligonucleotide according to any one of items 1 to 26, which shows at least 50% mRNA inhibition at an oligonucleotide concentration of 20 nM when determined using a cell assay and compared to control cells. (Item 28) An oligonucleotide according to any one of items 1 to 26, which shows at least 60% mRNA inhibition at an oligonucleotide concentration of 20 nM when determined using a cell assay and compared to control cells. (Item 29) When determined using a cell assay, an oligonucleotide according to any one of items 1 to 26, which shows at least 70% mRNA inhibition at an oligonucleotide concentration of 20 nM when compared with control cells. (Item 30) When determined using a cell assay, an oligonucleotide according to any one of items 1 to 26, which shows at least 85% mRNA inhibition at an oligonucleotide concentration of 20 nM when compared with control cells. (Item 31) When determined using a cell assay, an oligonucleotide according to any one of items 1 to 26, which shows at least 50% mRNA inhibition at 2 nM when compared with control cells. (Item 32) When determined using a cell assay, an oligonucleotide according to any one of items 1 to 26, which shows at least 60% mRNA inhibition at an oligonucleotide concentration of 2 nM when compared with control cells. (Item 33) When determined using a cell assay, an oligonucleotide according to any one of items 1 to 26, which shows at least 70% mRNA inhibition at an oligonucleotide concentration of 2 nM when compared with control cells. (Item 34) When determined using a cell assay, an oligonucleotide according to any one of items 1 to 26, which shows at least 85% mRNA inhibition at an oligonucleotide concentration of 2 nM when compared with control cells. (Item 35) An oligonucleotide according to any one of items 1 to 34, comprising at least one alternative internucleoside linkage. (Item 36) The oligonucleotide according to item 35, wherein the at least one alternative internucleoside linkage is a phosphorothioate internucleoside linkage. (Item 37) The oligonucleotide according to item 35, wherein the at least one alternative internucleoside linkage is a 2'-alkoxy internucleoside linkage. (Item 38) The oligonucleotide according to item 35, wherein the at least one alternative internucleoside linkage is an alkyl phosphate internucleoside linkage. (Item 39) An oligonucleotide according to any one of items 1 to 38, comprising at least one alternative nucleobase. (Item 40) The oligonucleotide according to item 39, wherein the substituted nucleobase is 5'-methylcytosine, pseudouridine or 5-methoxyuridine. (Item 41) The modified oligonucleotide according to any one of items 1 to 40, comprising at least one substituted sugar moiety. (Item 42) The modified oligonucleotide according to item 41, wherein the substituted sugar moiety is 2'-OMe or a bicyclic nucleic acid. (Item 43) The oligonucleotide according to any one of items 1 to 42, further comprising a ligand conjugated to the 5'-end or 3'-end of the oligonucleotide via a monovalent or branched divalent or trivalent linker. (Item 44) The oligonucleotide according to any one of items 1 to 43, comprising a region complementary to at least 17 consecutive nucleotides of the MSH3 gene. (Item 45) The oligonucleotide according to any one of items 1 to 43, comprising a region complementary to at least 19 consecutive nucleotides of the MSH3 gene. (Item 46) The oligonucleotide according to any one of items 1 to 43, comprising a region complementary to 19 to 23 consecutive nucleotides of the MSH3 gene. (Item 47) The oligonucleotide according to any one of items 1 to 43, comprising a region complementary to 19 consecutive nucleotides of the MSH3 gene. (Item 48) The oligonucleotide according to any one of items 1 to 43, comprising a region complementary to 20 consecutive nucleotides of the MSH3 gene. (Item 49) The oligonucleotide according to any one of items 1 to 43, which is about 15 to 25 nucleoside in length. (Item 50) The oligonucleotide according to any one of items 1 to 43, which is 20 nucleoside in length. (Item 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) A composition comprising one or more of the oligonucleotides according to any one of items 1 to 50 and lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles or liposomes. (Item 53) A method of inhibiting the transcription of MSH3 in a cell, the method comprising contacting the cell with one or more of the 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 a time sufficient to obtain degradation of the mRNA transcript of the MSH3 gene, thereby inhibiting the expression of the MSH3 gene in the cell. (Item 54) A method of treating, preventing or delaying the progression of a trinucleotide repeat expansion disorder in a subject in need thereof, the method comprising administering to the subject one or more of the oligonucleotides according to any one of items 1 to 50, the pharmaceutical composition according to item 51 or the composition according to item 52. (Item 55) A method of reducing the level and / or activity of MSH3 in a cell of a subject identified as having a trinucleotide repeat expansion disorder, the method comprising contacting the cell with one or more of the oligonucleotides according to any one of items 1 to 50, the pharmaceutical composition according to item 51 or the composition according to item 52. (Item 56) A method for inhibiting the expression of the MSH3 gene in a cell, the method comprising contacting the cell with one or more of the oligonucleotides according to any one of items 1 to 50, the pharmaceutical composition according to item 51 or the composition according to item 52, and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of the MSH3 gene, thereby inhibiting the expression of the MSH3 gene in the cell. (Item 57) A method of reducing trinucleotide repeat expansion in a cell, the method comprising contacting the cell with one or more of the oligonucleotides according to any one of items 1 to 50, the pharmaceutical composition according to item 51 or the composition according to item 52. (Item 58) The method according to item 56 or 57, wherein the cell is in a subject. (Item 59) The method according to any one of items 54, 55 and 58, wherein the subject is human. (Item 60) The method according to any one of items 54 to 58, wherein the cell is a cell of the central nervous system or a muscle cell. (Item 61) The method according to any one of items 54, 55, and 58 to 60, wherein the subject is identified as having a trinucleotide repeat expansion disorder. (Item 62) The method according to any one of items 54, 55, and 57 to 61, wherein the trinucleotide repeat expansion disorder is a polyglutamine disease. (Item 63) The method according to 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) The method according to any one of items 54 to 61, wherein the trinucleotide repeat expansion disorder is a non-polyglutamine disease. (Item 65) The method according to 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 insufficiency, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy. (Item 66) 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 trinucleotide repeat expansion disorders. (Item 67) 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 insufficiency, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy. (Item 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) The oligonucleotide, pharmaceutical composition or composition according to item 66 or 67, wherein the trinucleotide repeat expansion disorder is Friedreich's ataxia. (Item 70) 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) The oligonucleotide, pharmaceutical composition or composition according to any one of items 66 to 70, wherein the modified oligonucleotide, pharmaceutical composition or composition is administered into the subarachnoid space. (Item 72) The oligonucleotide, pharmaceutical composition or composition according to any one of items 66 to 70, wherein the modified oligonucleotide, pharmaceutical composition or composition is administered into the cerebral ventricle. (Item 73) The oligonucleotide, pharmaceutical composition or composition according to any one of items 66 to 70, which is administered intramuscularly. (Item 74) A method for treating, preventing or delaying the progression of a disorder in a subject in need thereof, comprising the steps of: the subject suffering from a trinucleotide repeat expansion disorder; and administering to the subject one or more of the oligonucleotides according to any one of items 1 to 50, the pharmaceutical composition according to item 51 or the composition according to item 52. (Item 75) The method according to item 74, further comprising the step of administering a further therapeutic agent. (Item 76) The method according to item 75, wherein the further therapeutic agent is another oligonucleotide that hybridizes to the mRNA encoding the huntingtin gene. (Item 77) A method for preventing or delaying the progression of a trinucleotide repeat expansion disorder in a subject, comprising the step of administering to the subject one or more of the oligonucleotides according to any one of items 1 to 50, the pharmaceutical composition according to item 51 or the composition according to item 52 in an amount effective to delay the progression of the trinucleotide repeat expansion disorder of the subject. (Item 78) The method according to 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 insufficiency, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy. (Item 79) The method according to item 77 or 78, wherein the trinucleotide repeat expansion disorder is Huntington's disease. (Item 80) The method according to item 77 or 78, wherein the trinucleotide repeat expansion disorder is Friedreich's ataxia. (Item 81) The method according to item 77 or 78, wherein the trinucleotide repeat expansion disorder is myotonic dystrophy type 1. (Item 82) The method according to item 77 or 78, further comprising the step of administering an additional therapeutic agent. (Item 83) The method according to item 82, wherein the additional therapeutic agent is an oligonucleotide that hybridizes to the mRNA encoding the huntingtin gene. (Item 84) The method according to any one of items 77 to 83, wherein the progression of the trinucleotide repeat expansion disorder is delayed by at least 120 days, for example, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years or longer when compared to the predicted progression. (Item 85) 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 preventing or delaying the progression of trinucleotide repeat expansion disorder in a subject. (Item 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 insufficiency, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy. (Item 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) The oligonucleotide, pharmaceutical composition or composition according to item 85 or 86, wherein the trinucleotide repeat expansion disorder is Friedreich's ataxia. (Item 89) 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) The oligonucleotide, pharmaceutical composition or composition according to any one of items 85 to 89, wherein the progression of the trinucleotide repeat expansion disorder is delayed by at least 120 days, for example, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years or longer when compared to the predicted progression.

Brief Description of the Drawings

[0096]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0097] Detailed Description The inventors have found that inhibition or depletion of MSH3 levels and / or activity in cells is effective in the treatment of trinucleotide repeat expansion disorders. Accordingly, useful compositions and methods for treating trinucleotide repeat expansion disorders in a subject in need thereof 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 such disorders, the number of repeats exceeds the normal stable threshold number of the gene and has expanded to a 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 trinucleotides other than CAG that can be in either coding or non-coding regions, such as the CGG repeat expansion underlying fragile X syndrome.

[0100] Trinucleotide repeat expansion disorders are dynamic in the sense that the number of repeats can vary between generations or even between cells within the same individual. Repeat expansion is thought to be caused by polymerase "slipping" during DNA replication. Tandem repeats in the DNA sequence can "loop out" while maintaining complementary base pairing between the parental and daughter strands. When a loop structure is formed from the daughter strand, the number of repeats increases.

[0101] Conversely, when a loop structure is formed from the parental strand, 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 worse disease severity. Thus, trinucleotide repeat expansion disorders are prone to "anticipation", which means worsening of symptom severity and / or onset age across generations of affected families due to 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 trinucleotide repeats of genes generally 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 a trinucleotide repeat expansion disorder can be increased or suppressed in a population with a trinucleotide repeat expansion disorder compared to a population lacking the trinucleotide repeat expansion disorder. Differences at the protein level can be evaluated using proteomic techniques including, but not limited to, Western blot, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, a protein associated with a trinucleotide repeat expansion disorder can be identified by obtaining a gene expression profile of the gene encoding the protein 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 of 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 the expansion of trinucleotide repeats. Recent genome-wide association (GWA) analyses have led to the identification of loci harboring genetic variants that modify the age of neurological onset in Huntington's disease (HD) (GEM-HD Consortium, Cell. 2015 Jul 30;162(3):516-26). This study identified MLH1, a human homolog of the E. coli DNA mismatch repair gene mutL. Subsequent GWA studies in polyglutamine disease patients have found significant associations between the age of onset, when all polyglutamine diseases (HD and SCA) are grouped together, and DNA repair genes as a group, as well as significant associations between specific SNPs in FAN1 and PMS2 and the disease (Bettencourt et al., (2016) Ann. Neurol., 79: 983-990). These results are consistent with those from an initial study comparing differences in repeat expansion in two different mouse models of Huntington's disease that identified Mlh1 and Mlh3 as novel and important 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 has been found to be reduced in transgenic mice lacking 8-oxoguanine glycosylase (OGG1), another member of the mismatch repair pathway, suggesting that OGG1 is also involved in expansion (Kovtun I. V. et al. (2007) Nature 447, 447-452). However, another study found that human subjects carrying the Ser326Cys polymorphism in hOGG1, which results in reduced OGG1 activity, generate increased mutant huntingtin (Coppede et al., (2009) Toxicol., 278: 199-203).Similarly, complete inactivation of Fan1, another component of the DNA repair pathway, in the mouse HD model results in somatic CAG expansions (Long et al. (2018) J. Hum Genet., 103: 1-9). MSH3, another component of the mismatch repair pathway, has been reported to be associated with 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 shown to be mediators of strain-specific differences in CTG·CAG repeat instability in mice (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 studies where shRNA knockdown of either MSH2 or MSH3, and ectopic expression of either MSH2 or MSH3, slowed down the 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 the 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 of the inconsistent results provided above, there is strong evidence that the MMR pathway plays some role in trinucleotide repeat expansions in various disorders. Furthermore, they were the first to recognize that inhibition of the MMR pathway offers treatment or prevention of these repeat expansion disorders; however, treatments that modulate MMR for the purpose of treating or preventing these repeat expansion disorders are not currently available or in development.

[0106] III. Oligonucleotide Agents Agents described herein that reduce the level and / or activity of MSH3 in a cell can be, for example, polynucleotides, such as oligonucleotides. These agents reduce the level of an activity associated with MSH3, or an effect downstream of the association, or 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, for example, a single-stranded oligonucleotide that acts via an RNase H-mediated pathway. Oligonucleotides include DNA and DNA / RNA chimeric molecules typically about 10 to 30 nucleotides in length that recognize a polynucleotide target sequence or sequence portion via hydrogen-bonding interactions with the nucleotide bases of the target sequence (e.g., MSH3). The oligonucleotide molecule can reduce the expression level of MSH3 (e.g., protein level or mRNA level). For example, oligonucleotides include oligonucleotides that target full-length MSH3. In some embodiments, the oligonucleotide molecule recruits an RNase H enzyme, resulting in target mRNA degradation.

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

[0109] In some embodiments, the oligonucleotide reduces the level and / or activity or function of MSH3. In some embodiments, the oligonucleotide inhibits the 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] The oligonucleotides include oligonucleotides having a complementary region (e.g., a contiguous nucleobase region) complementary to at least a portion of the mRNA formed in the expression of the MSH3 gene. The complementary region can be about 30 nucleotides in length or less (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19 or 18 nucleotides in length or less). Upon contact with cells expressing the MSH3 gene, the oligonucleotide can inhibit the expression of the MSH3 gene (e.g., human, primate, non-primate or avian MSH3 gene) by, for example, a method based on PCR or branched DNA (bDNA), or by a protein-based method, for example, immunofluorescence analysis using Western blotting or flow cytometry techniques, etc., by at least about 10% when assayed.

[0111] Similarly, the region of complementarity to the target sequence can have a linked nucleoside length between 10 and 30, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, or between 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22. Intermediate ranges and lengths between the ranges and lengths listed above are also contemplated.

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

[0113] Oligonucleotide compounds can be prepared using solution phase or solid phase organic synthesis or both. Organic synthesis provides the advantage that oligonucleotides containing non-natural or alternative nucleotides can be readily prepared. Single-stranded oligonucleotides can be prepared using solution phase or solid phase organic synthesis or both.

[0114] In one aspect, the oligonucleotide 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 at least 10 contiguous nucleotides of the MSH3 gene. In some aspects, the oligonucleotide comprises a sequence complementary to at least 17 contiguous nucleotides, 19 to 23 contiguous nucleotides, 19 contiguous nucleotides or 20 contiguous nucleotides of the MSH3 gene. The oligonucleotide sequence can be selected from the group of sequences provided in any one of SEQ ID NOs: 6 to 2545.

[0115] In one aspect, the array is substantially complementary to the sequence of the mRNA produced in the expression of the MSH3 gene. In some aspects, the region of at least 10 nucleobases is complementary to the MSH3 gene corresponding to the sequence of the reference mRNA NM_002439.4 at one or more positions among positions 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, 2262-2293, 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 of the MSH3 gene. In one aspect, the region of at least 10 nucleobases is complementary to the MSH3 gene corresponding to the sequence of the reference mRNA NM_002439.4 at one or more positions among positions 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, 2304-2329, 2371-2410, 2433-2458, 2494-2521, 2539-2647, 2679-2713, 2727-2753, 2767-2920, 2933-3000, 3046-3072, 3132-3245, 3266-3303, 3397-3484, 3528-3575, 3591-3617, 3753-3792, 3901-3936, 4076-4101 and 4281-4319 of the MSH3 gene.In one aspect, 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 positions among positions 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, 2265 - 2293, 2378 - 2410, 2433 - 2458, 2494 - 2521, 2539 - 2647, 2679 - 2712, 2727 - 2753, 2767 - 2919, 2934 - 3000, 3046 - 3071, 3144 - 3183, 3220 - 3245, 3397 - 3484, 3534 - 3575, 3591 - 3616, 3901 - 3931 and 4281 - 4306 of the MSH3 gene. In one aspect, 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 positions among 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 aspect, 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 positions among 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 aspect, 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 positions among 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, and 3151 to 3183 of the MSH3 gene.

[0116] In one aspect, the oligonucleotide comprises any one of the nucleic acid base sequences of SEQ ID NOs: 6 to 2545. In one aspect, the oligonucleotide is SEQ ID NO: 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 to 560, 562, 582 to 585, 588 to 591, 603 to 604, 611, 613 to 616, 659, 661, 699 to 700, 702, 705 to 707, 724 to 725, 770 to 771, 812 to 816, 838 to 842, 845 to 852, 856, 883 to 885, 889, 893 to 897, 936, 940 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 to 1242, 1244 to 1249, 1251 to 1252, 1254 to 1259, 1268, 1316, 1318 to 1322, 1328 to 1329, 1373 to 1375, 1379 to 1383, 1386 to 1387, 1407 to 1408, 1433 to 1435, 1450 to 1451, 1454 to 1461, 1476 to 1477, 1496 to 1499, 1532, 1538 to 1541, 1565 to 1566, 1579, 1581 to 1589, 1591, 1600 to 1607, 1610, 1625, 1627 to 1629, 1631 to 1639, 1643, 1650 to 1660, 1663 to 1665, 1668 to 1675, 1713 to 1714, 1716 to 1722, 1724, 1727 to 1731, 1741, 1745 to 1747, 1751 to 1755, 1799 to 1801, 1859 to 1866, 1868 to 1869, 1894 to 1896, 1905 to 1908, 1954, 1964 to 1966, 1969, 2066 to 2070, 2075 to 2079, 2108, 2138,It contains any one of the nucleotide sequences of 2143~2147, 2157~2160, 2193~2194, 2299~2300, 2312~2313, 2385, 2388, 2390~2395, 2416~2418, 2460, 2462 and 2463. In one aspect, the oligonucleotide has the sequences of SEQ ID NO: 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, 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, 1694~1966, 2066~2070,It contains any one of the nucleic acid base sequences of 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 aspect, the oligonucleotide has the sequences of SEQ ID NO: 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, 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, 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, 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,It contains any one of the nucleic acid base sequences of 2390 and 2460. In one aspect, the oligonucleotide contains any one of the nucleic acid base sequences of SEQ ID NO: 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 contains any one of the nucleic acid base sequences of SEQ ID NO: 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 aspect, the oligonucleotide contains any one of the nucleic acid base sequences of SEQ ID NO: 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 one aspect, the nucleobase sequence of the oligonucleotide consists of any one of SEQ ID NOs: 6 to 2545. In one aspect, the oligonucleotide is SEQ ID NO: 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 to 560, 562, 582 to 585, 588 to 591, 603 to 604, 611, 613 to 616, 659, 661, 699 to 700, 702, 705 to 707, 724 to 725, 770 to 771, 812 to 816, 838 to 842, 845 to 852, 856, 883 to 885, 889, 893 to 897, 936, 940 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 to 1242, 1244 to 1249, 1251 to 1252, 1254 to 1259, 1268, 1316, 1318 to 1322, 1328 to 1329, 1373 to 1375, 1379 to 1383, 1386 to 1387, 1407 to 1408, 1433 to 1435, 1450 to 1451, 1454 to 1461, 1476 to 1477, 1496 to 1499, 1532, 1538 to 1541, 1565 to 1566, 1579, 1581 to 1589, 1591, 1600 to 1607, 1610, 1625, 1627 to 1629, 1631 to 1639, 1643, 1650 to 1660, 1663 to 1665, 1668 to 1675, 1713 to 1714, 1716 to 1722, 1724, 1727 to 1731, 1741, 1745 to 1747, 1751 to 1755, 1799 to 1801, 1859 to 1866, 1868 to 1869, 1894 to 1896, 1905 to 1908, 1954, 1964 to 1966, 1969, 2066 to 2070, 2075 to 2079, 2108,It consists of any one of the nucleotide sequences of 2138, 2143 to 2147, 2157 to 2160, 2193 to 2194, 2299 to 2300, 2312 to 2313, 2385, 2388, 2390 to 2395, 2416 to 2418, 2460, 2462 and 2463. In one aspect, the oligonucleotide has the sequences of SEQ ID NO: 20, 22, 25 to 29, 31 to 32, 81 to 82, 115, 130, 132 to 134, 144, 145, 147, 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 506, 508 to 512, 543 to 550, 552 to 560, 562, 582 to 585, 589 to 591, 603 to 604, 611, 613 to 616, 659, 661, 699 to 700, 702, 705 to 707, 724, 770 to 771, 812 to 816, 838 to 842, 845 to 852, 856, 883 to 885, 889, 893 to 897, 936, 940 to 941, 945, 948, 950, 955, 959 to 961, 965 to 968, 972 to 973, 1041 to 1045, 1047, 1170, 1172, 1216, 1222, 1235, 1241 to 1242, 1244 to 1249, 1251 to 1252, 1254 to 1259, 1268, 1316, 1318 to 1319, 1321 to 1322, 1328, 1373, 1379 to 1383, 1386 to 1387, 1408, 1433 to 1435, 1450 to 1451, 1454 to 1461, 1476 to 1477, 1496 to 1499, 1532, 1538 to 1541, 1565 to 1566, 1579, 1581 to 1582, 1584 to 1589, 1591, 1601 to 1607, 1610, 1625, 1627 to 1629, 1631 to 1638, 1650 to 1655, 1659, 1665, 1668 to 1675, 1713 to 1714, 1716 to 1722, 1727 to 1731, 1745, 1747, 1751 to 1755, 1799 to 1800, 1859, 1861 to 1862, 1865 to 1866, 1868 to 1869, 1895 to 1896, 1905 to 1908, 1954, 1694 to 1966, 2066 to 2070,It consists of any one of the nucleotide sequences of 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 aspect, the oligonucleotide has the sequences of SEQ ID NO: 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, 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, 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, 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,It consists of any one of the nucleic acid base sequences of 2390 and 2460. In one aspect, the oligonucleotide consists of any one of the nucleic acid base sequences of SEQ ID NO: 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 NO: 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 aspect, the oligonucleotide consists of any one of the nucleic acid base sequences of SEQ ID NO: 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.,

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

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

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

[0121] The length of the 5' flanking sequence region can be at least 2 nucleoside lengths (e.g., at least 2, at least 3, at least 4, at least 5 or more nucleoside lengths). The length of the 3' flanking sequence region can be at least 2 nucleoside lengths (e.g., at least 2, at least 3, at least 4, at least 5 or more nucleoside lengths). 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 containing 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 adjacent to the DNA core sequence are alternative nucleosides, e.g., 2'-alternative nucleosides. The DNA core sequence comprises a continuous stretch of nucleotides capable of recruiting RNase H when the oligonucleotide is in a duplex with the MSH3 target nucleic acid. In some embodiments, the DNA core sequence comprises a continuous stretch of 5 to 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, the gapmer comprises a region complementary to at least 17 contiguous nucleotides, 19 to 23 contiguous nucleotides or 19 contiguous nucleotides of the MSH3 gene. The gapmer is complementary to the MSH3 target nucleic acid and can thus be a continuous nucleoside region of the oligonucleotide.

[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' flanking sequence and the 3' flanking sequence contain one MOE nucleoside. In some embodiments, all of the nucleosides in the flanking sequence are MOE nucleosides. In other embodiments, the flanking sequence may contain both MOE nucleosides and other nucleosides, such as DNA nucleosides and / or non-MOE alternative nucleosides, such as bicyclic nucleosides (BNA) (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 continuous sequence of at least 5 RNase H recruiting nucleosides (e.g., 5 to 16 DNA nucleosides) flanked by affinity-enhancing alternative nucleosides, such as MOE nucleosides, at the 5' and 3' ends.

[0124] In other embodiments, the 5' and / or 3' flanking sequences comprise, contain or consist of 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 BNA. In some embodiments, all nucleosides in the flanking sequences are BNA. In other embodiments, the flanking sequences can comprise both BNA 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 5 RNase H mobilizing nucleosides (e.g., 5 - 16 DNA nucleosides) flanked at the 5' and 3' ends by affinity enhancing alternative nucleosides, e.g., BNA, e.g., LNA, e.g., beta-D-oxy-LNA.

[0125] The 5' flank attached to the 5' end of the DNA core sequence comprises, contains or consists of at least one alternative sugar moiety (e.g., at least 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 - 7 alternative nucleobases, e.g., 2 - 6 alternative nucleobases, e.g., 2 - 5 alternative nucleobases, e.g., 2 - 4 alternative nucleobases, e.g., 1 - 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' adjacent sequence linked 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 adjacent 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 adjacent 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 certain embodiments, one or more or all of the alternative sugar moieties in the adjacent sequence are 2'-alternative sugar moieties.

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

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

[0130] In some embodiments, one or more alternative internucleoside linkages in the adjacent arrays 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 alkyl phosphate internucleoside linkages.

[0131] The DNA core sequence can comprise, contain or consist of at least 5 to 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 of the DNA core sequence are DNA, for example, at least 60%, at least 70% or at least 80% or at least 90% are DNA. In some embodiments, all of the nucleosides of the DNA core sequence are RNA units.

[0132] The oligonucleotide contains a contiguous region complementary to the target nucleic acid. In some embodiments, the oligonucleotide can further comprise additional linked nucleosides positioned 5' and / or 3' to either the 5' and 3' adjacent arrays. These additional linked nucleosides can each be linked to the 5' end of the 5' adjacent array or the 3' end of the 3' adjacent array. The additional nucleosides can, in some embodiments, form part of a contiguous sequence complementary to the target nucleic acid or, in other embodiments, can be non-complementary to the target nucleic acid.

[0133] Including additional nucleosides in either or both of the 5’ and 3’ flanking sequences may independently include 1, 2, 3, 4, or 5 additional nucleotides that may be complementary or non-complementary to the target nucleic acid. In this embodiment, the oligonucleotide may, in some embodiments, include a contiguous sequence capable of modulating a target to which additional nucleotides are adjacent at the 5’ and / or 3’ ends. Such additional nucleosides may function as nuclease-sensitive, biocleavable linkers and can thus 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 ease of synthesis.

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

[0135] In other embodiments, the oligonucleotide utilizes a “hemimer” design and includes a single 2’-modified flanking sequence adjacent to (either side of the 5’ or 3’ side of the DNA core sequence) 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 herein.

[0136] In some embodiments, the oligonucleotide has a nucleic acid sequence having 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 any one of the nucleic acid sequences of SEQ ID NOs: 6 - 2545. In some embodiments, the oligonucleotide has a nucleic acid sequence having at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 6 - 2545.

[0137] The sequences in SEQ ID NOs: 6 - 2545 are described as unmodified and / or unconjugated sequences, but it will be understood that the nucleosides of the oligonucleotide, e.g., the oligonucleotide, can include any one of the sequences shown in any one of SEQ ID NOs: 6 - 2545 that are alternative nucleosides and / or conjugated as described in detail below.

[0138] Those skilled in the art are well aware that oligonucleotides having a structure between about 18 and 20 base pairs can be particularly effective in inducing RNase H-mediated degradation. However, it can be understood that shorter or longer oligonucleotides can be effective. In the above aspect, due to the nature of the oligonucleotide sequences provided herein, the oligonucleotides described herein can include shorter or longer oligonucleotide sequences. It can be reasonably expected that only some linked nucleosides on one or both ends of the shorter oligonucleotide minus can be equally effective compared to the above oligonucleotide. Therefore, having a sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive linked nucleosides derived from one of the sequences provided herein, but inhibiting the expression of the MSH3 gene by about 5, 10, 15, 20, 25 or 30% or less from the oligonucleotide containing the full sequence, those oligonucleotides with different abilities are intended to be within the scope.

[0139] The oligonucleotides described herein can function through nuclease-mediated degradation of the target nucleic acid, where the oligonucleotide can recruit a nuclease, such as an endonuclease-like endoribonuclease (RNase) (e.g., RNase H). Examples of oligonucleotide designs that operate through a nuclease-mediated mechanism typically include regions of at least 5 or 6 DNA nucleosides, with affinity-enhancing alternative nucleosides adjacent to one or both sides, such as 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 having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers having phosphorothioate linkages between all monomers in the oligonucleotide, is used, and when using the methodology provided by Examples 91 - 95 of WO01 / 23613 (which is hereby incorporated by reference herein), if the oligonucleotide has an initial rate, measured in pmol / l / min, that is at least 5%, for example at least 10%, or greater than 20% of the initial rate determined when using the methodology provided by Examples 91 - 95 of WO01 / 23613 (which is hereby incorporated by reference herein), this oligonucleotide is considered capable of recruiting RNase H.

[0141] Furthermore, the oligonucleotides described herein identify sites (s) in the MSH3 transcript that are sensitive to RNase H - mediated cleavage. As used herein, an oligonucleotide is said to target within a specific site of an RNA transcript if the oligonucleotide promotes cleavage of the transcript at any location 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 to a selected sequence in the MSH3 gene.

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

[0143] Several types of systematic tests designed for the optimization of inhibitory oligonucleotide sequences can be performed according to the teachings provided herein. Considerations in designing interfering oligonucleotides include, but are not limited to, biophysical, thermodynamic and structural considerations, base preferences at specific positions, and homology. The production and use of inhibitory therapeutic agents based on non-coding oligonucleotides are also known in the art.

[0144] The various software packages and guidelines presented herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can be taken where a "window" or "mask" of a given size (e.g., 21 nucleotides as a non-limiting example) exists literally or metaphorically (including, e.g., in silico) on the target RNA sequence to identify sequences within the size range that can function as target sequences. Subsequent potential target sequences can be identified by progressively moving the sequence "window" one nucleotide upstream or downstream of the initial target sequence position until the complete set of possible sequences for any given selected target size is identified. This process, coupled with the systematic synthesis and testing of the identified sequences (using assays described herein or known in the art), can identify the RNA sequence that mediates the best inhibition of target gene expression when targeted with an oligonucleotide agent. Accordingly, while the sequences identified herein represent effective target sequences, it is contemplated that further optimization of inhibitory efficiency can be achieved by progressively "walking the window" one nucleotide upstream or downstream of a given sequence to identify sequences having equal or better inhibitory characteristics.

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

[0146] Furthermore, such optimized arrays can be adjusted by introduction of alternative nucleosides, alternative sugar moieties and / or alternative internucleoside linkages described herein or known in the art, for further optimizing the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes). The oligonucleotide agents described herein may contain one or more mismatches with the target sequence. In one aspect, the oligonucleotides described herein contain three or fewer mismatches. When an oligonucleotide contains a mismatch with the target sequence, in some aspects, the region of the mismatch is not located in the middle of the region of complementarity. When an oligonucleotide contains a mismatch with the target sequence, in some aspects, the mismatch should be restricted to within the last 5 nucleotides from either the 5' or 3' end of the region of complementarity. For example, for an oligonucleotide agent of 30 linked nucleosides, the region of contiguous nucleobases complementary to a region of the MSH3 gene generally contains no mismatches within the central 5 to 10 linked nucleosides. The methods described herein or known in the art can be used to determine whether an oligonucleotide containing a mismatch with the target sequence is effective in inhibiting the expression of the MSH3 gene. Consideration of the effectiveness of an oligonucleotide with a mismatch in inhibiting the expression of the MSH3 gene is particularly important when it is known that a particular region of complementarity in the MSH3 gene has polymorphic sequence variant forms within the population.

[0147] The construction of vectors for the expression of polynucleotides can be achieved using conventional techniques that do not require detailed explanation for those skilled in the art. For the generation of efficient expression vectors, it is necessary to have regulatory sequences that control the expression of the polynucleotide. These regulatory sequences include promoter and enhancer sequences, which are affected by specific cytokines that interact with these sequences and are well known in the art.

[0148] A. Alternative oligonucleosides In one aspect, 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 aspect, 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, certain modifications are thought to be able to increase nuclease resistance and / or serum stability or decrease immunogenicity. For example, the oligonucleotide can include nucleotides (e.g., adenine, thymidine, guanosine, cytidine, uridine, or inosine) found to occur naturally in DNA or RNA, or can include alternative nucleosides or internucleoside linkages having one or more chemical modifications to one or more components of the nucleotide (e.g., nucleobase, sugar, or phospho-linker moiety). The oligonucleotides can be linked to each other via naturally occurring phosphodiester bonds or can include alternative linkages (e.g., phosphorothioate (e.g., Sp phosphorothioate or Rp phosphorothioate), 3'-methylene phosphonate, 5'-methylene phosphonate, 3'-phosphoroamidate, 2'-5' phosphodiester, guanidinium, S-methylthiourea, 2'-alkoxy, alkyl phosphate, or covalently linked via a peptide bond).

[0149] In some embodiments, substantially all of the nucleosides or internucleoside linkages of the oligonucleotide are alternative nucleosides. In other embodiments, all of the nucleosides or internucleoside linkages of the oligonucleotide are alternative nucleosides. An oligonucleotide in which "substantially all of the nucleosides are alternative nucleosides" is mostly but not completely modified and may contain 5, 4, 3, 2, or 1 or fewer naturally occurring nucleosides. In still other embodiments, the oligonucleotide may contain 5, 4, 3, 2, or 1 or fewer alternative nucleosides.

[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, S. L. et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated by reference. Alternative nucleotides and nucleosides include, for example, terminal modifications such as 5′-terminal modifications (phosphorylation, conjugation, inverse ligation) or 3′-terminal modifications (conjugation, DNA nucleotides, inverse ligation, etc.); base modifications such as replacement of a base with a base that base pairs with an extended repertoire of partners, a stabilized base, a destabilized base, or removal of a base (abasic nucleotide), or a conjugated base; sugar modifications (e.g., at the 2′ or 4′ position) or replacement of the sugar; and / or backbone modifications including modifications or replacements of the phosphodiester linkage. Nucleobases can be isonucleosides in which the nucleobase is moved to a different position (e.g., C2, C3, C4, or C5) from the C1 position of the sugar moiety. Specific examples of oligonucleotide compounds useful in the embodiments described herein include, but are not limited to, alternative nucleosides that include a modified backbone or do not include native internucleoside linkages. Nucleotides and nucleosides with modified backbones include, inter alia, those having no phosphorus atoms in the backbone. For the purposes of this specification and as sometimes referred to in the art, alternative RNAs having no phosphorus atoms in their internucleoside backbones can be considered oligonucleosides. In some embodiments, the oligonucleotide has a phosphorus atom in its internucleoside backbone.

[0151] Alternative internucleoside linkages include, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'-aminophosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, and boranophosphate having the normal 3'-5' linkage, their 2'-5' linked analogs, and those having the reverse polarity with adjacent pairs of nucleoside units linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts and free acid forms.

[0152] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Reissue Patent No. 39464, the entire contents of each of which are hereby incorporated by reference herein.

[0153] Alternative internucleoside linkages that do not contain phosphorus atoms therein have a backbone 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 nucleoside linkages. These include those having a morpholino linkage (partially formed from the sugar moiety of the nucleoside); a siloxane backbone; 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 having mixed N, O, S and CH2 component parts.

[0154] Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are hereby incorporated by reference herein.

[0155] In other embodiments, suitable oligonucleotides include oligonucleotides in which both the sugar of the nucleotide unit and the internucleoside linkage, i.e., the backbone, are replaced. The base units are maintained for hybridization to a suitable nucleic acid target compound. One such oligomeric compound, a mimic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar of the nucleoside is replaced by an amide-containing backbone, specifically, an aminoethylglycine backbone. The nucleobases are retained and are attached directly or indirectly to the azanitrogen atoms of the amide portions of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated 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] One aspect includes oligonucleotides having a phosphorothioate backbone, as well as oligonucleotides having a heteroatom backbone, in particular, -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [also known as methylene(methylimino) or MMI backbone] of U.S. Patent No. 5,489,677 mentioned above, -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2- and -N(CH3)-CH2-CH2- [wherein the native phosphodiester backbone is shown as -O-P-O-CH2-] and oligonucleotides having an amide backbone of U.S. Patent No. 5,602,240 mentioned above. In some aspects, the oligonucleotides characterized herein have the morpholino backbone structure of U.S. Patent No. 5,034,506 mentioned above. In other aspects, the oligonucleotides described herein include phosphorodiamidate morpholino oligomers (PMOs), where, as described in Summerton, et al., Antisense Nucleic Acid Drug Dev. 1997, 7:63-70, the deoxyribose moiety is replaced by a morpholine ring and the charged phosphodiester subunit linkage is replaced by an uncharged phosphorodiamidate linkage.

[0157] Alternative nucleosides and nucleotides can include one or more substituted sugar moieties. Oligonucleotides, such as those characterized herein, can include at the 2'-position one of the following: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl are substituted or unsubstituted C1-C 10 alkyl or C2-C 10 alkenyl and alkynyl can be. Exemplary suitable modifications include -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n -NH2, -O(CH2)n CH3, -O(CH2) n -ONH2 and -O(CH2) n -ON[(CH2) n CH3]2 is included, where n and m are from 1 to about 10. In other embodiments, the oligonucleotide contains, at the 2'-position, one of the following: C1-C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavage group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of the oligonucleotide, or a group for improving the pharmacodynamic properties of the oligonucleotide, and other substituents having similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O-CH2CH2OCH3) (Martin et al., Helv. Chin. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group is included. MOE nucleosides confer to the oligonucleotide several beneficial properties 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 is the 2'-dimethylaminooxyethoxy, i.e., the -O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, described in the following examples herein, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethyl or 2'-DMAEOE), i.e., 2'-O-(CH2)2-O-(CH2)2-N(CH3)2. Further exemplary alternatives include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers in these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0159] Other alternatives include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can be made at other positions on the nucleosides and nucleotides of the oligonucleotide, particularly at the 3' position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked oligonucleotide, and at the 5' position of the 5'-terminal nucleotide. The oligonucleotide can have a sugar mimic such as a cyclobutyl moiety instead of a pentofuranosyl sugar. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, of which certain ones are related to the present application by co-ownership. The entire contents of each of the above are hereby incorporated herein by reference.

[0160] Oligonucleotides can contain nucleobase (often simply referred to as "base" in the art) alternatives (e.g., modifications or substitutions). Unmodified or natural nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Alternative nucleobases include other synthetic and natural nucleobases, such as 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, pyrrolocytidine, dideoxycytidine, uridine, 5-methoxyuridine, 5-hydroxydeoxyuridine, dihydrouridine, 4-thiourdine, pseudouridine, 1-methyl-pseudouridine, deoxyuridine, 5-hydroxybutynl-2'-deoxyuridine, xanthine, hypoxanthine, 7-deaza-xanthine, thienoguanine, 8-aza-7-deazaguanosine, 7-methylguanosine, 7-deazaguanosine, 6-aminomethyl-7-deazaguanosine, 8-aminoguanine, 2,2,7-trimethylguanosine, 8-methyladenine, 8-azidoadenine, 7-methyladenine, 7-deazaadenine, 3-deazaadenine, 2,6-diaminopurine, 2-aminopurine, 7-deaza-8-aza-adenine, 8-amino-adenine, thymine, dideoxythymine, 5-nitroindole, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouridine, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluridine and cytidine, 6-azouridine, 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. Patent No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, 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, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of oligonucleotides. These include 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, 5-substituted pyrimidines, 6-azapyrimidines, as well as N-2, N-6 and 0-6 substituted purines. 5-Methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6 to 1.2 °C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and even more particularly, when combined with 2'-O-methoxyethyl sugar modifications, is an exemplary base substitution.

[0161] Representative U.S. patents that teach the preparation of specific ones of the foregoing alternative nucleobases as well as other alternative nucleobases include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

[0162] In other embodiments, the sugar moiety in the nucleotide can be a ribose molecule having, as necessary, a 2'-O-methyl, 2'-O-MOE, 2'-F, 2'-amino, 2'-O-propyl, 2'-aminopropyl or 2'-OH modification.

[0163] An oligonucleotide can include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a bridge of two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes 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 the 2'-carbon of the sugar ring. Thus, in some embodiments, an oligonucleotide can include one or more locked nucleic acids. A locked nucleic acid is a nucleoside having a modified ribose moiety that includes an extra bridge connecting the 2' and 4' carbons of the ribose moiety. In other words, a locked nucleic acid is a nucleoside that includes a bicyclic sugar moiety including a 4'-CH2-O-2' bridge. This structure efficiently "locks" the ribose in the structural conformation of the 3' end. The addition of locked nucleic acids to oligonucleotides has been shown to increase oligonucleotide stability in serum and to 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 include a bridge between the 4' ribosyl ring atom and the 2' ribosyl ring atom. In some embodiments, a polynucleotide agent includes one or more bicyclic nucleosides that include a 4' to 2' bridge.Examples of such bicyclic nucleosides cross-linked from the 4' to the 2' position 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(CH2OCH3)-O-2' (and its analogs; see, for example, U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and its analogs; see, for example, U.S. Patent No. 8,278,283); 4'-CH2-N(OCH3)-2' (and its analogs; see, for example, U.S. Patent No. 8,278,425); 4'-CH2-O-N(CH3)2-2' (see, for example, U.S. Patent Application Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2', wherein R is H, C1-C. 12 alkyl or a protecting group (see, for example, U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, for example, Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and its analogs; see, for example, U.S. Patent No. 8,278,426), but are not limited thereto. The entire content of each of the above is hereby incorporated herein by reference.

[0164] Further representative U.S. patents and U.S. patent application publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to: U.S. Patent No. 6,268,490; No. 6,525,191; No. 6,670,461; No. 6,770,748; No. 6,794,499; No. 6,998,484; No. 7,053,207; 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, the entire contents of each of which are hereby incorporated by reference herein.

[0165] For example, any of the above bicyclic nucleosides having one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose can be prepared (see WO99 / 14226).

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

[0167] An oligonucleotide can include one or more “conformationally restricted nucleosides” (“CRNs”). A CRN is a nucleoside analog having a linker connecting the C2’ and C4’ carbons of the ribose or the C3 and -C5’ carbons of the ribose. The CRN locks the ribose ring into a stable conformation and increases the hybridization affinity for mRNA. The linker is of sufficient length to place oxygen in an optimal position for stability and affinity and results in less ribose ring packing.

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

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

[0170] Representative U.S. publications teaching 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] Ribose molecules can be modified with cyclopropane rings to produce tricyclodeoxy nucleic acids (tricyclo DNA). The ribose moiety can be replaced with another sugar, such as 1,5-anhydrohexitol, threose for producing threose nucleosides (TNA), or arabinose for producing arabinonucleosides. Ribose molecules can be replaced with non-sugars, such as cyclohexene for producing cyclohexene nucleosides, or glycol for producing glycol nucleosides.

[0172] Potentially stabilizing modifications to the termini of nucleoside molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3''-phosphate, reverse base dT (idT), and the like. The disclosure of this modification can be found in PCT Application Publication No. WO2011 / 005861.

[0173] Other alternative chemistries of 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 by reference herein.

[0174] Exemplary oligonucleotides include nucleosides having alternative sugar moieties and may include DNA or RNA nucleosides. In some embodiments, the oligonucleotide includes nucleosides having alternative sugar moieties and DNA nucleosides. Incorporation of alternative nucleosides into the oligonucleotide can enhance the affinity of the oligonucleotide for the target nucleic acid. In that case, the alternative nucleoside can be referred to as an affinity-enhancing alternative nucleotide.

[0175] In some embodiments, the oligonucleotide includes at least one alternative nucleoside, for example, 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 includes from 1 to 10 alternative nucleosides, for example, from 2 to 9 alternative nucleosides, for example, from 3 to 8 alternative nucleosides, for example, from 4 to 7 alternative nucleosides, for example, 6 or 7 alternative nucleosides. In one embodiment, the oligonucleotide can include 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 includes one or more nucleosides having alternative sugar moieties, for example, 2'-sugar substituted nucleosides. In some embodiments, the oligonucleotide includes one or more 2'-sugar substituted nucleosides 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., LNA), e.g., at least 2, e.g., at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 of the alternative nucleosides are BNA. In still further embodiments, all of the alternative nucleosides are BNA.

[0177] In further embodiments, the oligonucleotide comprises at least one alternative internucleoside linkage. In some embodiments, the internucleoside linkage within a contiguous nucleotide sequence is a phosphorothioate or boranophosphate internucleoside linkage. In some embodiments, all of the internucleoside linkages in a contiguous 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 these 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 2'-sugar modified nucleosides and DNA units. In some embodiments, the oligonucleotide or a contiguous nucleotide region thereof is a gapmer oligonucleotide.

[0179] B. Oligonucleotides Conjugated to LigandsAn oligonucleotide can 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 such as 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 such as beryl-S-tritylthiol (Manoharan et al., (1992) Ann. N.Y. Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), thiocolesterol (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), polyamines 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), the palmitoyl moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237), or the octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937), but are not limited thereto.

[0180] In one aspect, the ligand modifies the distribution, targeting or lifespan of the incorporated oligonucleotide agent. In some aspects, the ligand provides, for example, an enhanced affinity for a selected target, such as a molecule, cell or cell type, compartment, such as a cellular or organ compartment, tissue, organ or region of the body, as 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 globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or lipids. Ligands can 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 polyamino acids, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) 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, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salts of polyamines, or alpha helix peptide.

[0182] The ligand may contain a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid or protein, such as an antibody that binds to a specific cell type, such as kidney cells. The targeting group may be thyrotropin, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine (gulucosamine), multivalent mannose, multivalent fucose, glycosylated polyamino acid, 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., psoralen, 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, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl) lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphates, aminos, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamines, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu3+ complex of tetraaza macrocycle), dinitrophenyl, HRP or AP.

[0184] A ligand can be a protein, such as a glycoprotein or a peptide, a molecule having specific affinity for a co-ligand, or an antibody that binds to a specific cell type such as a hepatocyte. Hormones and hormone receptors can be included in the ligands. These can include non-peptidic species such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose or polyvalent fucose.

[0185] A 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, such as by disrupting the microtubules, microfilaments and / or intermediate filaments of the cell. The drug can be, for example, a taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine or myoservin.

[0186] In some embodiments, the ligand attached to the oligonucleotide described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipid-soluble drugs, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glyceride, diacyl glyceride, phospholipid, sphingolipid, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins, and thus short oligonucleotides containing multiple phosphorothioate linkages in the backbone, such as oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, are also suitable as ligands (e.g., as PK modulating ligands). In addition, 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 (as described below) by use of oligonucleotides having pendant reactive functionality, such as those resulting from the attachment of a linking molecule to the oligonucleotide. This reactive oligonucleotide can be reacted directly with a commercially available ligand, a synthesized ligand having any of a variety of protecting groups, or a ligand to which a linking moiety is attached.

[0188] The oligonucleotides used in the conjugate can be readily and conventionally made via well-known techniques of solid-phase synthesis. Apparatus 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 phosphorothioate and alkylated derivatives.

[0189] In ligand-conjugated oligonucleotides, such as sequence-specifically linked nucleosides bearing a ligand molecule, the 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, the synthesis of the array-specific linked nucleosides 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 nucleosides are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to standard and non-standard phosphoramidites commonly used in commercial and oligonucleotide synthesis.

[0191] i. Lipid conjugate In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such lipids or lipid-based molecules can bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of the conjugate to target tissues, such as non-renal target tissues of the body. Lipids or lipid-based ligands can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into target cells or cell membranes, and / or (c) be used to modulate binding to serum proteins, such as HSA.

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

[0193] ii. Cell penetrating agent In another aspect, the ligand is a cell penetrating agent, e.g., a helical cell penetrating agent. In one aspect, the agent is amphiphilic. Exemplary agents are peptides, e.g., tat or antennopedia. If the agent is a peptide, it can be modified, including the use of peptidomimetics, invertomers, non-peptides or pseudo-peptide linkages, and D-amino acids. In one aspect, the helical agent is an alpha-helical agent that can have a lipophilic phase and a lipophobic phase.

[0194] The ligand can be a peptide or a peptidomimetic drug. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The conjugation of peptides and peptidomimetics to oligonucleotide agents can affect the pharmacokinetic distribution of the oligonucleotide, e.g., by enhancing cell recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, e.g., 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 amphiphilic peptide or a hydrophobic peptide (e.g., mainly composed of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, a constrained peptide or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having 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 that can cross the cell membrane and transport large polar molecules including peptides, oligonucleotides and proteins. For example, sequences derived from the HIV Tat protein (GRKKRRQRRRPPQ) and the Drosophila antennapedia protein (RQIKIWFQNRRMKWKK) have been found to be capable of functioning as delivery peptides. The peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage display library or a 1-bead 1-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of peptides or peptidomimetics tethered to an oligonucleotide agent via incorporated monomer units for cell targeting purposes are arginine-glycine-aspartic acid (RGD) peptides or RGD mimetics. The peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moiety can have structural modifications, for example, to increase stability or to direct conformational properties. Any of the structural modifications described below can be utilized.

[0196] RGD peptides 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, as well as synthetic RGD mimetics. In addition to RGD, other moieties that target integrin ligands can be used. Some conjugates of this ligand target PECAM-1 or VEGF.

[0197] Cell-penetrating peptides are capable of penetrating cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-permeable peptides can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensin, β-defensin or bactenecin), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can include a nuclear localization signal (NLS). For example, cell-penetrating peptides can be amphipathic peptides, 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 conjugate In some aspects of the compositions and methods described herein, the oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated oligonucleotides are advantageous for in vivo delivery of nucleic acids, as well as for compositions suitable for in vivo therapeutic use, as described herein. As used herein, "carbohydrate" is a compound consisting of itself of one or more monosaccharide units having at least 6 carbon atoms with oxygen, nitrogen or sulfur atoms bonded to each carbon atom (which may be linear, branched or cyclic); or a compound having as part of it a carbohydrate moiety (which may be linear, branched or cyclic) consisting of one or more monosaccharide units each having at least 6 carbon atoms with oxygen, nitrogen or sulfur atoms bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8 or 9 monosaccharide units), as well as polysaccharides such as starch, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include sugars having C5 and above (e.g., C5, C6, C7 or C8); disaccharides and trisaccharides include sugars having 2 or 3 monosaccharide units (e.g., C5, C6, C7 or C8).

[0199] In one aspect, the carbohydrate conjugate for use in the compositions and methods described herein is a monosaccharide.

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

[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 embodiments, the conjugates or ligands described herein can be attached to an oligonucleotide using a variety of linkers that can be cleavable or non-cleavable.

[0203] Linkers typically are direct bonds or atoms, such as oxygen or sulfur, units, such as NR 8, C(O), C(O)NH, SO, SO2, SO2NH, or, for example, a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl (alkylhererocyclylalkynyl), alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl (alkynylhereroaryl), including a chain of atoms but not limited to these, where one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R 8 ), C(O), a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocyclic; wherein R 8is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1 and 24, 2 and 24, 3 and 24, 4 and 24, 5 and 24, 6 and 24, 6 and 18, 7 and 18, 8 and 18, 7 and 17, 8 and 17, 6 and 16, 7 and 17, 8 and 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 a group that is sufficiently stable outside the cell but is cleaved upon entry into the target cell to release the two moieties that the linker holds together. 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 can be selected to mimic or represent, for example, intracellular conditions) than in the blood of the subject or under a second reference condition (which can be selected to mimic or represent conditions found, for example, in blood or serum).

[0205] A cleavable linking group is sensitive to a cleaving agent, e.g., pH, redox potential, or the presence of a degradable molecule. Generally, the cleaving agent is more prevalent, or found at a higher level or activity, inside the cell than in serum or blood. Examples of such degradable agents include: for example, oxidizing or reducing enzymes or reducing agents present in the cell that can degrade a redox-cleavable linking group by reduction, e.g., mercaptans, redox agents that are selective for or lack substrate specificity for certain substrates; esterases; agents that can create an endosome, or an acidic environment, e.g., those that yield a pH of 5 or less; enzymes, peptidases (which can be substrate-specific) and phosphatases that can hydrolyze or degrade an acid-cleavable linking group by acting as a general acid.

[0206] Cleavable linking groups, such as disulfide bonds, can be sensitive to pH. The pH of human serum is 7.4, but the average intracellular pH is slightly lower, in the range of about 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of 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 the desired compartment of the cell.

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

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

[0209] In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linker. It may also be desirable to test candidate cleavable linkers for their ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, it is possible to determine the relative sensitivity to cleavage between at least two conditions, where at least one condition is selected to exhibit cleavage in target cells and another condition is selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. The 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 can be useful to perform an initial evaluation under cell-free or culture conditions and confirm it by further evaluation in whole animals. In some embodiments, a useful candidate compound is 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 linker In one aspect, 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 (-S-S-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or, for example, suitable for use with a particular oligonucleotide moiety and a particular targeting agent, attention can be paid to the methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the rate of cleavage observed in cells, such as target cells. A candidate can be evaluated under conditions selected to mimic blood or serum conditions. In one aspect, the candidate compound is cleaved by at most about 10% in blood. In other aspects, a useful candidate compound is at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster degraded 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 a candidate compound can be determined using standard enzyme kinetics assays under conditions selected to mimic intracellular media compared to conditions selected to mimic extracellular media.

[0211] b. Phosphate-based cleavable linking group In another aspect, the cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of agents that cleave phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are -O-P(O)(OR k )-O-, -O-P(S)(OR k )-O-, -O-P(S)(SR k )-O-, -S-P(O)(OR k )-O-, -O-P(O)(OR k )-S-, -S-P(O)(OR k )-S-, -O-P(S)(ORk )-S-, -S-P(S)(OR k )-O-, -O-P(O)(R k )-O-, -O-P(S)(R k )-O-, -S-P(O)(R k )-O-, -S-P(S)(R k )-O-, -S-P(O)(R k )-S-, -O-P(S)(R k )-S-. These candidates can be evaluated using methods similar to the above methods.

[0212] c. Acid-cleavable linking group In another aspect, 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 aspects, 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. In cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. The acid-cleavable group can have the general formula -C=NN-, C(O)O or -OC(O). In one aspect, the carbon is bonded 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 the above methods.

[0213] d. Ester-based linking group In another aspect, the cleavable linker comprises a cleavable linking group based on an ester. The cleavable linking group based on an ester is cleaved by enzymes such as esterases and amidases in cells. Examples of cleavable linking groups based on esters include, but are not limited to, esters of alkylene, alkenylene and alkynylene groups. The cleavable ester linking group has the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using a method similar to the above method.

[0214] e. Cleavage groups based on peptides In yet another aspect, the cleavable linker comprises a cleavable linking group based on a peptide. The cleavable linking group based on a peptide is cleaved by enzymes such as peptidases and proteases in cells. The cleavable linking group based on a peptide is a peptide bond formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The cleavable group based on a peptide does not include an amide group. The amide group can be formed between any alkylene, alkenylene or alkynelene. The peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The cleavage group based on a peptide is generally limited to a peptide bond (i.e., an amide bond) formed between amino acids to yield peptides and proteins and does not include the entire amide functional group. The cleavable linking group based on a peptide has the general formula -NHCHR A C(O)NHCHR B C(O)-, wherein R A and R B are the R groups of two adjacent amino acids. These candidates can be evaluated using a method similar to the above method.

[0215] In one aspect, the oligonucleotide is conjugated to a carbohydrate via a linker. The linker includes divalent and trivalent branched-chain linker groups. Linkers for oligonucleotide carbohydrate conjugates include, but are not limited to, those described in Formulas 24-35 of PCT Application Publication No. WO2018 / 195165.

[0216] Representative U.S. patents that teach the preparation of oligonucleotide conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which are hereby incorporated by reference herein.

[0217] Not all positions in a given compound need to be uniformly modified. Indeed, more than one of the above-described modifications can be incorporated in a single compound or even in a single nucleoside within an oligonucleotide. Oligonucleotide compounds that are chimeric compounds are also contemplated. Chimeric oligonucleotides typically include at least one region in which the RNA is modified to impart to the oligonucleotide an increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for a target nucleic acid. A further region of the oligonucleotide can function as a substrate for an enzyme capable of cleaving RNA:DNA. By way of example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, 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 as compared to phosphorothioate deoxynucleotides that hybridize to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, optionally, by related nucleic acid hybridization techniques known in the art.

[0218] In certain cases, the nucleotides of the oligonucleotide can be modified by non-ligand groups. Some non-ligand molecules are conjugated to the oligonucleotide to enhance the activity, cellular distribution or cellular uptake of the oligonucleotide, and procedures for performing 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. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), thiocolesterol (Oberhauser et al., Nucl. Acids Res., 1992, 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), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxy cholesterol 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. Typical conjugation protocols involve the synthesis of oligonucleotides bearing amino linkers at one or more positions in the sequence. The amino groups are then reacted with the conjugated molecule using appropriate coupling or activating reagents. The conjugation reaction can be carried out using oligonucleotides still attached to the solid support or after cleavage of the oligonucleotide in solution phase. Purification of the oligonucleotide conjugate by HPLC typically yields a pure conjugate.

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

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

[0221] Based on the above methods, an oligonucleotide, or a composition comprising such an oligonucleotide, is contemplated for use in therapy, or for use as a medicament, or for use in treating a disorder associated with DNA mismatch repair, such as a repeat expansion disorder, in a subject in need thereof, or for use in reducing the level of MSH3 in cells of a subject identified as having a trinucleotide repeat expansion disorder, or for use in inhibiting the expression of MSH3 in cells of a subject, or for use in reducing trinucleotide repeat expansion in cells. These uses include contacting the cells with an oligonucleotide in an amount effective to inhibit the expression of MSH3 in the cells, thereby inhibiting the expression of MSH3 in the cells. In connection with the methods described herein, the aspects described below are also applicable to these further aspects.

[0222] Contacting a cell with an oligonucleotide can be performed in vitro or in vivo. Contacting a cell with an oligonucleotide in vivo includes contacting a cell or group of cells within a subject, such as a human subject, with the oligonucleotide. Combinations of in vitro and in vivo methods of contacting a cell are also possible. Contacting a cell can be direct or indirect, as discussed above. Further, contacting a cell can be achieved via a targeting ligand that includes 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 the oligonucleotide to the site of interest. The cell can include a cell of the central nervous system or a muscle cell.

[0223] Inhibiting the expression of the MSH3 gene includes inhibiting any level of the MSH3 gene, such as at least partial suppression of the expression of the MSH3 gene, such as 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] The expression of the MSH3 gene can be evaluated based on the level of any variable associated with MSH3 gene expression, such as the MSH3 mRNA level or the MSH3 protein level.

[0225] Inhibition can be evaluated by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, for example, a baseline level before dosing, or a level determined from a similar subject, cell or sample treated with an untreated or control (e.g., a control of buffer only or an inert agent control).

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

[0227] In some embodiments of the method, the 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 until below the level of detection of the assay. In some embodiments, the method comprises a clinically appropriate inhibition of the expression of MSH3, as demonstrated by a clinically appropriate outcome after treatment of a subject with an agent that reduces the expression of MSH3.

[0228] Inhibition of the expression of the MSH3 gene can be demonstrated by a reduction in the amount of mRNA expressed by a first cell or group of cells that is substantially identical to a second cell or group of cells that have not been so treated (control cells not treated with an oligonucleotide or not treated with an oligonucleotide targeted to the gene of interest), but in which the expression of the MSH3 gene is inhibited compared to the second cell or group of cells. The degree of inhibition can be expressed in terms of:

Number

[0229] In other embodiments, inhibition of MSH3 gene expression can be evaluated with respect to parameters functionally associated with MSH3 gene expression, such as reduction in MSH3 protein expression or MSH3 signaling pathways. Silencing of the MSH3 gene 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 demonstrated 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 described above, for the assessment of mRNA inhibition, inhibition of protein expression levels in treated cells or groups of cells can be similarly expressed as a percentage of the level of protein in control cells or groups of cells.

[0231] Control cells or groups of cells that can be used to evaluate inhibition of MSH3 gene expression include cells or groups of cells that have not yet been contacted with the oligonucleotide. For example, control cells or groups of cells can be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with the 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 aspect, the level of expression of MSH3 in a sample is determined by detecting the 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 / guanidinium thiocyanate extraction (RNAzol B; Biogenesis), RNEASY™ RNA preparation kits (Qiagen), or PAXgene (PreAnalytix, Switzerland). Typical assay formats that utilize 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 WO2012 / 177906, the entire contents of which are hereby incorporated by reference herein. In some aspects, the level of expression of MSH3 is determined using a nucleic acid probe. The term "probe" as used herein refers to any molecule capable of selectively binding to a specific MSH3 sequence, e.g., mRNA or polypeptide. Probes can be synthesized by those of skill in the art or derived from appropriate biological preparations. Probes can be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0233] 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, for example, by electrophoresing the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose, and contacting it with the probe. In an alternative embodiment, the probe(s) is 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 readily adapt known mRNA detection methods for use in determining the level of MSH3 mRNA.

[0234] Alternative methods for determining the level of MSH3 expression in a sample include, for example, RT-PCR (experimental protocol shown in Mullis, 1987, U.S. Patent 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 system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-beta replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques well known to those skilled in the art, for example, the process of nucleic acid amplification of mRNA in a sample and / or reverse transcriptase (to prepare cDNA) is involved. 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 MSH3 expression is determined by quantitative fluorescence-generating RT-PCR (i.e., TAQMAN (trademark) System) or DUAL-GLO (registered trademark) Luciferase assay.

[0235] The expression level of MSH3 mRNA can be monitored using a membrane blot (e.g., used in hybridization assays such as Northern, Southern, dot, etc.), or using a microwell, sample tube, gel, bead or fiber (or any solid support containing bound nucleic acid). 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. Determination of the MSH3 expression level can include using a nucleic acid probe in solution.

[0236] In some embodiments, the level of mRNA expression is evaluated using branched DNA (bDNA) assay or real-time PCR (qPCR). The use of such PCR methods is described and exemplified in the examples presented herein. Such methods can be used for the detection of MSH3 nucleic acids.

[0237] The level of MSH3 protein expression can be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), ultrafiltration chromatography, fluid or gel precipitation reactions, 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, and the like. Such assays can be used for the detection of proteins that indicate the presence or duplication of MSH3 protein.

[0238] In some embodiments of the methods described herein, the oligonucleotide is administered to a subject such that the oligonucleotide is delivered to a specific site within the subject. Inhibition of MSH3 expression can be evaluated using measurement of the level or change in level of MSH3 mRNA or MSH3 protein in a sample derived from a specific site within the subject. In some embodiments, the method includes clinically appropriate inhibition of MSH3 expression, as demonstrated by clinically appropriate outcomes following treatment of the subject with an agent that reduces the expression of MSH3.

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

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

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

[0242] A. Delivery of Anti-MSH3 Agents Delivery of oligonucleotides to cells, e.g., cells in a subject, e.g., a human subject, e.g., a subject in need of delivery of an oligonucleotide, e.g., cells in a subject having a trinucleotide repeat expansion disorder, can be accomplished in several different ways. For example, delivery can be effected by contacting the cells with the oligonucleotide either in vitro or in vivo. In vivo delivery can be effected directly by administering to the subject a composition comprising the oligonucleotide. These alternative methods are discussed further below.

[0243] In general, any method for delivering nucleic acid molecules (either 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, which are incorporated herein by reference in their entirety). For in vivo delivery, factors to consider for delivering oligonucleotide molecules include, for example, the biological stability of the molecule being delivered, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. 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 the preparation. Local administration to the site of treatment maximizes the local concentration of the drug, limits exposure of the drug to systemic tissues that could otherwise be damaged or degraded by the drug, and allows for a lower total dose of the oligonucleotide to be administered.

[0244] For systemic administration of oligonucleotides for the treatment of diseases, the oligonucleotides can include alternative nucleobases, alternative sugar moieties and / or alternative internucleoside linkages, or alternatively, can be delivered using a drug delivery system; both methods act to prevent the rapid degradation of the oligonucleotides in vivo by endonucleases and exonucleases. Modification of the oligonucleotide or pharmaceutical carrier can enable targeting of the oligonucleotide composition to the target tissue and avoid unwanted off-target effects. Oligonucleotide molecules can be modified by chemical conjugation to lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation. In an alternative embodiment, the oligonucleotide can 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 facilitate the binding of the oligonucleotide molecule (negatively charged) and enhance the interaction with the negatively charged cell membrane to enable efficient uptake of the oligonucleotide by the cell. Cationic lipids, dendrimers or polymers can be conjugated to the oligonucleotide or induced to form vesicles or micelles that encapsulate the oligonucleotide. The formation of vesicles or micelles further prevents the degradation of the oligonucleotide when administered systemically. In general, any method of nucleic acid delivery known in the art can be adaptable for the delivery of the oligonucleotides described herein. Methods for making and administering cationic oligonucleotide complexes are well within the skill of 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) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487) and polyamidoamine (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 oligonucleotide forms a complex with cyclodextrin for systemic administration. Methods and pharmaceutical compositions for the administration of oligonucleotides and cyclodextrin can be found in U.S. Patent 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 the administration of oligonucleotides and polyplex nanoparticles and lipoplex nanoparticles can be found in U.S. Patent Application Nos. 2017 / 0121454; 2016 / 0369269; 2016 / 0279256; 2016 / 0251478; 2016 / 0230189; 2015 / 0335764; 2015 / 0307554; 2015 / 0174549; 2014 / 0342003; 2014 / 0135376; and 2013 / 0317086, which are hereby incorporated by reference in their entirety.

[0245] i. Membrane Molecular Assembly Delivery Method Oligonucleotides can be delivered using a variety of membranous molecular assembly delivery methods, including polymeric biodegradable microparticles or microcapsule delivery devices known in the art. For example, colloidal dispersions can be used for the targeted delivery of the oligonucleotide agents described herein. Colloidal dispersions include lipid-based systems such as polymer complexes, nanocapsules, microspheres, beads, as well as water-in-oil emulsions, micelles, mixed micelles and liposomes. Liposomes are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. Large unilamellar vesicles (LUVs) in the size range of 0.2 - 4.0 μm have been shown to be able to encapsulate a substantial percentage of an aqueous buffer containing large polymers. Liposomes are useful for the translocation and delivery of active ingredients to the site of action. Since the liposome membrane is structurally similar to biological membranes, when liposomes are applied to tissues, the liposome bilayer fuses with the cell membrane bilayer. As the integration of liposomes and cells progresses, the internal aqueous contents containing the oligonucleotide are delivered into the cell, where the oligonucleotide can specifically bind to the target RNA and mediate RNase H-mediated gene silencing. In some cases, liposomes are also specifically targeted, for example, to direct oligonucleotides to specific cell types. The composition of liposomes is usually a combination of phospholipids, usually in combination with a steroid, particularly 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 such that micelles are formed with the lipid components. For example, the lipid components can be amphiphilic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholate, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. Next, the oligonucleotide preparation is added to the micelles containing the lipid components. The cationic groups on the lipid interact with the oligonucleotide and condense around the oligonucleotide to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposome preparation of the oligonucleotide.

[0247] Optionally, a carrier compound that aids in condensation can be added during the condensation 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 be favorable for condensation.

[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 WO96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation can include one or more aspects of the exemplary methods described in Feigner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Patent No. 4,897,355; U.S. Patent No. 5,171,678; Bangham et al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75: 4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, for example, Mayer et al., (1986) Biochim. Biophys. Acta 858:161). Microfluidization can be used if consistently small (50-200 nm) relatively uniform aggregates are desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169). These methods are readily adaptable for packaging 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 complexes bind to the negatively charged cell surface and are internalized into endosomes. Due to the acidic pH within endosomes, the liposomes rupture and release 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. Since both the nucleic acids and the lipids are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acids are encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).

[0251] One major type of liposome composition contains phospholipids other than naturally derived phosphatidylcholine. Neutral liposome compositions can be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are mainly formed from dioleoyl phosphatidylethanolamine (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] Examples of other methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent No. 5,283,185; U.S. Patent 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, particularly those containing nonionic surfactants and cholesterol, have also been tested to determine their usefulness in drug delivery 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 cyclosporin-A into the dermis of mouse skin. The results showed that such nonionic liposome systems were effective in promoting the deposition of cyclosporin A into different layers of the skin (Hu et al., (1994) S.T.P. Pharma. Sci., 4(6):466).

[0254] Liposomes can be sterically stabilized liposomes containing one or more special lipids that result in an enhanced circulation lifetime compared to liposomes lacking such special lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome is (A) one or more glycolipids, such as monosialoganglioside G M1which comprises, or (B) is a liposome 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 results from 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] A variety of liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64) reported the ability of monosialoganglioside G M1 , galactosylcerebroside sulfate and phosphatidylinositol to improve the blood half-life of liposomes. These findings were explained by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., (1988), 85:6949). U.S. Patent No. 4,837,028 and WO88 / 04924 to Allen et al. both disclose liposomes containing (1) sphingomyelin and (2) ganglioside G M1 or galactosylcerebroside sulfate ester. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO97 / 13499 (Lim et al.).

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

[0257] Further advantages of liposomes include the following: Liposomes obtained from natural phospholipids are biocompatible and biodegradable; Liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs; Liposomes can protect oligonucleotides encapsulated within 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 capable of fusing with the negatively charged lipids of the cell membrane of tissue culture cells to effect delivery of oligonucleotides (see, for example, Feigner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417 and U.S. Patent No. 4,897,355 for descriptions of its use with DOTMA and DNA).

[0259] The DOTMA analog, 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), can be used in combination with phospholipids to form vesicles that complex DNA. LIPOFECTIN™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells, consisting of positively charged DOTMA liposomes that interact spontaneously 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 bind spontaneously to the negatively charged cell surface, fuse with the plasma membrane, and efficiently deliver functional nucleic acids, for example, into tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonio)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, for example, compounds conjugated to one of two types of lipids, such as 5-carboxyspermidine dioctaoleylamide (“DOGS”) (TRANSFECTAM™, Promega, Madison, Wis.) and compounds conjugated to various moieties that include carboxyspermine, such as dipalmitoylphosphatidylethanolamine 5-carboxyspermidine-amide (“DPPES”) (see, for example, U.S. Patent No. 5,171,678).

[0261] Other cationic lipid conjugates include cholesterol-derivatized lipids (``DC-Chol'') formulated into liposomes in combination with DOPE (see Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolyllysine, prepared 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 exhibit several advantages over other formulations. Such advantages include a reduction in side effects associated with high systemic absorption of the administered drug, an increase in the accumulation of the administered drug at 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 the penetration of oligonucleotides into dermal tissue, such as in the skin. For example, liposomes can be topically applied. Topical delivery of drugs formulated as liposomes to the skin has been reported (see, e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2, 405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. and Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

[0263] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have also been tested to determine their usefulness in drug delivery to the skin. Nonionic liposomal 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 having oligonucleotides are useful for treating dermatological disorders.

[0264] Targeting of liposomes is also possible, for example, based on organ specificity, cell specificity, and organelle specificity, and is known in the art. In the case of liposomal targeted delivery systems, lipid groups can be incorporated into the lipid bilayer of the liposome to stably associate the targeting ligand with the liposome bilayer. Various linking groups can be used to conjugate the lipid chain to the targeting ligand. Further methods are known in the art and are described, for example, in U.S. Patent Application Publication No. 20060058255, which is hereby incorporated by reference herein.

[0265] Liposomes containing oligonucleotides can be highly deformable. Such deformability can enable the liposomes to penetrate through pores smaller than the average radius of the liposomes. For example, transferosomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates as drug delivery vehicles. Transferosomes can be described as highly deformable lipid droplets that can readily penetrate through pores smaller than the droplets. Transferosomes can be prepared by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Transferosomes containing oligonucleotides can be delivered subcutaneously, for example, by infection, to deliver the oligonucleotides to keratinocytes in the skin. To cross intact mammalian skin, the lipid vesicles must pass through a series of fine pores, each having a diameter of less than 50 nm, under the influence of an appropriate transdermal gradient. Further, due to their lipid properties, these transferosomes can be self-optimizing (e.g., adapting to the shape of the pores in the skin), self-healing, can frequently reach their targets without fragmentation, and can often be self-filling. Transferosomes have been used to deliver serum albumin to the skin. Transferosome-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 Nos. 61 / 018,616, filed Jan. 2, 2008; 61 / 018,611, filed Jan. 2, 2008; 61 / 039,748, filed Mar. 26, 2008; 61 / 047,087, filed Apr. 22, 2008; and 61 / 051,528, filed May 8, 2008. PCT Application No. PCT / US2007 / 080331, filed Oct. 3, 2007, also describes suitable ones. 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 use of the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0267] Surfactant molecules are classified as nonionic surfactants when not ionized. Nonionic surfactants find wide application in pharmaceutical products and cosmetics and can be used over a wide range of pH values. Generally, their HLB values range from 2 to about 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers are also included in this class. Polyoxyethylene surfactants are the most common members of the class of nonionic surfactants.

[0268] When surfactant molecules carry a negative charge when dissolved or dispersed in water, this surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acylamides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates, and sulfosuccinates, as well as phosphates. The most important members of the class of anionic surfactants are alkyl sulfates and soaps.

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

[0270] When surfactant molecules have the ability to carry either a positive or a negative charge, this surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines, and phosphatides.

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

[0272] Oligonucleotides for use in methods can be provided as micellar formulations. Micelles are a particular type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all of the hydrophobic portions of the molecules face inward while keeping the hydrophilic portions in contact with the surrounding aqueous phase. In a hydrophobic environment, the reverse arrangement exists.

[0273] ii. Delivery methods based on lipid nanoparticles Oligonucleotides can be fully encapsulated in lipid formulations, such as lipid nanoparticles (LNPs), or other nucleic acid-lipid particles. LNPs exhibit an extended circulation lifetime after intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically distant from the administration site), making them highly useful for systemic applications. LNPs include "pSPLP" containing encapsulated condensing agent-nucleic acid complexes as shown in PCT Application Publication No. WO00 / 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 non-toxic. Further, nucleic acids are resistant to nuclease degradation in aqueous solution when present in nucleic acid-lipid particles. 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. WO96 / 40964.

[0274] In one aspect, the ratio of lipid to drug (mass / mass ratio) (e.g., the ratio of lipid to oligonucleotide) 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. Intermediate ranges within the ranges listed above 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-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy (Dilinolenyloxy)-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy (Dilinoleyoxy)-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.(Cl), 1,2-dilinoleoyl-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-dilinoleyloxy-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 an analog thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyltetrahydro(dienyetetrahydro)-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl didodecane(yeethylazanediyedidodecan)-2-ol (Tech G1), or a mixture thereof. The cationic lipid can constitute, for example, about 20 mol% to about 50 mol%, or about 40 mol% of the total lipids present in the particles.

[0276] Ionizable / non-cationic lipids can be anionic or neutral lipids including, but not limited to, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. The non-cationic lipid can be, for example, about 5 mol% to about 90 mol%, about 10 mol%, or about 60 mol% of the total lipid present in the particles when cholesterol is included.

[0277] Conjugated lipids that inhibit aggregation of the particles can be polyethylene glycol (PEG)-lipids including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryl oxypropyl (C 12 )), PEG-dimyristyl oxypropyl (C 14 ), PEG-dipalmityl oxypropyl (C 16 ) or PEG-distearyl oxypropyl (C 18) It can be. The 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, for example, about 10 mol% to about 60 mol%, or about 50 mol% cholesterol of the total lipid present in the particle.

[0279] B. Combination Therapy The oligonucleotide can be used alone or in combination with at least one additional therapeutic agent, such as another agent that treats trinucleotide repeat expansion disorders or symptoms associated therewith, or in combination with another type of treatment for treating trinucleotide repeat expansion disorders. In combination treatment, the dosage of one or more of the therapeutic compounds can be reduced from the standard dosage when administered alone. For example, the dosage can be determined empirically from drug combinations and sequences, or can be estimated by isobolographic analysis (e.g., Black et al., Neurology 65:S3-S6 (2005)). In this case, the dosage of the compounds in combination should provide a 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, which is an additional therapeutic agent, comprises one or more phosphorothioate internucleotide linkages. In some embodiments, the modified oligonucleotide comprises one or more 2'-MOE moieties. In some embodiments, the oligonucleotide, which 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 herein by reference.

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

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

[0283] In any of the combinatorial embodiments described herein, two or more therapeutic agents are administered simultaneously or sequentially in any order. For example, the first therapeutic agent can be administered immediately before or after one or more of the additional therapeutic agents, or before or after one or more of the additional therapeutic agents, up to a maximum of 1 hour, up to a maximum of 2 hours, up to a maximum of 3 hours, up to a maximum of 4 hours, up to a maximum of 5 hours, up to a maximum of 6 hours, up to a maximum of 7 hours, up to a maximum of 8 hours, up to a maximum of 9 hours, up to a maximum of 10 hours, up to a maximum of 11 hours, up to a maximum of 12 hours, up to a maximum of 13 hours, up to a maximum of 14 hours, up to a maximum of 16 hours, up to a maximum of 17 hours, up to a maximum of 18 hours, up to a maximum of 19 hours, up to a maximum of 20 hours, up to a maximum of 21 hours, up to a maximum of 22 hours, up to a maximum of 23 hours, up to a maximum of 24 hours, or up to a maximum of 1-7, 1-14, 1-21, or 1-30 days before or after one or more of the additional therapeutic agents.

[0284] V. Pharmaceutical Composition 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 bases, salts, solvates, or prodrugs. All forms are within the scope of the methods described herein. According to the methods described herein, the oligonucleotides or salts, solvates, or prodrugs thereof described can be administered to a patient in various forms depending on the selected route of administration, as will be understood by those skilled in the art. The compounds described herein can be administered, for example, orally, parenterally, intrathecally, intracerebroventricularly, intrasubstantially, buccally, sublingually, nasally, rectally, by patch, pump, or transdermally, and by appropriately formulated pharmaceutical compositions. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, intracerebroventricularly, intrasubstantially, rectal, and topical modes of administration. Parenteral administration can be by continuous infusion over a selected period.

[0286] The compounds described herein can be administered orally, for example, with an inert diluent or an absorbable edible carrier, or encapsulated in hard or soft shell gelatin capsules, or compressed into tablets, or directly incorporated with foods or diets. For oral therapeutic administration, the compounds described herein can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, 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 suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can be prepared in glycerol, liquid polyethylene glycol, DMSO, and mixtures thereof, with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations can contain preservatives to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2012, 22nd ed.) and 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 immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy administration via syringe is possible. 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 substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in sterile form as a single dose or multiple doses in a sealed container that can take the form of a cartridge or refill for use with a nebulizing device. Alternatively, the sealed container can be a unit-dose 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 which can be a compressed gas such as compressed air, or an organic spray agent such as a 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, where the active ingredient is formulated using carriers such as sugar, acacia, tragacanth, gelatin and glycerin. Compositions for rectal administration are conveniently in the form of suppositories which contain conventional suppository bases such as cocoa butter.

[0287] As noted herein, the compounds described herein can be administered to animals such as humans, either alone or in combination with a pharmaceutically acceptable carrier, the proportion being determined by the solubility and chemical nature of the compound, the chosen route of administration, and standard pharmaceutical practice.

[0288] VI. Dosage The dosage of the compositions described herein (e.g., compositions comprising oligonucleotides) can vary depending on many factors such as the pharmacodynamic properties of the compound; the mode of administration; the age, health status and weight of the recipient; the nature and extent of the symptoms; the frequency of treatment, and the type of concurrent treatment, if any; and the clearance rate of the compound in the animal being treated. The compositions described herein can be initially administered at an appropriate dosage which can be adjusted as needed depending on the clinical response. In some embodiments, the dosage of the composition (e.g., compositions comprising oligonucleotides) is a prophylactically 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. EXAMPLES

[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, GC content of 20 - 60%, no G homopolymer of 4 or longer, and no A, T, or C homopolymer of 6 or longer. These selected or "preferred" oligonucleotides were further evaluated for specificity (off-target scoring, below).

[0292] Off-target scoring: The specificity of the preferred ASOs was evaluated via alignment to all unspliced RefSeq transcripts (the "NM" model for human, mouse, and rat; the "NM" and "XM" models for cynomolgus monkey) using the FASTA algorithm with a 1000 E-value cutoff. The number of mismatches (by species) between each ASO and each transcript was tabulated. 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 preferred ASOs was selected for screening according to both specificity and information maximizing the ASO:mRNA (target) hybridization energy as follows. All candidate ASOs were evaluated for the predicted delta G (ΔG 全体 ) of hybridization to the target mRNA secondary structure according to Xu and Mathews (Methods Mol Biol. 1490:15 - 34 (2016)). Next, two subsets of ASOs were selected: First, 69 ASOs that matched human, cynomolgus monkey, and mouse target transcripts had at least one off-target score in the three species and had a negative ΔG overall ; Second, 411 ASOs that matched human and cynomolgus monkey target transcripts had at least two off-target scores in both species and the ΔG 全体 was less than -9.5 °C.

[0294] The sequences of each ASO, their positions in the human transcript, and conservation in other species and off-target scores are given in Table 2. When indicated as "NC", the ASO did not match the MSH3 gene in that species and thus no off-target score was generated.

[0295] The ASOs were synthesized as 5-10-5 "flanking sequence-DNA core sequence-flanking sequence" antisense oligonucleotides having 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’ where · Nm: 2'-MOE residue (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" within the DNA core (positions 6-15) are 5'-methyl-2'-MOE-dC · All "T" at positions 1-5 or 16-20 are 5'-methyl-2'-MOE-U. Desalted oligonucleotides were used for the primary screening at 2 nM and 20 nM. For detailed characterization of a subset of the 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 3-1

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

Table 3-19

Table 3-20

[0297] (Example 3) In vitro screening for reduced elongation The elongation of DNA triplet repeats can be reproduced in vitro using patient-derived cell lines and DNA-damaging agents. Human fibroblasts from Huntington's (GM04281, GM04687, and GM04212) or Friedreich's ataxia patients (GM03816 and GM02153) or myotonic dystrophy type 1 (GM04602, GM03987, and GM03989) were purchased from Coriell Cell Repositories and maintained in 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 elongation in vitro, fibroblasts were treated with oxidizing agents such as hydrogen peroxide (H2O2), potassium chromate (K2CrO4), or potassium bromate (KBrO3) for up to 2 hours (Kovtum et al., ibid). Cells were washed, the medium was replaced, and the cells were allowed to recover for 3 days. The treatment was repeated up to 2 more times, after which the cells were harvested and DNA was isolated. The CAG repeat length was 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 is purified using standard proteinase K digestion and extracted using DNAzol (Invitrogen) according to the manufacturer's instructions. The CAG repeat length is determined by the previously described small pool-PCR analysis (Mario Gomes-Pereira and Darren Monckton, 2017, Front Cell Neuro 11:153). Briefly, the DNA is digested with HindIII, diluted to a final concentration between 1 - 6 pg / μl, and approximately 10 pg is used in subsequent PCR reactions. Primers flanking exon 1 of human HTT are used to amplify the CAG alleles, and the PCR products are resolved by electrophoresis. Subsequently, Southern blot hybridization is performed, and the CAG alleles are observed by autoradiography or visualized by ethidium bromide staining. The CAG length can be directly measured by sequencing on a MiSeQ or appropriate machine. Changes in the CAG repeat number in various treatment groups compared to controls are calculated using simple descriptive statistics (e.g., mean ± standard deviation).

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

[0303] (Example 5) Mouse studies Natural history study in HD mouse models: The HD mouse R6 / 2 line is transgenic for the 5’ end of the human HD gene (HTT) carrying approximately 120 CAG repeat expansions. HTT is ubiquitously expressed. The transgenic mice exhibit a progressive neurological phenotype that mimics many of the pathological features of HD, including chorea-like movements, involuntary stereotyped movements, tremors and seizure-like episodes, as well as non-motor components including abnormal vocalizations. They urinate frequently and show loss of body weight and muscle mass over the course of the disease. Neurologically, these mice develop nuclear inclusion bodies (NIIs) that contain both huntingtin protein and ubiquitin protein. These NIIs, previously unknown, were subsequently identified in HD patients. The onset age of HD symptoms in R6 / 2 mice has been reported to be between 9 and 11 weeks (Mangiarini et al., 1996 Cell 87: 493-506).

[0304] Somatic expansions were 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 with 120 CAG repeats. Their genotypes and the length of the CAG expansion were determined. R6 / 2 mice at 4, 8, 12 and 16 weeks of age (4 male and 4 female mice per age group) were sacrificed. The striatum, cerebellum, cortex, liver, kidney, heart, spleen, lung, duodenum, colon, quadriceps muscle, CSF and plasma were collected and snap frozen in liquid nitrogen. Genomic DNA was extracted, the length of the CAG repeat was measured and the 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 expansion as measured by the instability index ( **** p<0.0001, one-way ANOVA) (Figure 1). In the R6 / 2 mouse cerebellum, no changes in somatic expansion were observed over all ages (Figure 2).

[0305] Mouse models that recapitulate many of the features of trinucleotide repeat expansion diseases, including HD, FA, and DM1, are readily available from 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 conducted in accordance with local IACUC guidelines. Three examples of different affected mouse models and how they can be used to investigate the utility of pharmacological intervention against MSH3 for somatic expansion are included below.

[0306] In Huntington's 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 with 144 copies of the CAG repeat (Mangiarini et al., 1996 Cell 87: 493-506), while the HdhQ111 model was generated by replacing mouse HTT exon 1 with a human exon 1 containing 111 copies of the CAG repeat (Wheeler et al., 2000 Hum Mol Genet 9:503-513). Both the R6 / 2 model and the HdhQ111 model recapitulate many of the features of human HD, including motor and behavioral deficits, neurodegeneration, and CAG repeat expansion 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] Genotype the R6 / 2 mice using DNA derived from a tail snip at weaning and determine the CAG repeat size. Randomize the mice at 4 weeks of age at weaning into groups (n = 12 / group) and dose them by monthly (at 4 and 8 weeks) ICV injection with either PBS (control) or an oligo targeting up to 500 μg dose of MSH3. A series of oligos targeting different regions of MSH3 are tested to identify the most effective oligo sequence in vivo. At 12 weeks of age, euthanize the mice and extract tissues for analysis. The list of tissues includes, but is not limited to, striatum, cortex, cerebellum, and liver. Extract genomic DNA and measure the length of the CAG repeat as described below. Collect CSF and plasma for biomarker analysis. Additional appropriate mouse models of HD can be considered.

[0308] In Friedreich 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 via the insertion of a human YAC transgenic containing in the background of null FRDA mice. The YG8 model demonstrates somatic expansion of GAA triplet repeats in neural tissues, with only mild motor deficits. Genotype the YG8 FRDA mice using DNA derived from a tail snip at weaning and determine the CAG repeat size using the method. To determine whether MSH3 plays a role in the somatic expansion of disease alleles, administer ICV to hemizygous YG8 FRDA animals an oligo targeting the knockdown of MSH3 identified above.

[0309] Approximately 2 months later, euthanize the animals and collect tissues for molecular analysis. Appropriate tissues are heart, quadriceps, dorsal root ganglia (DRG), cerebellum, kidney, and liver. Extract genomic DNA and measure the length of the CAG repeat as described in Example 4 above.

[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 line in which most of exon 1 and part of intron 1 on one allele of the huntingtin gene (i.e., HTT or the Huntington's disease gene) are replaced with human DNA containing approximately 111 CAG repeats. In this example, an ASO that knockdowns MSH3 activity or level is administered. After the treatment period, brain tissue (e.g., striatal tissue) is isolated from treated or untreated mice and analyzed using qRT-PCR as previously described to determine the RNA level of MSH3. Huntingtin gene repeat analysis is performed using a human-specific PCR assay in which the HTT CAG repeats from the knock-in allele are amplified but the mouse sequence (i.e., wild-type allele) is not, using mouse tissue (e.g., striatal tissue) after the treatment period. In this protocol, the forward primer is fluorescently labeled (e.g., with 6-FAM as previously described in, for example, 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. The repeat size is determined from the peak with the greatest intensity from control tissue (e.g., mouse tail tissue) and affected tissue (e.g., brain striatal t...

Claims

Claim 1: A single-stranded oligonucleotide 20 nucleosides in length, comprising a region of at least 10 consecutive nucleobases having at least 95% complementarity to the MSH3 gene, wherein said oligonucleotide (a) a DNA core sequence comprising linked deoxyribonucleosides; (b) a 5' adjacent sequence comprising linked nucleosides; and (c) a 3' adjacent sequence comprising linked nucleosides are included, wherein said DNA core comprises a region of at least 10 consecutive nucleobases positioned between said 5' adjacent sequence and said 3' adjacent sequence; wherein said 5' adjacent sequence and said 3' adjacent sequence each comprise at least two linked nucleosides; and at least one nucleoside of each adjacent sequence comprises an alternative nucleoside, The oligonucleotides are SEQ ID NO: 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, 1581-1589, 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 nucleotide sequences of 2385, 2388, 2390 to 2395, 2416 to 2418, 2460, 2462, or 2463. Claim 2 A composition for inhibiting the 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 consecutive nucleobases having at least 95% complementarity to the MSH3 gene, wherein said oligonucleotide (a) a DNA core sequence comprising linked deoxyribonucleosides; (b) a 5' adjacent sequence comprising linked nucleosides; and (c) a 3' adjacent sequence comprising linked nucleosides are included, wherein said DNA core comprises a region of at least 10 consecutive nucleobases positioned between said 5' adjacent sequence and said 3' adjacent sequence; wherein said 5' adjacent sequence and said 3' adjacent sequence each comprise at least two linked nucleosides; and at least one nucleoside of each adjacent sequence comprises an alternative nucleoside, The oligonucleotides are SEQ ID NO: 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, 1581-1589, 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 nucleobase sequences of 2385, 2388, 2390 to 2395, 2416 to 2418, 2460, 2462, or 2463. Claim 3 The oligonucleotide according to claim 1 or the composition according to claim 2, wherein said oligonucleotide comprises at least one alternative internucleoside linkage. Claim 4 The oligonucleotide or composition according to claim 3, wherein said at least one alternative internucleoside linkage is a phosphorothioate internucleoside linkage. Claim 5 The oligonucleotide or composition according to claim 3, wherein said at least one alternative internucleoside linkage is a 2'-alkoxy internucleoside linkage. Claim 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. One or more of the oligonucleotides according to claim 1, or the composition according to claim 2, and a composition comprising lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles or liposomes.

18. A composition comprising one or more of the 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 a method of inhibiting the transcription of MSH3 in a cell, wherein the method comprises contacting the cell with one or more of the 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 a time sufficient to obtain degradation of the mRNA transcript of the MSH3 gene, thereby inhibiting the expression of the MSH3 gene in the cell.

19. A composition comprising one or more of the 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 treating, preventing or delaying the progression of trinucleotide repeat expansion disorder in a subject in need thereof.

20. A composition comprising one or more of the 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 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, wherein the method comprises contacting the cell with one or more of the 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.

21. A composition comprising one or more of the 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 a method for inhibiting the expression of the MSH3 gene in a cell, wherein the method comprises contacting the cell with one or more of the 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, and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of the MSH3 gene, thereby inhibiting the expression of the MSH3 gene in the cell.

22. A composition comprising one or more of the 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 a method for reducing trinucleotide repeat expansion in a cell, wherein the method comprises contacting the cell with one or more of the 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.

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

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

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

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

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

28. The composition according to claim 27, wherein the polyglutamine disease is selected from the group consisting of dentatorubral - pallidoluysian atrophy, Huntington's disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 17, and Huntington's disease - like 2.

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

30. The composition according to 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 insufficiency, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy.

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

32. The composition according to 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 insufficiency, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy.

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

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

35. The composition according to 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, wherein 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, wherein the composition is administered intramuscularly.

39. A composition comprising one or more of the 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 treating, preventing or delaying the progression of a disorder in a subject in need thereof, wherein the subject has a trinucleotide repeat expansion disorder.

40. The composition according to claim 39, wherein the composition is administered in combination with a further therapeutic agent.

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

42. A composition comprising one or more of the 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 preventing or delaying the progression of a trinucleotide repeat expansion disorder in a subject.

43. The composition according to 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 insufficiency, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy.

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

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

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

47. The composition according to claim 39 or 40, wherein the composition is administered in combination with a further therapeutic agent.

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

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

50. 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 trinucleotide repeat expansion disorder in a subject.

51. The composition according to 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 insufficiency, FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy.

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

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

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

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

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