Antisense oligonucleotide targeting tdp-43 mRNA or pre-mRNA

Abstract

In the description of the present invention, provided is an antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, which comprises 15-22 nucleotides, and which is complementary to a nucleic acid including at least 15 consecutive bases in a target region selected from the group consisting of, from 5' end of the base sequence of SEQ ID NO: 1, position 1 to position 102, position 159 to position 842, position 879 to position 1822, and position 1874 to position 4182.

Description

Antisense oligonucleotides targeting TDP-43 mRNA or pre-mRNA

[0001] The present invention relates to an antisense oligonucleotide targeting TDP-43 mRNA or pre-mRNA, or a pharmaceutically acceptable salt thereof or a hydrate thereof, a pharmaceutical composition comprising the antisense oligonucleotide or a pharmaceutically acceptable salt thereof or a hydrate thereof, and the like.

[0002] TAR DNA-binding protein-43 (TDP-43) is a highly conserved, ubiquitously expressed RNA / DNA-binding protein belonging to the heterogeneous nuclear ribonucleoprotein (hnRNP) family (Non-Patent Document 1). Accumulation of TDP-43 is known to be involved in many neurodegenerative diseases (called "TDP-43 proteinopathies"), including amyotrophic lateral sclerosis (ALS). For TDP-43 proteinopathies, antisense oligonucleotides that regulate the expression level of TDP-43 (Patent Document 1, Patent Document 2, Non-Patent Document 2) and antisense oligonucleotides that enhance alternative splicing of intron 6 of TDP-43 mRNA (Patent Document 3) have been studied, but no effective treatment exists.

[0003] International Publication No. 2019 / 013141 International Publication No. 2022 / 120410 International Publication No. 2022 / 113799

[0004] de Boer EMJ et al. , J. Neurol Neurosurg Psychiatry, 2021, Vol. 92(1), pp. 86-95T Takeuchi et al. , Molecular Therapy Nucleic Acids, 2023, Vol. 31, pp. 353-366

[0005] Under these circumstances, it is desired to provide a new therapeutic agent for TDP-43 proteinopathy.

[0006] The present invention provides the following antisense oligonucleotides that target TDP-43 mRNA or pre-mRNA, or pharmaceutically acceptable salts thereof, or hydrates thereof, and pharmaceutical compositions containing the antisense oligonucleotides, or pharmaceutically acceptable salts thereof, or hydrates thereof.

[0007] (1-1) An antisense oligonucleotide consisting of 15 to 22 nucleotides, or a pharmaceutically acceptable salt thereof, which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 1 to 102, 159 to 842, 879 to 1822, and 1874 to 4182 from the 5' end of the base sequence of SEQ ID NO: 1. (1-2) An antisense oligonucleotide consisting of 15 to 30 nucleotides, or a pharmaceutically acceptable salt thereof, which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 1 to 102, 159 to 842, 879 to 1822, and 1874 to 4182 from the 5' end of the base sequence of SEQ ID NO: 1. (1-3) An antisense oligonucleotide consisting of 15 to 25 nucleotides, or a pharmaceutically acceptable salt thereof, which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 1 to 102, 159 to 842, 879 to 1822, and 1874 to 4182 from the 5' end of the base sequence of SEQ ID NO: 1. (1-4) The antisense oligonucleotide according to any of (1-1) to (1-3), or a pharmaceutically acceptable salt thereof, which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 1 to 100, 161 to 840, 881 to 1820, and 1876 to 4180 from the 5' end of the base sequence of SEQ ID NO: 1.(In the above (1-1) to (1-4), it is preferable to exclude an antisense oligonucleotide having a sequence consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 65, 436 to 440, 444 to 449, 451 to 452, and 454 to 474, or a pharmaceutically acceptable salt thereof; and it is preferable to exclude an antisense oligonucleotide having a sequence consisting of the nucleotide sequence of SEQ ID NO: 450, or a pharmaceutically acceptable salt thereof, in which positions 2 to 3, 5 to 6, 10 to 14, 16, 18, and 20 from the 5' end of the nucleotide sequence of SEQ ID NO: 450 are ENA: 2'-O,4'-C-Ethylenebridged Nucleic Acid, and positions 1, 4, 7 to 9, 15, 17, and 19 are ribonucleotides containing a 2'-OMe group; and / or (2) (i) a base sequence selected from the group consisting of SEQ ID NOs: 2 to 64, 66 to 224, 422 to 423, 426 to 427, and 432; (ii) a base sequence selected from the group consisting of SEQ ID NOs: 2 to 64, 66 to 224, 422 to 423, 426 to 427, and 432, in which one or several bases have been added, deleted, or substituted; or (iii) a base sequence having 90% or more sequence identity to a base sequence selected from the group consisting of SEQ ID NOs: 2 to 64, 66 to 224, 422 to 423, 426 to 427, and 432, or a pharmaceutically acceptable salt thereof, or a hydrate thereof.(3-1) positions 1 to 102, 159 to 622, 639 to 842, 879 to 1442, 1459 to 1497, 1499 to 1522, 1539 to 1702, 1719 to 1762, 1779 to 1802, 1874 to 1937, 1939 to 2302, 2319 to 3162, 3199 to 3242, and 3279 from the 5' end of the base sequence of SEQ ID NO: 1 The antisense oligonucleotide according to any one of (1-1) to (1-3), or a pharmaceutically acceptable salt thereof, is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 3399 to 3482, 3539 to 3562, 3579 to 3602, 3619 to 3662, 3679 to 3702, and 3759 to 4182. (3-2) positions 1 to 100, 161 to 620, 641 to 840, 881 to 1440, 1461 to 1495, 1501 to 1520, 1541 to 1700, 1721 to 1760, 1781 to 1800, 1876 to 1935, 1941 to 2300, 2321 to 3160, 3201 to 3240 from the 5' end of the base sequence of SEQ ID NO: 1, The antisense oligonucleotide according to (3-1), or a pharmaceutically acceptable salt thereof, is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 3281 to 3380, 3401 to 3480, 3541 to 3560, 3581 to 3600, 3621 to 3660, 3681 to 3700, and 3761 to 4180.(4) (i) SEQ ID NOs: 2 to 30, 32 to 64, 66 to 72, 74 to 75, 77, 79 to 86, 88 to 89, 91, 93 to 96, 98 to 102, 104 to 120, 122 to 146, 148 to 155, 157 to 158, 160 to 173, 176 to 177, 180 to 184, 186 to 189, 193, 195, 197 to 198, 200, and 204 to 224; (ii) SEQ ID NOs: 2 to 30, 32 to 64, 66 to 72, 74 to 75, 77, 79 to 86, 88 to 89, 91, 93 to 96, 98 to 102, 104 to 120, 122 to 146, 148 to 155, 157 to 158, 160 to 173, 176 to 177, 180 to 184, 186 to 189, 193, 195, 197 to 198, 200, and 204 to 224. A base sequence in which one or several bases are added, deleted, or substituted, or (iii) The antisense oligonucleotide according to (3-1) or (3-2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, comprising a base sequence having 90% or more sequence identity to a base sequence selected from the group consisting of SEQ ID NOs: 2 to 30, 32 to 64, 66 to 72, 74 to 75, 77, 79 to 86, 88 to 89, 91, 93 to 96, 98 to 102, 104 to 120, 122 to 146, 148 to 155, 157 to 158, 160 to 173, 176 to 177, 180 to 184, 186 to 189, 193, 195, 197 to 198, 200, and 204 to 224.(5-1) positions 19 to 42, 79 to 102, 159 to 182, 199 to 622, 639 to 662, 679 to 762, 779 to 802, 819 to 842, 879 to 1002, 1019 to 1402, 1419 to 1442, 1459 to 1497, 1499 to 1522, 1559 to 1642, 1659 to 1702, 1779 to 1802, 1874 to 1902, 1914 to 1937, 1959 to 2002, 2019 from the 5' end of the base sequence of SEQ ID NO: 1 The antisense oligonucleotide according to any one of (1-1) to (1-3), which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 1 to 2302, 2319 to 2342, 2359 to 2802, 2819 to 3142, 3199 to 3222, 3299 to 3322, 3339 to 3362, 3399 to 3422, 3799 to 3842, 3879 to 3982, 4019 to 4042, and 4119 to 4142, or a pharmaceutically acceptable salt thereof. (5-2) positions 21 to 40, 81 to 100, 161 to 180, 201 to 620, 641 to 660, 681 to 760, 781 to 800, 821 to 840, 881 to 1000, 1021 to 1400, 1421 to 1440, 1461 to 1495, 1501 to 1520, 1561 to 1640, 1661 to 1700, 1781 to 1800, 1876 to 1900, 1916 to 1935, and 1961 to 2000 from the 5' end of the base sequence of SEQ ID NO: 1 , 2021 to 2300, 2321 to 2340, 2361 to 2800, 2821 to 3140, 3201 to 3220, 3301 to 3320, 3341 to 3360, 3401 to 3420, 3801 to 3840, 3881 to 3980, 4021 to 4040, and 4121 to 4140. The antisense oligonucleotide according to (5-1), or a pharmaceutically acceptable salt thereof, is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of:(6) (i) SEQ ID NOs: 3, 6 to 7, 9 to 30, 32, 34 to 37, 39, 41 to 48, 50 to 64, 66 to 69, 72, 74 to 75, 77, 80 to 83, 85 to 86, 91, 93 to 94, 96, 99 to 100, 102, 104 to 120, 122, 124 to 143, 145 to 146, 148 to 149, 151, 154 to 155, 157 to 158, 160 to 165, 167 to 172, 176, 181, 183, 186, 206 to 207, 210 to 214, 217, and 222; (ii) SEQ ID NOs: 3, 6 to 7, 9 to 30, 32, 34 to 37, 39, 41 to 48, 50 to 64, 66 to 69, 72, 74 to 75, 77, 80 to 83, 85 to 86, 91, 93 to 94, 96, 99 to 100, 102, 104 to 120, 122, 124 to 143, 145 to 146, 148 to 149, 151, 154 to 155, 157 to 158, 160 to 165, 167 to 172, 176, 181, 183, 186, 206 to 207, 210 to 214, 217, and 222. A base sequence in which one or several bases are added, deleted, or substituted in a base sequence selected from the group consisting of: (iii) SEQ ID NOs: 3, 6-7, 9-30, 32, 34-37, 39, 41-48, 50-64, 66-69, 72, 74-75, 77, 80-83, 85-86, 91, 93-94, 96, 99-100, 102, 104-120, 122, 124-143, 145-146, 148-149, 151, 154-155, 157-158, 160-165, 167-172, 176, 181, 183, 186, 206-207, 210-214, 217, and 222 A base sequence having 90% or more sequence identity to a base sequence selected from the group consisting of The antisense oligonucleotide according to (5-1) or (5-2), or a pharmaceutically acceptable salt thereof, or a hydrate of these, comprising:(7-1) From the 5' end of the base sequence of SEQ ID NO: 1, positions 19 to 42, 159 to 182, 239 to 282, 319 to 342, 359 to 482, 499 to 602, 639 to 662, 679 to 762, 779 to 802, 819 to 842, 879 to 902, 919 to 1002, 1039 to 1342, 1359 to 1382, 1419 to 1442, 1459 to 1482, 1499 to 1522, 1559 to 1642, 1659 to 1702, 1779 to 1802, 1879 to 1902, 1 The antisense oligonucleotide according to any one of (1-1) to (1-3), which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 914 to 1937, positions 1979 to 2002, positions 2019 to 2042, positions 2059 to 2302, positions 2319 to 2342, positions 2359 to 2422, positions 2446 to 2542, positions 2559 to 2622, positions 2639 to 2702, positions 2939 to 2962, positions 3019 to 3042, positions 3119 to 3142, and positions 3199 to 3222, or a pharmaceutically acceptable salt thereof. (7-2) Positions 21 to 40, 161 to 180, 241 to 280, 321 to 340, 361 to 480, 501 to 600, 641 to 660, 681 to 760, 781 to 800, 821 to 840, 881 to 900, 921 to 1000, 1041 to 1340, 1361 to 1380, 1421 to 1440, 1461 to 1480, 1501 to 1520, 1561 to 1640, 1661 to 1700, 1781 to 1800, 1881 to 1900, 1901 to 2000, 2001 to 2000, 2001 to 2000, 2002 to 2002, 2003 to 2004, 2005 to 2006, 2007 to 2008, 2009 to 21009, 2110 to 2120, 2121 to 2130, 2130 to 2140, 2141 to 2150, 2151 to 2160, 2161 to 2170, 21781 to 2180, 2179 to 2200, 2182 to 2290, 2183 to 2291, 2184 to 2292, 2185 to 2293, 2186 to 2294, 2187 to 2295, 2188 to 2296 00, 1916 to 1935, 1981 to 2000, 2021 to 2040, 2061 to 2300, 2321 to 2340, 2361 to 2420, 2448 to 2540, 2561 to 2620, 2641 to 2700, 2941 to 2960, 3021 to 3040, 3121 to 3140, and 3201 to 3220. The antisense oligonucleotide according to (7-1), or a pharmaceutically acceptable salt thereof, is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 00, 1916 to 1935, 1981 to 2000, 2021 to 2040, 2061 to 2300, 2321 to 2340, 2361 to 2420, 2448 to 2540, 2561 to 2620, 2641 to 2700, 2941 to 2960, 3021 to 3040, 3121 to 3140, and 3201 to 3220.(8) (i) SEQ ID NOs: 3, 7, 11-12, 15, 17-22, 24-29, 32, 34-37, 39, 41-42, 44-48, 51-64, 66, 68, 72, 74, 77, 80-83, 85-86, 91, 94, 96, 100, 102, 105-120, 122, 124-126, 129-133, 136-138, 140, 142-143, 162, 167, 172, and 176; (ii) SEQ ID NOs: 3, 7, 11 to 12, 15, 17 to 22, 24 to 29, 32, 34 to 37, 39, 41 to 42, 44 to 48, 51 to 64, 66, 68, 72, 74, 77, 80 to 83, 85 to 86, 91, 94, 96, 100, 102, 105 to 120, 122, 124 to 126, 129 to 133, 136 to 138, 140, 142 to 143, 162, 167, 172, and 176. A base sequence in which one or several bases are added, deleted, or substituted, or (iii) The antisense oligonucleotide according to (7-1) or (7-2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, comprising a base sequence having 90% or more sequence identity to a base sequence selected from the group consisting of SEQ ID NOs: 3, 7, 11 to 12, 15, 17 to 22, 24 to 29, 32, 34 to 37, 39, 41 to 42, 44 to 48, 51 to 64, 66, 68, 72, 74, 77, 80 to 83, 85 to 86, 91, 94, 96, 100, 102, 105 to 120, 122, 124 to 126, 129 to 133, 136 to 138, 140, 142 to 143, 162, 167, 172, and 176.(9-1) Positions 239 to 262, 359 to 402, 419 to 462, 519 to 542, 559 to 602, 699 to 722, 819 to 842, 879 to 902, 919 to 982, 1091 to 1122, 1139 to 1162, 1179 to 1202, 1239 to 1262, 1279 to 1342, 1359 to 1382, 1459 to 1482, 1499 to 1522, 1559 to 1602, 1659 to 1682 from the 5' end of the base sequence of SEQ ID NO: 1 the antisense oligonucleotide according to any one of (1-1) to (1-3), which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 1779 to 1802, 1879 to 1902, 1979 to 2002, 2019 to 2042, 2059 to 2122, 2139 to 2216, 2219 to 2242, 2259 to 2302, 2446 to 2469, 2479 to 2502, and 2659 to 2682, or a pharmaceutically acceptable salt thereof. (9-2) positions 241 to 260, 361 to 400, 421 to 460, 521 to 540, 561 to 600, 701 to 720, 821 to 840, 881 to 900, 921 to 980, 1093 to 1120, 1141 to 1160, 1181 to 1200, 1241 to 1260, 1281 to 1340, 1361 to 1380, 1461 to 1480, 1501 to 1520, 1561 to 1600, 1661 The antisense oligonucleotide according to (9-1), or a pharmaceutically acceptable salt thereof, is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 1680 to 1780, 1881 to 1900, 1981 to 2000, 2021 to 2040, 2061 to 2120, 2141 to 2214, 2221 to 2240, 2261 to 2300, 2448 to 2467, 2481 to 2500, and 2661 to 2680.(10) (i) a base sequence selected from the group consisting of SEQ ID NOs: 11, 17-18, 20-21, 25, 27-29, 35, 41-42, 44, 46-47, 54-55, 57, 59, 62, 64, 66, 68, 74, 77, 80-81, 85, 91, 94, 100, 102, 105-108, 110-114, 116, 118-120, 129, 131, and 142; (ii) a base sequence selected from the group consisting of SEQ ID NOs: 11, 17-18, 20-21, 25, 27-29, 35, 41-42, 44, 46-47, 54-55, 57, 59, 62, 64, 66, 68, 74, 77, 80-81, 85, 91, 94, 100, 102, 105-108, 110-114, 116, 118-120, 129, 131, and 142, in which one or several bases are added, deleted, or substituted; or (iii) The antisense oligonucleotide according to (9-1) or (9-2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, comprising a base sequence having 90% or more sequence identity to a base sequence selected from the group consisting of SEQ ID NOs: 11, 17-18, 20-21, 25, 27-29, 35, 41-42, 44, 46-47, 54-55, 57, 59, 62, 64, 66, 68, 74, 77, 80-81, 85, 91, 94, 100, 102, 105-108, 110-114, 116, 118-120, 129, 131, and 142. (11) The antisense oligonucleotide according to any one of (2), (4), (6), (8), and (10), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, comprising the base sequence of (i).(12-1) An antisense oligonucleotide consisting of 15 to 22 nucleotides, which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 194 to 217, 350 to 452, 500 to 612, 629 to 684, 689 to 732, 821 to 844, 874 to 972, 1089 to 1177, 1277 to 1338, 1349 to 1392, 1444 to 1537, 1544 to 1592, 1644 to 1697, 1769 to 1812, 1969 to 2012, 2051 to 2317, and 2469 to 2512 from the 5' end of the base sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof. (12-2) positions 196 to 215, 352 to 374, 376 to 413, 416 to 450, 502 to 610, 631 to 650, 653 to 682, 691 to 730, 823 to 842, 876 to 970, 1091 to 1175, 1279 to 1336, 1351 to 1390, and 1446 to 1535 from the 5' end of the base sequence of SEQ ID NO: 1 , 1546 to 1590, 1646 to 1695, 1771 to 1810, 1971 to 2010, 2053 to 2250, 2252 to 2315, and 2471 to 2510, or a pharmaceutically acceptable salt thereof.(In the above (12-1) to (12-2), it is preferable to exclude an antisense oligonucleotide having a sequence consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 436 to 438, 440 to 449, 451 to 452, 454 to 455, and 472 to 474, or a pharmaceutically acceptable salt thereof; and it is preferable to exclude an antisense oligonucleotide having a sequence consisting of the nucleotide sequence of SEQ ID NO: 450, or a pharmaceutically acceptable salt thereof, in which positions 2 to 3, 5 to 6, 10 to 14, 16, 18, and 20 from the 5' end of the nucleotide sequence of SEQ ID NO: 450 are ENA: 2'-O,4'-C-Ethylenebridged Nucleic Acid, and positions 1, 4, 7 to 9, 15, 17, and 19 are ribonucleotides containing a 2'-OMe group, and / or (13) (i) a base sequence selected from the group consisting of SEQ ID NOs: 231 to 419, 424 to 425, 428 to 431, 433 to 435, and 475 to 502; (ii) a base sequence in which one or several bases are added, deleted, or substituted in a base sequence selected from the group consisting of SEQ ID NOs: 231 to 419, 424 to 425, 428 to 431, 433 to 435, and 475 to 502; or (iii) a base sequence having 90% or more sequence identity to a base sequence selected from the group consisting of SEQ ID NOs: 231 to 419, 424 to 425, 428 to 431, 433 to 435, and 475 to 502. The antisense oligonucleotide according to (12-1) or (12-2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof.(14-1) From the 5' end of the base sequence of SEQ ID NO: 1, positions 194 to 217, 350 to 452, 500 to 612, 629 to 679, 689 to 732, 821 to 844, 874 to 912, 924 to 972, 1089 to 1177, 1277 to 1338, 1349 to 1392, 1449 to 1484, 1504 to 1532, 154 The antisense oligonucleotide according to (12-1), or a pharmaceutically acceptable salt thereof, is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 4 to 1592, positions 1644 to 1697, positions 1769 to 1812, positions 1969 to 2012, positions 2051 to 2252, positions 2264 to 2317, and positions 2476 to 2512. (14-2) positions 196 to 215, 352 to 450, 502 to 610, 631 to 650, 653 to 677, 691 to 730, 823 to 842, 876 to 910, 926 to 970, 1091 to 1175, 1279 to 1336, 1351 to 1390, 1451 to 1482, 1506 to 1530 from the 5' end of the base sequence of SEQ ID NO: 1 The antisense oligonucleotide according to (14-1), or a pharmaceutically acceptable salt thereof, is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 1546 to 1590, 1646 to 1695, 1771 to 1810, 1971 to 2010, 2053 to 2250, 2266 to 2315, and 2478 to 2510.(15) (i) a base sequence selected from the group consisting of SEQ ID NOs: 231 to 242, 244, 246 to 268, 270 to 271, 273 to 285, 288, 290 to 292, 296 to 297, 299 to 390, 392 to 408, 412 to 417, 419, 475 to 476, 478 to 497, and 499 to 502; (ii) SEQ ID NOs: 231 to 242, 244, 246 to 268, 270 to 271, 273 to 285, 288, 290 to 292, 296 to 297, 299 to 390, 392 to 408, 412 to 417, 419, 475 to 476, 478 to 497, and 499 to 502. A base sequence in which one or several bases are added, deleted, or substituted, or (iii) SEQ ID NOs: 231 to 242, 244, 246 to 268, 270 to 271, 273 to 285, 288, 290 to 292, 296 to 297, 299 to 390, 392 to 408, 412 to 417, 419, 475 to 476, 478 to 497, and 499 to 502. The antisense oligonucleotide according to (14-1) or (14-2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, contains a base sequence having 90% or more sequence identity to the base sequence selected from the group consisting of: (16-1) From the 5' end of the base sequence of SEQ ID NO: 1, positions 351 to 376, 384 to 452, 509 to 552, 569 to 607, 656 to 679, 821 to 844, 874 to 912, 927 to 952, 1109 to 1177, 1277 to 1338, 1364 to 1392, 1449 to 1484, 1504 to 1532, 1544 to 1587, 164 The antisense oligonucleotide according to (12-1), or a pharmaceutically acceptable salt thereof, is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 6 to 1697, positions 1774 to 1812, positions 1984 to 2007, positions 2054 to 2127, positions 2131 to 2215, positions 2229 to 2252, positions 2264 to 2312, and positions 2478 to 2512.(16-2) From the 5' end of the base sequence of SEQ ID NO: 1, positions 353 to 374, 386 to 450, 511 to 550, 571 to 605, 658 to 677, 823 to 842, 876 to 910, 929 to 950, 1111 to 1175, 1279 to 1336, 1366 to 1390, 1451 to 1482, 1506 to 1530, 1546 to 1585, 164 The antisense oligonucleotide according to (16-1), or a pharmaceutically acceptable salt thereof, is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 8 to 1695, positions 1776 to 1810, positions 1986 to 2005, positions 2056 to 2125, positions 2133 to 2213, positions 2231 to 2250, positions 2266 to 2310, and positions 2480 to 2510.(17) (i) SEQ ID NOs: 232-234, 236-238, 240-241, 244, 247-255, 258, 262-264, 268, 274-285, 290, 292, 296-297, 299-300, 302, 306-312, 314-316, 318-320, 323, 328 a base sequence selected from the group consisting of: (ii) SEQ ID NOs: 232-234, 236-238, 240-241, 244, 247-255, 258, 262-264, 268, 274-285, 290, 292, 296-297, 299-300, 302, 306-312, 314-316, 318-320, 323, 328-340, 342-350, 353-3 A base sequence selected from the group consisting of 59, 361-377, 380-382, 384-387, 390, 392-399, 402-407, 414-415, 419, 478, 482-483, 485-487, 489-496, and 499-502, in which one or several bases have been added, deleted, or substituted; or (iii) SEQ ID NOs: 232-234, 236-238, 240-241, 244, 247-255, 258, 262-264, 268, 274-285, 290, 292, 296-297, 299-300, 302, 306-312, 314-316, 318-320, 323, 328-340, 342-350, 353-359, 361-377, 380-382, 384-387 , 390, 392 to 399, 402 to 407, 414 to 415, 419, 478, 482 to 483, 485 to 487, 489 to 496, and 499 to 502. The antisense oligonucleotide according to (16-1) or (16-2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, comprising a base sequence having 90% or more sequence identity to a base sequence selected from the group consisting of:(18-1) The antisense oligonucleotide according to (12-1), which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 409 to 452, 509 to 548, 656 to 679, 874 to 907, 927 to 950, 1129 to 1172, 1294 to 1333, 1449 to 1484, 1504 to 1532, 1549 to 1587, 1646 to 1697, 1784 to 1807, 2057 to 2117, 2131 to 2215, 2266 to 2312, and 2478 to 2501 from the 5' end of the base sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof. (18-2) The antisense oligonucleotide according to (18-1), which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 411 to 450, 511 to 546, 658 to 677, 876 to 905, 929 to 948, 1131 to 1170, 1296 to 1331, 1451 to 1482, 1506 to 1530, 1551 to 1585, 1648 to 1695, 1786 to 1805, 2059 to 2115, 2133 to 2213, 2268 to 2310, and 2480 to 2499 from the 5' end of the base sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.(19) (i) SEQ ID NOs: 240-241, 244, 249-252, 254-255, 268, 275, 277-281, 284, 290, 292, 296-297, 300, 302, 306-312, 315-316, 319, 329-331, 334-339, 343-349, 353 a base sequence selected from the group consisting of 359, 361-365, 367-371, 375-377, 381-382, 384, 386-387, 393-394, 396-399, 402-405, 407, 414, 478, 482, 487, 489, 490, 493, 495, and 502; (ii) SEQ ID NOs: 240-241, 244, 249-252, 254-255, 268, 275, 277-281, 284, 290, 292, 296-297, 300, 302, 306-312, 315-316, 319, 329-331, 334-339, 343-349, 353-359, 361-365, 367-3 A base sequence in which one or several bases are added, deleted, or substituted in a base sequence selected from the group consisting of 71, 375-377, 381-382, 384, 386-387, 393-394, 396-399, 402-405, 407, 414, 478, 482, 487, 489, 490, 493, 495, and 502, or (iii) SEQ ID NOs: 240-241, 244, 249-252, 254-255, 268, 275, 277-281, 284, 290, 292, 296-297, 300, 302, 306-312, 315-316, 319, 329-331, 334-339, 343-349, 353-359, 361-365, 367-371, 375-377, 381-382, 384, 386 The antisense oligonucleotide according to (18-1) or (18-2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, comprising a base sequence having 90% or more sequence identity to a base sequence selected from the group consisting of 393-394, 396-399, 402-405, 407, 414, 478, 482, 487, 489, 490, 493, 495, and 502.(20-1) The antisense oligonucleotide according to (12-1), which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 520 to 548, 880 to 903, 927 to 950, 1129 to 1172, 1461 to 1484, 1509 to 1532, 1549 to 1572, 1647 to 1697, 1784 to 1807, 2069 to 2117, 2131 to 2215, and 2269 to 2305 from the 5' end of the base sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof. (20-2) The antisense oligonucleotide according to (20-1), which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 522 to 546, 882 to 901, 929 to 948, 1131 to 1170, 1463 to 1482, 1511 to 1530, 1551 to 1570, 1649 to 1671, 1676 to 1695, 1786 to 1805, 2071 to 2115, 2133 to 2213, and 2271 to 2303 from the 5' end of the base sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.(21) (i) SEQ ID NOs: 250-251, 254-255, 280, 292, 297, 300, 307-310, 316, 319, 335-337, 343, 346, 349, 353-356, 359, 361-364, 367, 371, 375, 377, 381-382, 384, 387, 396-399, 402-403, 405, 482, 487, 489, and 490; (ii) SEQ ID NOs: 250 to 251, 254 to 255, 280, 292, 297, 300, 307 to 310, 316, 319, 335 to 337, 343, 346, 349, 353 to 356, 359, 361 to 364, 367, 371, 375, 377, 381 to 382, ​​384, 387, 396 to 399, 402 to 403, 405, 482, 487, 489, and 490. A base sequence in which one or several bases are added, deleted, or substituted in the base sequence selected from the group consisting of: (iii) SEQ ID NOs: 250-251, 254-255, 280, 292, 297, 300, 307-310, 316, 319, 335-337, 343, 346, 349, 353-356, 359, 361-364, 367, 371, 375, 377, 381-382, 384, 387, 396-399, 402-403, 405, 482, 487, 489, and 490. The antisense oligonucleotide according to (20-1) or (20-2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, comprising a base sequence having 90% or more sequence identity to the base sequence selected from the group consisting of: (22) The antisense oligonucleotide according to (12-1) or (12-2), which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 521 to 545, 881 to 900, 1461 to 1480, 1561 to 1580, 1649 to 1670, 1676 to 1695, 2061 to 2105, 2134 to 2213, 2271 to 2299, and 2481 to 2500 from the 5' end of the base sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.(23) (i) a base sequence selected from the group consisting of SEQ ID NOs: 25, 42, 74, 80, 105-107, 112, 119, 131, 250-251, 254, 307-309, 316, 337, 346, 354-356, 363-364, 371, 381, 387, 396, 398-399, and 402; (ii) a base sequence selected from the group consisting of SEQ ID NOs: 25, 42, 74, 80, 105-107, 112, 119, 131, 250-251, 254, 307-309, 316, 337, 346, 354-356, 363-364, 371, 381, 387, 396, 398-399, and 402, in which one or several bases are added, deleted, or substituted; or (iii) The antisense oligonucleotide according to (20-1) or (20-2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, comprising a base sequence having 90% or more sequence identity to a base sequence selected from the group consisting of SEQ ID NOs: 25, 42, 74, 80, 105-107, 112, 119, 131, 250-251, 254, 307-309, 316, 337, 346, 354-356, 363-364, 371, 381, 387, 396, 398-399, and 402. (24) The antisense oligonucleotide according to any one of (13), (15), (17), (19), (21), and (23), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, comprising the base sequence of (i). (25-1) The antisense oligonucleotide according to any one of (1) to (24), or a pharmaceutically acceptable salt thereof, or a hydrate of either, wherein the antisense oligonucleotide consists of 20 nucleotides. (25-2) The antisense oligonucleotide according to any one of (1) to (24), or a pharmaceutically acceptable salt thereof, or a hydrate of either, wherein the antisense oligonucleotide consists of 15 to 20 nucleotides. (26) The antisense oligonucleotide according to any one of (1) to (25), or a pharmaceutically acceptable salt thereof, or a hydrate of either, which reduces the expression level of the TDP-43 gene.(27) The antisense oligonucleotide according to any one of (1) to (26), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, wherein the sugar moiety and / or the phosphate linkage moiety of at least one nucleotide constituting the oligonucleotide is modified. (28-1) The antisense oligonucleotide according to (27), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, wherein the antisense oligonucleotide is a gapmer comprising a central gap region and two wing regions (5' wing region and 3' wing region) flanking the 5'-end and 3'-end of the gap region. (28-2) The antisense oligonucleotide according to (28-1), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, wherein the antisense oligonucleotide is composed, from the 5'-end to the 3'-end, of a 5' wing region 5 nucleotides in length, a gap region 10 nucleotides in length, and a 3' wing region 5 nucleotides in length. (29) The antisense oligonucleotide according to (28-1) or (28-2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, wherein all internucleoside bonds are phosphorothioate bonds. (30) The antisense oligonucleotide according to (28-1) or (28-2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, wherein one or more of the bonds between the second and third nucleosides, the third and fourth nucleosides, and the fourth and fifth nucleosides from the 5' side of the 5' wing region are phosphodiester bonds, and / or one or more of the bonds between the first and second nucleosides, the second and third nucleosides, and the third and fourth nucleosides from the 5' side of the 3' wing region are phosphodiester bonds, and all other internucleoside bonds are phosphorothioate bonds.(31) The antisense oligonucleotide according to (28-1) or (28-2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, wherein the bond between the second and third nucleosides and the bond between the fourth and fifth nucleosides from the 5' side of the 5' wing region are phosphodiester bonds, and / or the bond between the first and second nucleosides and the bond between the third and fourth nucleosides from the 5' side of the 3' wing region are phosphodiester bonds, and all other internucleoside bonds are phosphorothioate bonds. (32) The antisense oligonucleotide according to (28-1) or (28-2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, wherein the bond between the first and second nucleosides from the 5' side of the gap region is phosphodiester bond, and all other internucleoside bonds are phosphorothioate bonds. (33) The antisense oligonucleotide according to (28-1) or (28-2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, wherein the bond between the second and third nucleosides from the 5' side of the gap region is a phosphodiester bond, and all other internucleoside bonds are phosphorothioate bonds. (34) The antisense oligonucleotide according to (28-1) or (28-2), or a pharmaceutically acceptable salt thereof, or a hydrate thereof, wherein the bond between the second and third nucleosides from the 5' side of the 5' wing region and the bond between the fourth and fifth nucleosides are phosphodiester bonds, and / or the bond between the first and second nucleosides from the 5' side of the 3' wing region and the bond between the third and fourth nucleosides are phosphodiester bonds, and / or the bond between the first and second nucleosides from the 5' side of the gap region is phosphodiester bond, and all other internucleoside bonds are phosphorothioate bonds.(35) The antisense oligonucleotide according to (28-1) or (28-2), wherein the bond between the second and third nucleosides from the 5' side and the bond between the fourth and fifth nucleosides from the 5' side in the 5' wing region are phosphodiester bonds, and / or the bond between the first and second nucleosides from the 5' side and the bond between the third and fourth nucleosides from the 5' side in the 3' wing region are phosphodiester bonds, and / or the bond between the second and third nucleosides from the 5' side in the gap region is a phosphodiester bond, and all other internucleoside bonds are phosphorothioate bonds. (36) The antisense oligonucleotide according to (28-1) or (28-2), wherein the wing and / or gap region is 2'-OMe(2'-O-CH. ３ ) group and / or 2'-O-MOE(2'-O-CH ２ CH ２ OCH ３ (37) The antisense oligonucleotide according to any one of (28-1) to (35), or a pharmaceutically acceptable salt thereof, or a hydrate of the same, wherein the second nucleotide from the 5' end of the gap region is a 2'-OMe(2'-O-CH ３ ) group or 2'-O-MOE(2'-O-CH ２ CH ２ OCH ３(36) The antisense oligonucleotide according to (36), or a pharmaceutically acceptable salt thereof, or a hydrate of these, which is a ribonucleotide having a 2-amino-3-methyl-2-propanol group. (38) A pharmaceutical composition comprising the antisense oligonucleotide according to any of (1) to (37), or a pharmaceutically acceptable salt thereof, or a hydrate of these. (39) The antisense oligonucleotide according to any of (1) to (37), or a pharmaceutically acceptable salt thereof, or a hydrate of these, or the pharmaceutical composition according to (38), for treating and / or preventing TDP-43 proteinopathy. (40-1) A method for treating and / or preventing TDP-43 proteinopathy, comprising the step of administering to a subject the antisense oligonucleotide according to any of (1) to (37), or a pharmaceutically acceptable salt thereof, or a hydrate of these, or the pharmaceutical composition according to (38). (40-2) Use of the antisense oligonucleotide according to any one of (1) to (37) or a pharmaceutically acceptable salt thereof or a hydrate thereof, or the pharmaceutical composition according to (38), in the manufacture of a medicament for use in a method for treating and / or preventing TDP-43 proteinopathy. (40-3) The pharmaceutical composition according to (39), the method according to (40-1), or the use according to (40-2), wherein the TDP-43 proteinopathy is selected from the group consisting of amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Perry syndrome, and Lewy body disease.

[0008] The antisense oligonucleotide of the present invention preferably excludes (does not include) antisense oligonucleotides consisting of the following base sequences:

[0009] The present invention provides an antisense oligonucleotide that targets TDP-43 mRNA or pre-mRNA, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, as well as a composition comprising the antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, or a hydrate thereof.

[0010] In a preferred embodiment of the present invention, a therapeutic agent for TDP-43 proteinopathy with high therapeutic satisfaction can be provided, since it acts directly on the transcript of the TDP-43 gene, which is the causative gene for TDP-43 proteinopathy, thereby inhibiting TDP-43 expression. Since the present invention allows antisense oligonucleotides to be designed by targeting the transcript of the TDP-43 gene, which is the causative gene for TDP-43 proteinopathy, a preferred embodiment of the present invention can provide a therapeutic agent for TDP-43 proteinopathy with few side effects. According to one embodiment of the present invention, it is also possible to provide a tailored treatment based on the genetic information of an individual patient.

[0011] In one embodiment, the present invention relates to an antisense oligonucleotide consisting of 15 to 22 nucleotides, or a pharmaceutically acceptable salt or hydrate thereof, which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 1 to 102, 159 to 842, 879 to 1822, and 1874 to 4182 from the 5' end of the base sequence of SEQ ID NO: 1. Alternatively, in one embodiment, the present invention relates to an antisense oligonucleotide consisting of 15 to 30 nucleotides, or a pharmaceutically acceptable salt or hydrate thereof, which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 1 to 102, 159 to 842, 879 to 1822, and 1874 to 4182 from the 5' end of the base sequence of SEQ ID NO: 1. Alternatively, in one embodiment, the present invention relates to an antisense oligonucleotide consisting of 15 to 25 nucleotides, which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 1 to 102, 159 to 842, 879 to 1822, and 1874 to 4182 from the 5' end of the base sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt or hydrate thereof.

[0012] SEQ ID NO: 1 is the mRNA sequence of human TDP-43 (Gen Bank: NM_007375.4), which is a base sequence containing 4185 bases.

[0013] In one embodiment, the antisense oligonucleotides of the invention are complementary to a nucleic acid comprising or consisting of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, e.g., 20, consecutive bases in the target region.

[0014] In this specification, a base that is "complementary" to a certain base means a base that forms a base pair with the target base, and is not limited to bases that form Watson-Crick base pairs, but also includes bases that form wobble base pairs or Hoogsteen base pairs. Here, Watson-Crick base pairs refer to base pairs in which the acceptor of the hydrogen donated from the N3 position of the pyrimidine base in hydrogen bonds between adenine-thymine, adenine-uracil, and guanine-cytosine is the N1 position of the purine base, and wobble base pairs refer to base pairs in which hydrogen bonds are formed between guanine-uracil, inosine-uracil, inosine-adenine, and inosine-cytosine. The Hoogsteen base pair refers to a base pair in which the acceptor of the hydrogen donated from the N3 position of the pyrimidine base in the hydrogen bond between adenine-thymine, adenine-uracil, and guanine-cytosine is the N7 position of the purine base.

[0015] An example of an antisense oligonucleotide that is "complementary" to a certain nucleic acid is an antisense oligonucleotide that can hybridize to that nucleic acid, for example, under stringent conditions. As used herein, "stringent conditions" may refer to low stringency conditions, moderate stringency conditions, or high stringency conditions. "Low stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C. "Medium stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 42°C, or 5x SSC, 1% SDS, 50 mM Tris-HCl (pH 7.5), 50% formamide, and 42°C. "Highly stringent conditions" are, for example, conditions of 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 50°C, or 0.2x SSC, 0.1% SDS, and 65°C. Under these conditions, it is expected that the higher the temperature, the more efficiently base sequences with higher sequence identity can be obtained. However, several factors are thought to affect the stringency of hybridization, such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration, and those skilled in the art can achieve similar stringency by appropriately selecting these factors.

[0016] When using a commercially available kit for hybridization, for example, the AlkPhos Direct Labeling and Detection System (GE Healthcare) can be used. In this case, after incubation with the labeled probe overnight according to the protocol provided with the kit, the membrane can be washed with a primary wash buffer containing 0.1% (w / v) SDS at 55°C, and then hybridization can be detected. Alternatively, when preparing a probe based on a target sequence, if the probe is labeled with digoxigenin (DIG) using a commercially available reagent (e.g., PCR Labeling Mix (Roche Diagnostics)), hybridization can be detected using a DIG Nucleic Acid Detection Kit (Roche Diagnostics), etc.

[0017] In this specification, the identity of nucleotide sequences can be determined using the BLAST (Basic Local Alignment Search Tool) algorithm by Carlin and Altschul (Proc. Natl. Acad. Sci. USA, 1990, Vol. 87, pp. 2264-2268; Proc. Natl. Acad. Sci. USA, 1993, Vol. 90, pp. 5873-5877). Programs based on the BLAST algorithm, such as BLASTN and BLASTX, have been developed (Altschul S. F. et al., J. Mol. Biol., 1990, Vol. 215, pp. 403-410). When analyzing a base sequence using BLASTN, the parameters are, for example, score = 100 and word length = 12. When using BLAST and Gapped BLAST programs, the default parameters of each program are used.

[0018] In one embodiment, the antisense oligonucleotide may be, for example, 15 or more nucleotides, 16 or more nucleotides, 17 or more nucleotides, 18 or more nucleotides, 19 or more nucleotides, 20 or more nucleotides, 21 or more nucleotides, or 22 nucleotides in length, or, for example, 22 or less nucleotides, 21 or less nucleotides, 20 or less nucleotides, 19 or less nucleotides, 18 or less nucleotides, 17 or less nucleotides, 16 or less nucleotides, or 15 nucleotides in length. In one embodiment, the antisense oligonucleotide may consist of 15 to 22 nucleotides, 15 to 21 nucleotides, 15 to 20 nucleotides, 15 to 19 nucleotides, 15 to 18 nucleotides, 15 to 17 nucleotides, 15 to 16 nucleotides, 18 to 22 nucleotides, 19 to 21 nucleotides, for example, 20 nucleotides. In one embodiment, the antisense oligonucleotide may be, for example, 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides in length. In addition, the nucleotide length of the antisense oligonucleotide in one embodiment may be NLa to NLb. Here, NLa and NLb are natural numbers between 15 and 22, and NLa<NLb.

[0019] In another embodiment, the antisense nucleotide may be 23 or more nucleotides in length, 24 or more nucleotides in length, 25 or more nucleotides in length, 26 or more nucleotides in length, 27 or more nucleotides in length, 28 or more nucleotides in length, 29 or more nucleotides in length, or 30 nucleotides in length, or 30 or less nucleotides in length, 29 or less nucleotides in length, 28 or less nucleotides in length, 27 or less nucleotides in length, 26 or less nucleotides in length, 25 or less nucleotides in length, 24 or less nucleotides in length, or 23 or less nucleotides in length. In another embodiment, the antisense oligonucleotide may be, for example, 23 to 30 nucleotides, 23 to 25 nucleotides (e.g., 23 to 24 nucleotides, or 23 nucleotides). In another embodiment, the antisense oligonucleotide may be, for example, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In another embodiment, the nucleotide length of the antisense oligonucleotide may be NLc to NLd, where NLc and NLd are natural numbers from 23 to 30, and NLc < NLd.

[0020] Examples of pharmaceutically acceptable salts of the antisense oligonucleotides of the present invention include alkali metal salts such as sodium salt, potassium salt, and lithium salt, and alkaline earth metal salts such as calcium salt and magnesium salt; metal salts such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; ammonium salt; t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, Examples of suitable salts include organic amine salts such as piperazine salts, tetramethylammonium salts, and tris(hydroxymethyl)aminomethane salts; hydrohalide salts such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkane sulfonate salts such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonate salts such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate. Preferred examples of pharmaceutically acceptable salts of the antisense oligonucleotides of the present invention include triethylamine salts and sodium salts. These salts can be prepared by known methods. Alternatively, the antisense oligonucleotides of the present invention may be in the form of their hydrates.

[0021] The antisense oligonucleotides of the present invention are composed of nucleotides as building blocks, and such nucleotides may be ribonucleotides, deoxyribonucleotides, or modified nucleotides.

[0022] A modified nucleotide refers to a ribonucleotide or deoxyribonucleotide in which all or part of the nucleic acid base, sugar moiety, and phosphate linkage moiety that constitute the ribonucleotide or deoxyribonucleotide have been modified.

[0023] Examples of nucleic acid bases include adenine, guanine, hypoxanthine, cytosine, thymine, uracil, and modified bases thereof. Examples of such modified bases include pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (e.g., 5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (e.g., 6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 1-methyluracil, 1-methylhydroxy ... Examples of amino acids include xanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, indole, imidazole, and xanthine.

[0024] As used herein, thymine "T" and uracil "U" are interchangeable, and since the activity of the antisense oligonucleotide of the present invention is not essentially affected whether "T" or "U" is used, the base sequences shown herein include cases where "T" is replaced with "U," and are represented by the same SEQ ID NO. Also, as used herein, sequences containing modified bases are represented by the same SEQ ID NO as sequences not containing modified bases; for example, "cytosine" and "methylcytosine" are interchangeable, and when "cytosine" is replaced with "methylcytosine," the same SEQ ID NO is used.

[0025] Modifications of the sugar moiety include, for example, modifications of the 2'-position of ribose and modifications of other parts of the sugar. Modifications of the 2'-position of ribose include, for example, replacing the -OH group at the 2'-position of ribose with -OR, -OROR, -R, -R'OR, -SH, -SR, -NH ２ , -NHR, -NR ２ , -N ３ , —CN, —F, —Cl, —Br, —I, for example, —OMe(—O—CH ３ ) or -O-methoxyethyl (-O-MOE: -O-CH ２ CH ２ OCH ３ ) where R represents alkyl or aryl, and R' represents alkylene.

[0026] Modifications of other sugar moieties include, but are not limited to, substitution of the O at the 4' position of ribose or deoxyribose with S, and cross-linking of the 2' and 4' positions of the sugar, such as LNA (Locked Nucleic Acid) and ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acids). The cross-linking site may also be modified with urea or guanidine.

[0027] Modifications of the phosphate linkage moiety include, for example, modifications in which the phosphodiester bond is replaced with a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond, or a boranophosphate bond (see, for example, Enya et al., Bioorganic & Medicinal Chemistry, 2008, 18, pp. 9154-9160) (see, for example, Republished Patent Publication No. 2006 / 129594 and Republished Patent Publication No. 2006 / 038608).

[0028] In this specification, the alkyl is preferably a linear or branched alkyl having 1 to 6 carbon atoms. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, and isohexyl. The alkyl may be substituted, and examples of such substituents include halogen, alkoxy, cyano, and nitro, and the alkyl may be substituted with 1 to 3 of these.

[0029] In this specification, halogen includes fluorine, chlorine, bromine, and iodine.

[0030] In this specification, examples of alkoxy include straight-chain or branched-chain alkoxy having 1 to 6 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy, isohexyloxy, etc. In particular, alkoxy having 1 to 3 carbon atoms is preferred.

[0031] In this specification, the aryl is preferably an aryl having 6 to 10 carbon atoms. Specific examples include phenyl, α-naphthyl, and β-naphthyl. Phenyl is particularly preferred. The aryl may be substituted, and examples of such substituents include alkyl, halogen, alkoxy, cyano, and nitro, and the aryl may be substituted with 1 to 3 of these.

[0032] In this specification, the alkylene is preferably a linear or branched alkylene having 1 to 6 carbon atoms, such as methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, 2-(ethyl)trimethylene, and 1-(methyl)tetramethylene.

[0033] In one embodiment, the present invention provides a gapmer in which the antisense oligonucleotide comprises a central gap region and two wing regions (also referred to as the 5' wing region and the 3' wing region) flanking the 5'- and 3'-ends of the gap region. The gap region is a region recognized by RNase H, and at least its 5'- and 3'-ends are composed of deoxyribonucleotides with unmodified sugar moieties. The gap region may be entirely composed of deoxyribonucleotides with unmodified sugar moieties, or may contain one or more nucleotides with modified sugar moieties. For example, the gap region may contain one nucleotide with a modified sugar moiety at the second position from the 5'-end of the gap region, and all the remaining nucleotides may be deoxyribonucleotides with unmodified sugar moieties. The modified nucleotide may be, for example, a ribonucleotide modified at the 2'-position of the ribose, and may contain a 2'-OMe group and / or a 2'-O-MOE group. In one embodiment, the second nucleotide from the 5'-end of the gap region is 2'-OMe (2'-O-CH ３ ) group or 2'-O-MOE(2'-O-CH ２ CH ２ OCH ３ ) group.

[0034] The wing regions contain at least one modified nucleotide, for example, all of the nucleosides are modified nucleotides (e.g., ribonucleotides modified at the 2'-position of the ribose). In one embodiment, the nucleosides in the 5' wing region and the 3' wing region each contain at least one, for example, two or more, three or more, four or more, or five or more sugar moiety modifications, such as a 2'-OMe group and / or a 2'-O-MOE group. For example, all of the nucleosides in the 5' wing region and the 3' wing region may contain a 2'-OMe group and / or a 2'-O-MOE group, for example, a 2'-O-MOE group. Furthermore, the nucleosides in the 5' wing region and the 3' wing region may contain a modification in the base moiety, for example, at least one methylcytosine, for example, all of the cytosines contained may be methylcytosines.

[0035] The length of the gap region is not limited, but may be, for example, 5 to 15, 8 to 12, 9 to 11, or 10 bases long. The lengths of the 5' wing region and the 3' wing region are not limited, but may be, for example, each independently, 2 to 10, 3 to 8, 4 to 6, or 5 bases long. In one embodiment, the gap region is 10 bases long, and the 5' wing region and the 3' wing region are each 5 bases long; such a gapmer is referred to herein as a "5-10-5 gapmer."

[0036] The antisense oligonucleotide of the present invention may be a gapmer containing a base sequence in which 1 to 2 bases have been added, deleted, or substituted in the wing regions compared to any of the gapmers in the Examples herein, or may be a gapmer containing a base sequence in which 1 to 4 bases have been added, deleted, or substituted in the gap region.

[0037] The antisense oligonucleotide of the present invention may be a 5-10-5 gapmer containing a base sequence in which one to two bases have been substituted in the wing regions of any of the gapmers in the Examples herein, or a 5-10-5 gapmer containing a base sequence in which one to four bases have been substituted in the gap region. Furthermore, the antisense oligonucleotide of the present invention may be a 5-10-5 gapmer having at least three contiguous bases in the wing regions of a gapmer in the Examples herein, or a 5-10-5 gapmer having at least six contiguous bases in the gap region of a gapmer in the Examples herein.

[0038] In one embodiment, a gapmer of the invention comprises one or more phosphate linkage modifications, e.g., phosphorothioate linkages, e.g., one or more, two or more, three or more, four or more, five or more, ten or more, fifteen or more, e.g., all, of the internucleotide linkages may be phosphorothioate linkages. In one embodiment, a gapmer of the invention does not comprise any phosphate linkage modifications, and all of the internucleotide linkages may be phosphate linkages.

[0039] In one embodiment, in a gapmer of the present invention, one or more of the bond between the second and third nucleosides from the 5' side, the bond between the third and fourth nucleosides, and the bond between the fourth and fifth nucleosides from the 5' side of the 5' wing region are phosphodiester bonds, and / or one or more of the bond between the first and second nucleosides from the 5' side of the 3' wing region, the bond between the second and third nucleosides, and the bond between the third and fourth nucleosides from the 5' side of the 3' wing region are phosphodiester bonds, and all other internucleoside bond may be phosphorothioate bonds.

[0040] In one embodiment, in a gapmer of the present invention, the bond between the second and third nucleosides from the 5' side and the bond between the fourth and fifth nucleosides from the 5' side in the 5' wing region are phosphodiester bonds, and / or the bond between the first and second nucleosides from the 5' side and the bond between the third and fourth nucleosides from the 3' wing region are phosphodiester bonds, and all other internucleoside bonds may be phosphorothioate bonds.

[0041] In one embodiment, in a gapmer of the present invention, the bond between the first and second nucleosides from the 5' side of the gap region may be a phosphodiester bond, and all other internucleoside bonds may be phosphorothioate bond.

[0042] In one embodiment, in a gapmer of the present invention, the bond between the second and third nucleosides from the 5' side of the gap region may be a phosphodiester bond, and all other internucleoside bonds may be phosphorothioate bond.

[0043] In one embodiment, in a gapmer of the present invention, the bond between the second and third nucleosides from the 5' side and the bond between the fourth and fifth nucleosides from the 5' side in the 5' wing region are phosphodiester bonds, and / or the bond between the first and second nucleosides from the 5' side and the bond between the third and fourth nucleosides from the 5' side in the 3' wing region are phosphodiester bonds, and / or the bond between the first and second nucleosides from the 5' side in the gap region is phosphodiester bond, and all other internucleoside bonds may be phosphorothioate bonds.

[0044] In one embodiment, in a gapmer of the present invention, the bond between the second and third nucleosides from the 5' side and the bond between the fourth and fifth nucleosides from the 5' side in the 5' wing region are phosphodiester bonds, and / or the bond between the first and second nucleosides from the 5' side and the bond between the third and fourth nucleosides from the 5' side in the 3' wing region are phosphodiester bonds, and / or the bond between the second and third nucleosides from the 5' side in the gap region is a phosphodiester bond, and all other internucleoside bonds may be phosphorothioate bonds.

[0045] In one embodiment, the antisense oligonucleotide of the present invention is complementary to a nucleic acid comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 1 to 102, 159 to 842, 879 to 1822, and 1874 to 4182 from the 5' end of the base sequence of SEQ ID NO: 1.

[0046] In one embodiment, the antisense oligonucleotide of the present invention preferably excludes an antisense oligonucleotide having a sequence consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 65, 436-440, 444-449, 451-452, and 454-474, or a pharmaceutically acceptable salt thereof; and preferably excludes an antisense oligonucleotide having a sequence consisting of the nucleotide sequence of SEQ ID NO: 450, or a pharmaceutically acceptable salt thereof, in which positions 2-3, 5-6, 10-14, 16, 18, and 20 from the 5' end of the nucleotide sequence of SEQ ID NO: 450 are ENA: 2'-O,4'-C-Ethylenebridged Nucleic Acid, and positions 1, 4, 7-9, 15, 17, and 19 are ribonucleotides containing a 2'-OMe group; and / or Preferably, antisense oligonucleotides or pharmaceutically acceptable salts thereof having the base sequence of SEQ ID NO: 453, in which positions 1-2, 4, 6, 8-9, 11-12, and 16-20 from the 5' end of the base sequence of SEQ ID NO: 453 are ENA: 2'-O,4'-C-Ethylenebridged Nucleic Acid, and positions 3, 5, 7, 10, and 13-15 are ribonucleotides containing 2'-OMe groups, are preferably excluded. In one embodiment, unmodified antisense oligonucleotides or pharmaceutically acceptable salts thereof having the base sequence of SEQ ID NO: 450 or SEQ ID NO: 453 are preferably excluded.

[0047] In one embodiment, the antisense oligonucleotide of the present invention is complementary to a nucleic acid comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 1 to 100, 161 to 840, 881 to 1820, and 1876 to 4180 from the 5' end of the base sequence of SEQ ID NO: 1.

[0048] In one embodiment, the antisense oligonucleotide of the present invention is from the 1st to 20th positions, 21st to 40th positions, 41st to 60th positions, 61st to 80th positions, 81st to 100th positions, 161st to 180th positions, 181st to 200th positions, 201st to 220th positions, 221st to 240th positions, 241st to 260th positions, 261st to 280th positions, 281st to 300th positions, 301st to 320th positions, 321st to 340th positions, 341st to 360th positions, 361st to 380th positions, 381st to 400th positions, 401st to 420th positions, 421st to 440th positions, 441st to 460th positions, 461st to 480th positions, 481st to 500th positions, 501st to 520th positions, 521st to 540th positions, 541st to 560th positions, 561st to 580th positions, 574th to 593rd positions, 581st to 600th positions, 601st to 620th positions, 621st to 640th positions, 641st to 660th positions, 661st to 680th positions, 681st to 700th positions, 701st to 720th positions, 721st to 740th positions, 741st to 760th positions, 761st to 780th positions, 781st to 800th positions, 801st to 820th positions, 821st to 840th positions, 881st to 900th positions, 901st to 920th positions, 921st to 940th positions, 933rd to 952nd positions, 941st to 960th positions, 961st to 980th positions, 981st to 1000th positions, 1001st to 1020th positions, 1021st to 1040th positions, 1041st to 1060th positions, 1061st to 1080th positions, 1081st to 1100th positions, 1093rd to 1112nd positions, 1101st to 1120th positions, 1121st to 1140th positions, 1141st to 1160th positions, 1161st to 1180th positions, 1181st to 1200th positions, 1201st to 1220th positions, 1221st to 1240th positions, 1241st to 1260th positions, 1261st to 1280th positions, 1281st to 1300th positions, 1301st to 1320th positions, 1321st to 1340th positions, 1341st to 1360th positions, 1361st to 1380th positions, 1381st to 1400th positions, 1394th to 1413th positions,1741st to 1760th, 1761st to 1780th, 1781st to 1800th, 1801st to 1820th, 1876th to 1895th, 1881st to 1900th, 1901st to 1920th, 1916th to 1935th, 1921st to 1940th, 1941st to 1960th, 1961st 1980, 1981-2000, 2001-2020, 2021-2040, 2033-2052, 2041-2060, 2061-2080, 2073-2092, 2081-2100, 2101-2120, 2121-214 0, 2141-2160, 2153-2172, 2161-2180, 2181-2200, 2195-2214, 2201-2220, 2221-2240, 2241-2260, 2261-2280, 2275-2294, 2 281st to 2300th, 2301st to 2320th, 2321st to 2340th, 2341st to 2360th, 2361st to 2380th, 2381st to 2400th, 2401st to 2420th, 2421st to 2440th, 2441st to 2460th, 2448th to 2467th, 2461st to 2480, 2481 to 2500, 2501 to 2520, 2521 to 2540, 2528 to 2547, 2541 to 2560, 2561 to 2580, 2581 to 2600, 2601 to 2620, 2621 to 2640, 2641 to 2660 2647th to 2666th, 2661st to 2680th, 2681st to 2700th, 2686th to 2705th, 2701st to 2720th, 2721st to 2740th, 2733rd to 2752nd, 2741st to 2760th, 2761st to 2780th, 2766th to 2785th, 278 1st to 2800th, 2801st to 2820th, 2807th to 2826th, 2821st to 2840th, 2841st to 2860th, 2847th to 2866th, 2861st to 2880th, 2881st to 2900th, 2887th to 2906th, 2901st to 2920th, 2921st to 2 940, 2941-2960, 2961-2980, 2981-3000, 3001-3020, 3005-3024, 3021-3040, 3041-3060, 3061-3080, 3081-3100, 3101-3120,It is complementary to a nucleic acid containing at least 15, for example at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 3121 to 3140, 3141 to 3160, 3161 to 3180, 3181 to 3200, 3201 to 3220, 3221 to 3240, 3241 to 3260, 3261 to 3280, 3281 to 3300, 3301 to 3320, 3321 to 3340, 3341 to 3360, 3361 to 3380, 3381 to 3400, 3401 to 3420, 3421 to 3440, 3441 to 3460, 3461 to 3480, 3481 to 3500, 3501 to 3520, 3521 to 3540, 3541 to 3560, 3561 to 3580, 3581 to 3600, 3601 to 3620, 3621 to 3640, 3641 to 3660, 3661 to 3680, 3681 to 3700, 3701 to 3720, 3721 to 3740, 3741 to 3760, 3761 to 3780, 3781 to 3800, 3801 to 3820, 3821 to 3840, 3841 to 3860, 3861 to 3880, 3881 to 3900, 3901 to 3920, 3921 to 3940, 3941 to 3960, 3961 to 3980, 3981 to 4000, 4001 to 4020, 4021 to 4040, 4041 to 4060, 4061 to 4080, 4081 to 4100, 4101 to 4120, 4121 to 4140, 4141 to 4160, and 4161 to 4180.

[0049] Examples of antisense oligonucleotides that are complementary to nucleic acids containing at least 15 consecutive bases in the target sequence include: (i) a base sequence selected from the group consisting of SEQ ID NOs: 2 to 64, 66 to 224, 422 to 423, 426 to 427, and 432; (ii) a base sequence selected from the group consisting of SEQ ID NOs: 2 to 64, 66 to 224, 422 to 423, 426 to 427, and 432 in which one or several bases have been added, deleted, or substituted; or (iii) a base sequence that has 75% or more, 80% or more, 85% or more, preferably 90% or more, 95% or more, 98% or more, or 99% or more sequence identity to a base sequence selected from the group consisting of SEQ ID NOs: 2 to 64, 66 to 224, 422 to 423, 426 to 427, and 432, such as antisense oligonucleotides that contain or consist of the base sequence of (i).

[0050] In this specification, "several" in a base sequence in which one or several bases have been added, deleted, or substituted means two, three, four, five, six, seven, eight, nine, or ten bases.

[0051] In one embodiment, the antisense oligonucleotide of the present invention is selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention, which are selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention, and the antisense oligonucleotides of the present invention are selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention. The nucleic acid is complementary to a nucleic acid comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 3199 to 3162, 3199 to 3242, 3279 to 3382, 3399 to 3482, 3539 to 3562, 3579 to 3602, 3619 to 3662, 3679 to 3702, and 3759 to 4182.

[0052] In one embodiment, the antisense oligonucleotide of the present invention is selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention, which are selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention, and the antisense oligonucleotides of the present invention are selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention, which are selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention, and which are selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention. The nucleic acid is complementary to a nucleic acid comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 3160, 3201, 3240, 3281, 3380, 3401, 3480, 3541, 3560, 3581, 3600, 3621, 3660, 3681, 3700, and 3761, 4180.

[0053] In one embodiment, the antisense oligonucleotide of the present invention is from the 1st to 20th positions, 21st to 40th positions, 41st to 60th positions, 61st to 80th positions, 81st to 100th positions, 161st to 180th positions, 181st to 200th positions, 201st to 220th positions, 221st to 240th positions, 241st to 260th positions, 261st to 280th positions, 281st to 300th positions, 301st to 320th positions, 321st to 340th positions, 341st to 360th positions, 361st to 380th positions, 381st to 400th positions, 401st to 420th positions, 421st to 440th positions, 441st to 460th positions, 461st to 480th positions, 481st to 500th positions, 501st to 520th positions, 521st to 540th positions, 541st to 560th positions, 561st to 580th positions, 574th to 593rd positions, 581st to 600th positions, 601st to 620th positions, 641st to 660th positions, 661st to 680th positions, 681st to 700th positions, 701st to 720th positions, 721st to 740th positions, 741st to 760th positions, 761st to 780th positions, 781st to 800th positions, 801st to 820th positions, 821st to 840th positions, 881st to 900th positions, 901st to 920th positions, 921st to 940th positions, 933rd to 952nd positions, 941st to 960th positions, 961st to 980th positions, 981st to 1000th positions, 1001st to 1020th positions, 1021st to 1040th positions, 1041st to 1060th positions, 1061st to 1080th positions, 1081st to 1100th positions, 1093rd to 1112nd positions, 1101st to 1120th positions, 1121st to 1140th positions, 1141st to 1160th positions, 1161st to 1180th positions, 1181st to 1200th positions, 1201st to 1220th positions, 1221st to 1240th positions, 1241st to 1260th positions, 1261st to 1280th positions, 1281st to 1300th positions, 1301st to 1320th positions, 1321st to 1340th positions, 1341st to 1360th positions, 1361st to 1380th positions, 1381st to 1400th positions, 1394th to 1413th positions, 1401st to 1420th positions, 1421st to 1440th positions, 1461st to 1480th positions, 1476th to 1495th positions, 1501st to 1520th positions, 1541st to 1560th positions, 1561st to 1580th positions, 1581st to 1600th positions, 1601st to 1620th positions, 1621st to 1640th positions, 1641st to 1660th positions, 1661st to 1680th positions, 1681st to 1700th positions, 1721st to 1740th positions, 1741st to 1760th positions, 1781st to 1800th positions, 1876th to 1895th positions, 1881st to 1900th positions, 1901st to 1920th positions, from the 5'-end of the nucleotide sequence of SEQ ID NO: 1.1916th to 1935th, 1941st to 1960th, 1961st to 1980th, 1981st to 2000th, 2001st to 2020th, 2021st to 2040th, 2041st to 2060th, 2061st to 2080th, 2073rd to 2092nd, 2081st to 2100th, 2101st 2120, 2121-2140, 2141-2160, 2153-2172, 2161-2180, 2181-2200, 2195-2214, 2201-2220, 2221-2240, 2241-2260, 2261-228 0, 2275-2294, 2281-2300, 2321-2340, 2341-2360, 2361-2380, 2381-2400, 2401-2420, 2421-2440, 2441-2460, 2448-2467, 2 461st to 2480th, 2481st to 2500th, 2501st to 2520th, 2521st to 2540th, 2528th to 2547th, 2541st to 2560th, 2561st to 2580th, 2581st to 2600th, 2601st to 2620th, 2621st to 2640th, 2641st to 2660, 2647-2666, 2661-2680, 2681-2700, 2686-2705, 2701-2720, 2721-2740, 2741-2760, 2761-2780, 2766-2785, 2781-2800 2801st to 2820th, 2807th to 2826th, 2821st to 2840th, 2841st to 2860th, 2861st to 2880th, 2881st to 2900th, 2901st to 2920th, 2921st to 2940th, 2941st to 2960th, 2961st to 2980th, 298 1st to 3000th, 3001st to 3020th, 3005th to 3024th, 3021st to 3040th, 3041st to 3060th, 3061st to 3080th, 3081st to 3100th, 3101st to 3120th, 3121st to 3140th, 3141st to 3160th, 3201st to 3 220, 3221-3240, 3281-3300, 3301-3320, 3321-3340, 3341-3360, 3361-3380, 3401-3420, 3421-3440, 3441-3460, 3461-3480,3541st to 3560th, 3581st to 3600th, 3621st to 3640th, 3641st to 3660th, 3681st to 3700th, 3761st to 3780th, 3781st to 3800th, 3801st to 3820th , 3821st to 3840th, 3841st to 3860th, 3861st to 3880th, 3881st to 3900th, 3901st to 3920th, 3921st to 3940th, 3941st to 3960th, 3961st to 3980th , 3981 to 4000, 4001 to 4020, 4021 to 4040, 4041 to 4060, 4061 to 4080, 4081 to 4100, 4101 to 4120, 4121 to 4140, 4141 to 4160, and 4161 to 4180.

[0054] Examples of antisense oligonucleotides complementary to nucleic acids containing at least 15 consecutive bases in the target sequence include: (i) a base sequence selected from the group consisting of SEQ ID NOs: 2 to 30, 32 to 64, 66 to 72, 74 to 75, 77, 79 to 86, 88 to 89, 91, 93 to 96, 98 to 102, 104 to 120, 122 to 146, 148 to 155, 157 to 158, 160 to 173, 176 to 177, 180 to 184, 186 to 189, 193, 195, 197 to 198, 200, and 204 to 224; (ii) SEQ ID NOs: 2 to 30, 32 to 64, 66 to 72, 74 to 75, 77, 79 to 86, 88 to 89, 91, 93 to 96, 98 to 102, 104 to 120, 122 to 146, 148 to 155, 157 to 158, 160 to 173, 176 to 177, 180 to 184, 186 to 189, 193, 195, 197 to 198, 200, and 204 to 224. A base sequence in which one or several bases are added, deleted, or substituted, or (iii) A base sequence having 75% or more, 80% or more, 85% or more, preferably 90% or more, 95% or more, 98% or more, or 99% or more sequence identity to a base sequence selected from the group consisting of SEQ ID NOs: 2 to 30, 32 to 64, 66 to 72, 74 to 75, 77, 79 to 86, 88 to 89, 91, 93 to 96, 98 to 102, 104 to 120, 122 to 146, 148 to 155, 157 to 158, 160 to 173, 176 to 177, 180 to 184, 186 to 189, 193, 195, 197 to 198, 200, and 204 to 224, for example, an antisense oligonucleotide comprising or consisting of the base sequence of (i).

[0055] In one embodiment, the antisense oligonucleotide of the present invention is selected from those that exhibit a high rate of suppression of TDP-43 gene expression when administered to A204 cells, as measured according to the method described in Example 3 herein. For example, the antisense oligonucleotide may be selected from those that exhibit a ratio (average value) of 4.12 or less to the TDP-43 gene expression level when ANT-10 is administered as a comparative antisense oligonucleotide (here, "average value" refers to the average value of the results obtained when a primer set for detecting exons 2-3 (RNA expression level (exon 2-3)) and a primer set for detecting exons 5-6 (RNA expression level (exon 5-6))). Examples of such antisense oligonucleotides include those derived from the 5' end of the nucleotide sequence of SEQ ID NO: 1 at positions 19 to 42, 79 to 102, 159 to 182, 199 to 622, 639 to 662, 679 to 762, 779 to 802, 819 to 842, 879 to 1002, 1019 to 1402, 1419 to 1442, 1459 to 1497, 1499 to 1522, 1559 to 1642, 1659 to 1702, 1779 to 1802, 1874 to 1902, 1914 to 1937, 1959 to 1962, 1964 to 1972, 1970 to 1982, 1984 to 1992, 1996 to 2002, 2002 to 2004, 2006 to 2008, 2008 to 2102, 2008 to 2112, 2008 to 2116, 2008 to 2120, 2008 to 2132, 2008 to 2142, 2008 to 2152, 2008 to 2152, 2008 to 2162, 2008 to 2172, 2008 to 2182, 2008 to 2194, 2008 to 2196, 2008 to 2198, 2199 to 2202, 2199 to 2216, 2199 to and antisense oligonucleotides that are complementary to nucleic acids comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 2019 to 2002, 2019 to 2302, 2319 to 2342, 2359 to 2802, 2819 to 3142, 3199 to 3222, 3299 to 3322, 3339 to 3362, 3399 to 3422, 3799 to 3842, 3879 to 3982, 4019 to 4042, and 4119 to 4142.

[0056] In one embodiment, the antisense oligonucleotide of the present invention is selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention, which are selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention, and the antisense oligonucleotides of the present invention are selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention, which are selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention, and which are selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention. It is complementary to a nucleic acid comprising at least 15, for example at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 6 to 1935, 1961 to 2000, 2021 to 2300, 2321 to 2340, 2361 to 2800, 2821 to 3140, 3201 to 3220, 3301 to 3320, 3341 to 3360, 3401 to 3420, 3801 to 3840, 3881 to 3980, 4021 to 4040, and 4121 to 4140.

[0057] In one embodiment, the antisense oligonucleotide of the present invention is from the 21st to 40th, 81st to 100th, 161st to 180th, 201st to 220th, 221st to 240th, 241st to 260th, 261st to 280th, 281st to 300th, 301st to 320th, 321st to 340th, 341st to 360th, 361st to 380th, 381st to 400th, 401st to 420th, 421st to 440th, 441st to 460th, 461st to 480th, 481st to 500th, 501st to 520th, 521st to 540th, 541st to 560th, 561st to 580th, 574th to 593rd, 581st to 600th, 601st to 620th, 641st to 660th, 681st to 700th, 701st to 720th, 721st to 740th, 741st to 760th, 781st to 800th, 821st to 840th, 881st to 9*00th, 901st to 920th, 921st to 940th, 933rd to 952nd, 941st to 960th, 961st to 980th, 981st to 1000th, 1021st to 1040th, 1041st to 1060th, 1061st to 108*0th, 1081st to 1100th, 1093rd to 1112nd, 1101st to 1120th, 1121st to 1140th, 1141st to 1160th, 1161st to 1180th, 1181st to 1200th, 1201st to 1220th, 1221st to 1240th, 1241st to 1260th, 1261st to 1280th, 1281st to 1300th, 1301st to 1320th, 1321st to 1340th, 1341st to 1360th, 1361st to 1380th, 1381st to 1400th, 1421st to 1440th, 1461st to 1480th, 1476th to 1495th, 1501st to 1520th, 1561st to 1580th, *1581st to 1600th, 1601st to 1620th, 1621st to 1640th, 1661st to 1680th, 1681st to 1700th, 1781st to 1800th, 1876th to 1895th, *1881st to 1900th, 1916th to 1935th, 1961st to 1980th, 1981st to 2000th, 2021st to 2040th, 2041st to 2060th, 2061st to 2080th, 2073rd to 2092nd, 2081st to 2100th, 2101st to 2120th, 2121st to 2140th, 2141st to 2160th, 2153rd to 2172nd, 2161st to 2180th, 2181st to 2200th of the base sequence of SEQ ID NO: 1 counted from the 5'-end. Note: There seem to be some inconsistent numbering notations in the original text (e.g., "9*00th", "*1581st", "*188*1st" etc.). I've translated it as accurately as possible while keeping the original format. You may want to double-check the original text for accuracy.It is complementary to a nucleic acid containing at least 15, for example at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 2195 to 2214, 2201 to 2220, 2221 to 2240, 2241 to 2260, 2261 to 2280, 2275 to 2294, 2281 to 2300, 2321 to 2340, 2361 to 2380, 2381 to 2400, 2401 to 2420, 2421 to 2440, 2441 to 2460, 2448 to 2467, 2461 to 2480, 2481 to 2500, 2501 to 2520, 2521 to 2540, 2528 to 2547, 2541 to 2560, 2561 to 2580, 2581 to 2600, 2601 to 2620, 2621 to 2640, 2641 to 2660, 2647 to 2666, 2661 to 2680, 2681 to 2700, 2701 to 2720, 2721 to 2740, 2741 to 2760, 2761 to 2780, 2781 to 2800, 28​​Examples of antisense oligonucleotides complementary to nucleic acids containing at least 15 consecutive bases in the target sequence include: (i) a base sequence selected from the group consisting of SEQ ID NOs: 3, 6-7, 9-30, 32, 34-37, 39, 41-48, 50-64, 66-69, 72, 74-75, 77, 80-83, 85-86, 91, 93-94, 96, 99-100, 102, 104-120, 122, 124-143, 145-146, 148-149, 151, 154-155, 157-158, 160-165, 167-172, 176, 181, 183, 186, 206-207, 210-214, 217, and 222; (ii) SEQ ID NOs: 3, 6 to 7, 9 to 30, 32, 34 to 37, 39, 41 to 48, 50 to 64, 66 to 69, 72, 74 to 75, 77, 80 to 83, 85 to 86, 91, 93 to 94, 96, 99 to 100, 102, 104 to 120, 122, 124 to 143, 145 to 146, 148 to 149, 151, 154 to 155, 157 to 158, 160 to 165, 167 to 172, 176, 181, 183, 186, 206 to 207, 210 to 214, 217, and 222. A base sequence in which one or several bases are added, deleted, or substituted in a base sequence selected from the group consisting of: (iii) SEQ ID NOs: 3, 6-7, 9-30, 32, 34-37, 39, 41-48, 50-64, 66-69, 72, 74-75, 77, 80-83, 85-86, 91, 93-94, 96, 99-100, 102, 104-120, 122, 124-143, 145-146, 148-149, 151, 154-155, 15 and a base sequence having 75% or more, 80% or more, 85% or more, preferably 90% or more, 95% or more, 98% or more, or 99% or more sequence identity to a base sequence selected from the group consisting of base sequences 7 to 158, 160 to 165, 167 to 172, 176, 181, 183, 186, 206 to 207, 210 to 214, 217, and 222, such as an antisense oligonucleotide comprising or consisting of the base sequence of (i).

[0059] In one embodiment, the antisense oligonucleotide of the present invention is selected from those that exhibit a high rate of suppression of TDP-43 gene expression levels when administered to A204 cells, as measured according to the method described in Example 3 herein. For example, the antisense oligonucleotide may be selected from those that exhibit a ratio (average value) of 3 or less of the TDP-43 gene expression level when administered to A204 cells to the TDP-43 gene expression level when ANT-10 is administered as a comparative antisense oligonucleotide (here, "average value" refers to the average value of the results obtained when a primer set for detecting exons 2-3 (RNA expression level (exon 2-3))) and the results obtained when a primer set for detecting exons 5-6 (RNA expression level (exon 5-6)))). Examples of such antisense oligonucleotides include those at positions 19 to 42, 159 to 182, 239 to 282, 319 to 342, 359 to 482, 499 to 602, 639 to 662, 679 to 762, 779 to 802, 819 to 842, 879 to 902, 919 to 1002, 1039 to 1342, 1359 to 1382, 1419 to 1442, 1459 to 1482, 1499 to 1522, 1559 to 1642, 1659 to 1702, 1779 to 1802, 1819 to 1902, 1919 to 2002, 2019 to 2102, 2119 to 2202, 2219 to 2302, 2309 to 2402, 2419 to 2502, 2519 to 2602, 2619 to 2702, 2719 to 2802, 2819 to 3002, 2919 to 3102, 3219 to 3302, 3319 to 3402, 3419 to 3502, 3519 to 3602, 3619 to 3702, 3719 to 3802, 3819 to 3902, 3919 to 4002, 4019 to 4102, 4109 to 4202, 4219 to 4302, 4319 to 4 and antisense oligonucleotides that are complementary to nucleic acids comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 879 to 1902, 1914 to 1937, 1979 to 2002, 2019 to 2042, 2059 to 2302, 2319 to 2342, 2359 to 2422, 2446 to 2542, 2559 to 2622, 2639 to 2702, 2939 to 2962, 3019 to 3042, 3119 to 3142, and 3199 to 3222.

[0060] In one embodiment, the antisense oligonucleotide of the present invention is selected from the group consisting of positions 21 to 40, 161 to 180, 241 to 280, 321 to 340, 361 to 480, 501 to 600, 641 to 660, 681 to 760, 781 to 800, 821 to 840, 881 to 900, 921 to 1000, 1041 to 1340, 1361 to 1380, 1421 to 1440, 1461 to 1480, 1501 to 1520, 1561 to 1640, and 1661 to 1700 from the 5' end of the base sequence of SEQ ID NO: 1. It is complementary to a nucleic acid comprising at least 15, for example at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 1781 to 1800, 1881 to 1900, 1916 to 1935, 1981 to 2000, 2021 to 2040, 2061 to 2300, 2321 to 2340, 2361 to 2420, 2448 to 2540, 2561 to 2620, 2641 to 2700, 2941 to 2960, 3021 to 3040, 3121 to 3140, and 3201 to 3220.

[0061] In one embodiment, the antisense oligonucleotide of the present invention is from the 21st to the 40th, 161st to 180th, 241st to 260th, 261st to 280th, 321st to 340th, 361st to 380th, 381st to 400th, 401st to 420th, 421st to 440th, 441st to 460th, 461st to 480th, 501st to 520th, 521st to 540th, 541st to 560th, 561st to 580th, 574th to 593rd, 581st to 600th, 641st to 660th, 681st to 700th, 701st to 720th, 721st to 740th, 741st to 760th, 781st to 800th, 821st to 840th, 881st to 900th, 921st to 940th, 933rd to 952nd, 941st to 960th, 961st to 980th, 981st to 1000th, 1041st to 1060th, 1061st to 1080th, 1081st to 1100th, 1093rd to 1112nd, 1101st to 1120th, 1121st to 1140th, 1141st to 1160th, 1161st to 1180th, "1181st to 1200th", 1201st to 1220th, 1221st to 1240th, 1241st to 1260th, 1261st to 1280th, 1281st to 1300th, 1301st to 1320th, 1321st to 1340th, 1361st to 1380th, 1421st to 1440th, 1461st to 1480th, 1501st to 1520th, 1561st to 1580th, 1581st to 1600th, 1601st to 1620th, 1621st to 1640th, 1661st to 1680th, 1681st to 1700th, 1781st to 1800th, 1881st to 1900th, 1916th to 1935th, 1981st to 2000th, 2021st to 2040th, 2061st to 2080th, 2073rd to 2092nd, 2081st to 2100th, 2101st to 2120th, 2121st to 2140th, 2141st to 2160th, 2153rd to 2172nd, 2161st to 2180th, "2181st to 2200th", 2195th to 2214th, 2201st to 2220th, 2221st to 2240th, 2241st to 2260th, 2261st to 2280th, 2275th to 2294th, 2281st to 2300th, 2321st to 2340th, 2361st to 2380th, 2381st to 2400th, "2401st to 2420th", 2448th to 2467th, 2461st to 2480th, 2481st to 2500th of the nucleotide sequence of SEQ ID NO: 1 counted from the 5'-end. It should be noted that in the English translation, the ordinal numbers are written in full for clarity. Also, the text seems to be a bit repetitive in its structure, and the "1181st to 1200th", "2181st to 2200th", "2401st to 2420th" parts are added in quotes as they might be a bit inconsistent in the original Chinese text's format. If there are specific requirements or corrections regarding these parts, please let me know.It is complementary to a nucleic acid comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 2501 to 2520, 2521 to 2540, 2561 to 2580, 2581 to 2600, 2601 to 2620, 2641 to 2660, 2661 to 2680, 2681 to 2700, 2941 to 2960, 3021 to 3040, 3121 to 3140, and 3201 to 3220.

[0062] Examples of antisense oligonucleotides complementary to nucleic acids containing at least 15 consecutive bases in the target sequence include: (i) a base sequence selected from the group consisting of SEQ ID NOs: 3, 7, 11-12, 15, 17-22, 24-29, 32, 34-37, 39, 41-42, 44-48, 51-64, 66, 68, 72, 74, 77, 80-83, 85-86, 91, 94, 96, 100, 102, 105-120, 122, 124-126, 129-133, 136-138, 140, 142-143, 162, 167, 172, and 176; (ii) SEQ ID NOs: 3, 7, 11 to 12, 15, 17 to 22, 24 to 29, 32, 34 to 37, 39, 41 to 42, 44 to 48, 51 to 64, 66, 68, 72, 74, 77, 80 to 83, 85 to 86, 91, 94, 96, 100, 102, 105 to 120, 122, 124 to 126, 129 to 133, 136 to 138, 140, 142 to 143, 162, 167, 172, and 176. A base sequence in which one or several bases are added, deleted, or substituted, or (iii) A base sequence having 75% or more, 80% or more, 85% or more, preferably 90% or more, 95% or more, 98% or more, or 99% or more sequence identity to a base sequence selected from the group consisting of SEQ ID NOs: 3, 7, 11-12, 15, 17-22, 24-29, 32, 34-37, 39, 41-42, 44-48, 51-64, 66, 68, 72, 74, 77, 80-83, 85-86, 91, 94, 96, 100, 102, 105-120, 122, 124-126, 129-133, 136-138, 140, 142-143, 162, 167, 172, and 176, for example, an antisense oligonucleotide comprising or consisting of the base sequence of (i).

[0063] In one embodiment, the antisense oligonucleotide of the present invention is selected from those that exhibit a high rate of suppression of TDP-43 gene expression when administered to A204 cells, as measured according to the method described in Example 3 herein. For example, the antisense oligonucleotide may be selected from those that exhibit a ratio (average value) of 1.63 or less to the TDP-43 gene expression level when ANT-10 is administered as a comparative antisense oligonucleotide (here, "average value" refers to the average value of the results obtained when a primer set for detecting exons 2-3 (RNA expression level (exon 2-3)) and a primer set for detecting exons 5-6 (RNA expression level (exon 5-6))). Examples of such antisense oligonucleotides include those derived from the nucleotide sequence of SEQ ID NO: 1 at positions 239 to 262, 359 to 402, 419 to 462, 519 to 542, 559 to 602, 699 to 722, 819 to 842, 879 to 902, 919 to 982, 1091 to 1122, 1139 to 1162, 1179 to 1202, 1239 to 1262, 1279 to 1342, 1359 to 1382, 1459 to 1482, 1499 to 1522, 1559 to 1602, and 1603 from the 5' end. antisense oligonucleotides that are complementary to nucleic acids comprising at least 15, for example at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 2, 1659 to 1682, 1779 to 1802, 1879 to 1902, 1979 to 2002, 2019 to 2042, 2059 to 2122, 2139 to 2216, 2219 to 2242, 2259 to 2302, 2446 to 2469, 2479 to 2502, and 2659 to 2682.

[0064] In one embodiment, the antisense oligonucleotide of the present invention is located at positions 241 to 260, 361 to 400, 421 to 460, 521 to 540, 561 to 600, 701 to 720, 821 to 840, 881 to 900, 921 to 980, 1093 to 1120, 1141 to 1160, 1181 to 1200, 1241 to 1260, 1281 to 1340, 1361 to 1380, 1461 to 1480, 1501 to 1502, 1503 to 1504, 1505 to 1506, 1507 to 1508, 1509 to 1510, 1511 to 1512, 1513 to 1514, 1515 to 1516, 1517 to 1518, 1519 to 1520, 1521 to 1522, 1522 to 1524, 1523 to 1525, 1526 to 1528, 1529 to 1530, 1531 to 1532, 1533 to 1534, 1535 to 1536, 1537 to 1538, 1539 to 1540, 1541 to 1542, 1543 to 1544, 1545 to 1546, 1548 to 1549, 1550 to 1551, 1552 to 1553, 1554 to 1 It is complementary to a nucleic acid comprising at least 15, for example at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 20, 1561-1600, 1661-1680, 1781-1800, 1881-1900, 1981-2000, 2021-2040, 2061-2120, 2141-2214, 2221-2240, 2261-2300, 2448-2467, 2481-2500, and 2661-2680.

[0065] In one embodiment, the antisense oligonucleotide of the present invention is a nucleotide sequence of SEQ ID NO: 1, from the 5' end, at positions 241 to 260, 361 to 380, 381 to 400, 421 to 440, 441 to 460, 521 to 540, 561 to 580, 574 to 593, 581 to 600, 701 to 720, 821 to 840, 881 to 900, 921st to 940th, 941st to 960th, 961st to 980th, 1093rd to 1112th, 1101st to 1120th, 1141st to 1160th, 1181st to 1200th, 1241st to 1 260th, 1281st to 1300th, 1301st to 1320th, 1321st to 1340th, 1361st to 1380th, 1461st to 1480th, 1501st to 1520th, 1561st to 15th 80th place, 1581st to 1600th, 1661st to 1680th, 1781st to 1800th, 1881st to 1900th, 1981st to 2000th, 2021st to 2040th, 2061st to 20th 80th place, 2073rd to 2092nd, 2081st to 2100th, 2101st to 2120th, 2141st to 2160th, 2153rd to 2172nd, 2161st to 2180th, 2181st to 220th It is complementary to a nucleic acid comprising at least 15, for example at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 0, 2195-2214, 2221-2240, 2261-2280, 2275-2294, 2281-2300, 2448-2467, 2481-2500, and 2661-2680.

[0066] Examples of antisense oligonucleotides complementary to nucleic acids containing at least 15 consecutive bases in the target sequence include: (i) a base sequence selected from the group consisting of SEQ ID NOs: 11, 17-18, 20-21, 25, 27-29, 35, 41-42, 44, 46-47, 54-55, 57, 59, 62, 64, 66, 68, 74, 77, 80-81, 85, 91, 94, 100, 102, 105-108, 110-114, 116, 118-120, 129, 131, and 142; (ii) a base sequence selected from the group consisting of SEQ ID NOs: 11, 17-18, 20-21, 25, 27-29, 35, 41-42, 44, 46-47, 54-55, 57, 59, 62, 64, 66, 68, 74, 77, 80-81, 85, 91, 94, 100, 102, 105-108, 110-114, 116, 118-120, 129, 131, and 142, in which one or several bases are added, deleted, or substituted; or (iii) A base sequence having 75% or more, 80% or more, 85% or more, preferably 90% or more, 95% or more, 98% or more, or 99% or more sequence identity to a base sequence selected from the group consisting of SEQ ID NOs: 11, 17-18, 20-21, 25, 27-29, 35, 41-42, 44, 46-47, 54-55, 57, 59, 62, 64, 66, 68, 74, 77, 80-81, 85, 91, 94, 100, 102, 105-108, 110-114, 116, 118-120, 129, 131, and 142, for example, an antisense oligonucleotide comprising or consisting of the base sequence of (i).

[0067] In one embodiment, the antisense oligonucleotide of the present invention is selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1, and the antisense oligonucleotides of the present invention are ... It is complementary to a nucleic acid comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 1444 to 1537, 1544 to 1592, 1644 to 1697, 1769 to 1812, 1969 to 2012, 2051 to 2317, and 2469 to 2512.

[0068] In one embodiment, the antisense oligonucleotide of the present invention preferably excludes an antisense oligonucleotide having a base sequence selected from the group consisting of SEQ ID NOs: 436 to 438, 440 to 449, 451 to 455, and 472 to 474, or a pharmaceutically acceptable salt thereof; and preferably excludes an antisense oligonucleotide having a base sequence of SEQ ID NO: 450, or a pharmaceutically acceptable salt thereof, in which positions 2 to 3, 5 to 6, 10 to 14, 16, 18, and 20 from the 5' end of the base sequence of SEQ ID NO: 450 are ENA: 2'-O,4'-C-Ethylenebridged Nucleic Acid, and positions 1, 4, 7 to 9, 15, 17, and 19 are ribonucleotides containing a 2'-OMe group; and / or Preferably, antisense oligonucleotides or pharmaceutically acceptable salts thereof having the base sequence of SEQ ID NO: 453, in which positions 1-2, 4, 6, 8-9, 11-12, and 16-20 from the 5' end of the base sequence of SEQ ID NO: 453 are ENA: 2'-O,4'-C-Ethylenebridged Nucleic Acid, and positions 3, 5, 7, 10, and 13-15 are ribonucleotides containing 2'-OMe groups, are preferably excluded. In one embodiment, unmodified antisense oligonucleotides or pharmaceutically acceptable salts thereof having the base sequence of SEQ ID NO: 450 or SEQ ID NO: 453 are preferably excluded.

[0069] In one embodiment, the antisense oligonucleotide of the present invention is selected from the group consisting of the following: positions 196 to 215, 352 to 374, 376 to 413, 416 to 450, 502 to 610, 631 to 650, 653 to 682, 691 to 730, 823 to 842, 876 to 970, 1091 to 1175, 1279 to 1336, 1351 to 1358, 1401 to 1409, 1501 to 1551, 1601 to 1609, 1701 to 1709, 1801 to 1810, 1811 to 1812, 1812 to 1813, 1813 to 1814, 1814 to 1815, 1815 to 1816, 1816 to 1817, 1818 to 1819, 1820 to 1821, 1822 to 1823, 1824 to 1825, 1826 to 1828, 1828 to 1830, 1829 to 1831, 1832 to 1832, 1833 to 1834, 1835 to 1835, 1836 to 1836, 1837 to 1840, 1840 to 1841, 1841 to 1842, 1842 to 1843, 1843 to 1844, 1845 to 1845, 1846 to 1846, 18 It is complementary to a nucleic acid comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 1 to 1390, 1446 to 1535, 1546 to 1590, 1646 to 1695, 1771 to 1810, 1971 to 2010, 2053 to 2250, 2252 to 2315, and 2471 to 2510.

[0070] In one embodiment, the antisense oligonucleotide of the present invention is located at positions 196 to 215, 352 to 371, 353 to 372, 354 to 373, 355 to 374, 371 to 390, 376 to 395, 386 to 405, 391 to 410, 394 to 413, 406 to 425, 411 to 430, 416 to 435, 426 to 445, 431 to 450, 502 to 521, 506 to 525, 511 to 530, 516 to 535, 517 to 536, 518 to 537, 519 to 538, 520 to 539, 522 to 541, 523 to 542, 524 to 543, 525 to 544, 526 to 545, 527 to 546, 528 to 547, 529 to 548, 531 to 550, 551 to 570, 556 to 575, 566 to 585, 571 to 590, 577 to 596, 586 to 605, 591 to 610, 631 to 650, 653 to 672, 658 to 677, 663 to 682, 691 to 710, 696 to 715, 706 to 725, 711 to 730, 823 to 842, 876 to 895, 877 to 896, 878 to 897, 879 to 898, 880 to 899, 882 to 901, 883 to 902, 884 to 903, 885 to 904, 886 to 905, 891 to 910, 911 to 930, 926 to 945, 927 to 946, 928 to 947, 929 to 948, 931 to 950, 936 to 955, 951 to 970, 1091 to 1110, 1111 to 1130, 1126 to 1145, 1131 to 1150, 1136 to 1155, 1146 to 1165, 1151 to 1170, 1156 to 1175, 1279 to 1298, 1296 to 1315, 1306 to 1325, 1312 to 1331, 1317 to 1336, 1351 to 1370, 1356 to 1375, 1366 to 1385, 1371 to 1390, 1446 to 1465, 1451 to 1470, 1454 to 1473, 1463 to 1482, 1471 to 1490, 1491 to 1510 from the 5'-end of the nucleotide sequence of SEQ ID NO: 1.1496th to 1515th, 1506th to 1525th, 1511th to 1530th, 1516th to 1535th, 1546th to 1565th, 1551st to 1570th, 1556th to 1575th, 1566th to 1585th, 1571st to 1590th, 1646th to 1665th, 1647th 1650-1669, 1651-1670, 1652-1671, 1653-1672, 1654-1673, 1656-1675, 1666-1685, 1671-169 0, 1676-1695, 1771-1790, 1776-1795, 1786-1805, 1791-1810, 1971-1990, 1976-1995, 1986-2005, 1991-2010, 2053-2072, 2 054 to 2073, 2055 to 2074, 2056 to 2075, 2059 to 2078, 2060 to 2079, 2062 to 2081, 2063 to 2082, 2065 to 2084, 2070 to 2089, 2071 to 2090, 2072 to 2091, 2074-2093, 2075-2094, 2076-2095, 2077-2096, 2078-2097, 2080-2099, 2082-2101, 2084-2103, 2085-2104, 2086-2105 2091st to 2110th, 2092nd to 2111th, 2096th to 2115th, 2106th to 2125th, 2111th to 2130th, 2131st to 2150th, 2133rd to 2152nd, 2134th to 2153rd, 2135th to 2154th, 2136th to 2155th, 2137th to 2156th, 7th to 2156th, 2138th to 2157th, 2139th to 2158th, 2140th to 2159th, 2142nd to 2161st, 2143rd to 2162nd, 2144th to 2163rd, 2145th to 2164th, 2146th to 2165th, 2147th to 2166th, 2148th to 2 167, 2149-2168, 2150-2169, 2151-2170, 2152-2171, 2154-2173, 2155-2174, 2158-2177, 2159-2178, 2160-2179, 2162-2181,2167th to 2186th, 2168th to 2187th, 2171st to 2190th, 2176th to 2195th, 2177th to 2196th, 2179th to 2198th, 21 86th to 2205th, 2189th to 2208th, 2190th to 2209th, 2191st to 2210th, 2192nd to 2211th, 2194th to 2213th, 2211th ~2230th place, 2219th place - 2238th place, 2231st place - 2250th place, 2252nd place - 2271st place, 2266th place - 2285th place, 2268th place - 2287th place, 2269th place - 22nd 88th, 2270th to 2289th, 2271st to 2290th, 2272nd to 2291st, 2273rd to 2292nd, 2274th to 2293rd, 2278th to 2297th , 2279th to 2298th, 2280th to 2299th, 2282nd to 2301st, 2283rd to 2302nd, 2284th to 2303rd, 2286th to 2305th, 22 91st to 2310th, 2296th to 2315th, 2471st to 2490th, 2476th to 2495th, 2477th to 2496th, 2478th to 2497th, 2479th is complementary to a nucleic acid comprising at least 15, for example at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 2498 to 2499, 2482 to 2501, 2483 to 2502, 2484 to 2503, 2486 to 2505, and 2491 to 2510.

[0071] Examples of antisense oligonucleotides complementary to nucleic acids containing at least 15 consecutive bases in the target sequence include: (i) a base sequence selected from the group consisting of SEQ ID NOs: 231 to 419, 424 to 425, 428 to 431, 433 to 435, and 475 to 502; (ii) a base sequence in which one or several bases are added, deleted, or substituted in a base sequence selected from the group consisting of SEQ ID NOs: 231 to 419, 424 to 425, 428 to 431, 433 to 435, and 475 to 502; or (iii) a base sequence having 75% or more, 80% or more, 85% or more, preferably 90% or more, 95% or more, 98% or more, or 99% or more sequence identity to a base sequence selected from the group consisting of SEQ ID NOs: 231 to 419, 424 to 425, 428 to 431, 433 to 435, and 475 to 502; For example, an antisense oligonucleotide containing or consisting of the base sequence (i) can be mentioned.

[0072] In one embodiment, the antisense oligonucleotide of the present invention is selected from those that exhibit a high rate of suppression of TDP-43 gene expression levels when administered to A204 cells, as measured according to the method described in Example 5 herein. For example, the antisense oligonucleotide may be selected from those that exhibit a ratio (average value) of 2.56 or less to the TDP-43 gene expression level when ANT-10 is administered as a comparative antisense oligonucleotide (here, "average value" refers to the average value of the results obtained when a primer set for detecting exons 2-3 (RNA expression level (exon 2-3)) and a primer set for detecting 3'UTR (RNA expression level (3'UTR))). Examples of such antisense oligonucleotides include those at positions 194 to 217, 350 to 452, 500 to 612, 629 to 679, 689 to 732, 821 to 844, 874 to 912, 924 to 972, 1089 to 1177, 1277 to 1338, 1349 to 1392, 1449 to 1484, 1504 to 1505, 1510 to 1516, 1517 to 1518, 1519 to 1520, 1521 to 1522, 1523 to 1524, 1525 to 1526, 1527 to 1528, 1529 to 1530, 1531 to 1532, 1533 to 1534, 1535 to 1536, 1537 to 1538, 1540 to 1546, 1547 to 1548, 1549 to 1550, 1551 to 1552, 1552 to 1554, 1555 to 1556, 1557 to 1558, 1559 to 1560, 1561 to 1562, 1562 to 1564, 1563 to 1565, 1564 to 1572, 1573 to 1576, 1577 to 1578, 1579 to 1580, 1581 to 1582, 1582 to 1584, 1583 to 1585 and antisense oligonucleotides that are complementary to nucleic acids comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 32, 1544 to 1592, 1644 to 1697, 1769 to 1812, 1969 to 2012, 2051 to 2252, 2264 to 2317, and 2476 to 2512.

[0073] In one embodiment, the antisense oligonucleotide of the present invention is selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention. The antisense oligonucleotides of the present invention are selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention. The antisense oligonucleotides of the present invention are selected from the group consisting of the nucleotide sequences of SEQ ID NO: 1 and the antisense oligonucleotides of the present invention. It is complementary to a nucleic acid comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 1482 to 1530, 1546 to 1590, 1646 to 1695, 1771 to 1810, 1971 to 2010, 2053 to 2250, 2266 to 2315, and 2478 to 2510.

[0074] In one embodiment, the antisense oligonucleotide of the present invention is located at positions 196 to 215, 352 to 371, 353 to 372, 354 to 373, 355 to 374, 371 to 390, 376 to 395, 386 to 405, 391 to 410, 394 to 413, 411 to 430, 416 to 435, 426 to 445, 431 to 450, 502 to 521, 511 to 530, 517 to 536, 518 to 537, 519 to 538, 520 to 539, 522 to 541, 523 to 542, 524 to 543, 525 to 544, 526 to 545, 527 to 546, 528 to 547, 529 to 548, 531 to 550, 551 to 570, 556 to 575, 566 to 585, 571 to 590, 577 to 596, 586 to 605, 591 to 610, 631 to 650, 653 to 672, 658 to 677, 691 to 710, 696 to 715, 711 to 730, 823 to 842, 876 to 895, 877 to 896, 878 to 897, 879 to 898, 880 to 899, 882 to 901, 883 to 902, 884 to 903, 885 to 904, 886 to 905, 891 to 910, 926 to 945, 927 to 946, 928 to 947, 929 to 948, 931 to 950, 951 to 970, 1091 to 1110, 1111 to 1130, 1126 to 1145, 1131 to 1150, 1136 to 1155, 1146 to 1165, 1151 to 1170, 1156 to 1175, 1279 to 1298, 1296 to 1315, 1306 to 1325, 1312 to 1331, 1317 to 1336, 1351 to 1370, 1366 to 1385, 1371 to 1390, 1451 to 1470, 1454 to 1473, 1463 to 11649th to 1668th, 1650th to 1669th, 1651st to 1670th, 1652nd to 1671st, 1653rd to 1672nd, 1654th to 1673rd, 1656th to 1675th, 1666th to 1685th, 1671st to 1690th, 1676th to 1695th, 1771st 1790, 1776-1795, 1786-1805, 1791-1810, 1971-1990, 1976-1995, 1986-2005, 1991-2010, 2053-2072, 2054-2073, 2055-207 4, 2056-2075, 2059-2078, 2060-2079, 2062-2081, 2063-2082, 2065-2084, 2070-2089, 2071-2090, 2072-2091, 2074-2093, 2 075 to 2094, 2076 to 2095, 2077 to 2096, 2078 to 2097, 2080 to 2099, 2082 to 2101, 2084 to 2103, 2085 to 2104, 2086 to 2105, 2091 to 2110, 2092 to 2111, 2096-2115, 2106-2125, 2111-2130, 2131-2150, 2133-2152, 2134-2153, 2135-2154, 2136-2155, 2137-2156, 2138-2157 2139th to 2158th, 2140th to 2159th, 2142nd to 2161st, 2143rd to 2162nd, 2144th to 2163rd, 2145th to 2164th, 2146th to 2165th, 2147th to 2166th, 2148th to 2167th, 2149th to 2168th, 215 0th to 2169th, 2151st to 2170th, 2152nd to 2171st, 2154th to 2173rd, 2155th to 2174th, 2158th to 2177th, 2159th to 2178th, 2160th to 2179th, 2162nd to 2181st, 2167th to 2186th, 2168th to 2 187, 2171-2190, 2176-2195, 2177-2196, 2179-2198, 2186-2205, 2189-2208, 2190-2209, 2191-2210, 2192-2211, 2194-2213,2211th to 2230th, 2219th to 2238th, 2231st to 2250th, 2266th to 2285th, 2268th to 2287th, 2269th to 2288th, 2270th to 2289th, 2271st to 22nd 90th place, 2272nd to 2291st, 2273rd to 2292nd, 2274th to 2293rd, 2278th to 2297th, 2279th to 2298th, 2280th to 2299th, 2282nd to 2301st, 2283rd to 2302, 2284 to 2303, 2286 to 2305, 2291 to 2310, 2296 to 2315, 2478 to 2497, 2479 to 2498, 2480 to 2499, 2482 to 2501, 2483 to 2502, 2484 to 2503, and 2491 to 2510.

[0075] Examples of antisense oligonucleotides complementary to nucleic acids containing at least 15 consecutive bases in the target sequence include: (i) a base sequence selected from the group consisting of SEQ ID NOs: 231-242, 244, 246-268, 270-271, 273-285, 288, 290-292, 296-297, 299-390, 392-408, 412-417, 419, 475-476, 478-497, and 499-502; (ii) SEQ ID NOs: 231 to 242, 244, 246 to 268, 270 to 271, 273 to 285, 288, 290 to 292, 296 to 297, 299 to 390, 392 to 408, 412 to 417, 419, 475 to 476, 478 to 497, and 499 to 502. A base sequence in which one or several bases are added, deleted, or substituted, or (iii) SEQ ID NOs: 231 to 242, 244, 246 to 268, 270 to 271, 273 to 285, 288, 290 to 292, 296 to 297, 299 to 390, 392 to 408, 412 to 417, 419, 475 to 476, 478 to 497, and 499 to 502. A base sequence having 75% or more, 80% or more, 85% or more, preferably 90% or more, 95% or more, 98% or more, or 99% or more sequence identity to a base sequence selected from the group consisting of (i). For example, an antisense oligonucleotide comprising or consisting of the base sequence of (i) is exemplified.

[0076] In one embodiment, the antisense oligonucleotide of the present invention is selected from those that exhibit a high rate of suppression of TDP-43 gene expression levels when administered to A204 cells, as measured according to the method described in Example 5 herein. For example, the antisense oligonucleotide may be selected from those that exhibit a ratio (average value) of 2.05 or less to the TDP-43 gene expression level when ANT-10 is administered as a comparative antisense oligonucleotide (here, "average value" refers to the average value of the results obtained when a primer set for detecting exons 2-3 (RNA expression level (exon 2-3)) and a primer set for detecting 3'UTR (RNA expression level (3'UTR))). Examples of such antisense oligonucleotides include those derived from the nucleotide sequence of SEQ ID NO: 1 at positions 351 to 376, 384 to 452, 509 to 552, 569 to 607, 656 to 679, 821 to 844, 874 to 912, 927 to 952, 1109 to 1177, 1277 to 1338, 1364 to 1392, 1449 to 1484, 1504 to 1532, 1544 to 1552, and 1554 to 1556 from the 5' end. and antisense oligonucleotides that are complementary to nucleic acids comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 87, 1646 to 1697, 1774 to 1812, 1984 to 2007, 2054 to 2127, 2131 to 2215, 2229 to 2252, 2264 to 2312, and 2478 to 2512.

[0077] In one embodiment, the antisense oligonucleotide of the present invention is provided at positions 353 to 374, 386 to 450, 511 to 550, 571 to 605, 658 to 677, 823 to 842, 876 to 910, 929 to 950, 1111 to 1175, 1279 to 1336, 1366 to 1390, 1451 to 1482, 1506 to 1518, 1520 to 1524, 1525 to 1530, 1531 to 1532, 1533 to 1534, 1535 to 1536, 1537 to 1538, 1539 to 1540, 1541 to 1542, 1543 to 1544, 1545 to 1546, 1547 to 1548, 1549 to 1550, 1551 to 1552, 1553 to 1554, 1555 to 1556, 1557 to 1558, 1559 to 1560, 1561 to 1562, 1562 to 1564, 1563 to 1565, 1564 to 1566, 1565 to 1570, 1571 to 1572, 1573 to 1574, 1575 to 1576, 1577 to 1578, 1579 to 1580, 1581 to 1582, 1582 to 1584, 1585 to It is complementary to a nucleic acid comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 530, 1546-1585, 1648-1695, 1776-1810, 1986-2005, 2056-2125, 2133-2213, 2231-2250, 2266-2310, and 2480-2510.

[0078] In one embodiment, the antisense oligonucleotide of the present invention is from position 353 to position 372, from position 354 to position 373, from position 355 to position 374, from position 386 to position 405, from position 391 to position 410, from position 394 to position 413, from position 411 to position 430, from position 426 to position 445, from position 431 to position 450, from position 511 to position 530, from position 518 to position 537, from position 519 to position 538, from position 520 to position 539, from position 522 to position 541, from position 523 to position 542, from position 524 to position 543, from position 525 to position 544, from position 526 to position 545, from position 527 to position 546, from position 531 to position 550, from position 571 to position 590, from position 577 to position 596, from position 586 to position 605, from position 658 to position 677, from position 823 to position 842, from position 876 to position 895, from position 877 to position 896, from position 878 to position 897, from position 879 to position 898, from position 880 to position 899, from position 882 to position 901, from position 883 to position 902, from position 884 to position 903, from position 885 to position 904, from position 886 to position 905, from position 891 to position 910, from position 929 to position 948, from position 931 to position 950, from position 1111 to position 1130, from position 1126 to position 1145, from position 1131 to position 1150, from position 1146 to position 1165, from position 1151 to position 1170, from position 1156 to position 1175, from position 1279 to position 1298, from position 1296 to position 1315, from position 1306 to position 1325, from position 1312 to position 1331, from position 1317 to position 1336, from position 1366 to position 1385, from position 1371 to position 1390, from position 1451 to position 1470, from position 1463 to position 1482, from position 1506 to position 1525, from position 1511 to position 1530, from position 1546 to position 1565, from position 1551 to position 1570, from position 1566 to position 1585, from position 1648 to position 1667, from position 1649 to position 1668, from position 1650 to position 1669, from position 1651 to position 1670, from position 1652 to position 1671, from position 1653 to position 1672, from position 1654 to position 1673, from position 1666 to position 1685, from position 1671 to position 1690, from position 1676 to position 1695, from position 1776 to position 1795, from position 1786 to position 1805, from position 1791 to position 1810, from position 1986 to position 2005, from position 2056 to position 2075, from position 2059 to position 2078, from position 2060 to position 2079, from position 2062 to position 2081, from position 2063 to position 2082, from position 2065 to position 2084, from position 2070 to position 2089, from position 2071 to position 2090, from position 2072 to position 2091, from position 2074 to position 2093, from position 2075 to position 2094, from position 2076 to position 2095, from position 2077 to position 2096 of the base sequence of SEQ ID NO: 1, starting from the 5'-end.It is complementary to a nucleic acid containing at least 15, for example at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 2080 to 2099, 2082 to 2101, 2084 to 2103, 2085 to 2104, 2086 to 2105, 2091 to 2110, 2092 to 2111, 2096 to 2115, 2106 to 2125, 2133 to 2152, 2134 to 2153, 2135 to 2154, 2136 to 2155, 2137 to 2156, 2138 to 2157, 2139 to 2158, 2142 to 2161, 2143 to 2162, 2144 to 2163, 2145 to 2164, 2146 to 2165, 2147 to 2166, 2148 to 2167, 2149 to 2168, 2150 to 2169, 2151 to 2170, 2152 to 2171, 2154 to 2173, 2155 to 2174, 2158 to 2177, 2159 to 2178, 2160 to 2179, 2162 to 2181, 2171 to 2190, 2176 to 2195, 2177 to 2196, 2186 to 2205, 2189 to 2208, 2190 to 2209, 21{91} to 2210, 2192 to 2211, 2194 to 2213, 2231 to 2250, 2266 to 2285, 2268 to 2287, 2269 to 2288, 2270 to 2289, 2271 to 2290, 2272 to 2291, 2273 to 2292, 2274 to 2293, 2280 to 2299, 2282 to 2301, 2283 to 2302, 2284 to 2303, 2286 to 2305, 2291 to 2310, 2480 to 2499, 2482 to 2501, and 2491 to 2510.

[0079] Note: There is a possible typo in "21{91}" in the original text. It should probably be "2191". This translation assumes that's the case. If it's not a typo, more context would be needed to accurately translate it.Examples of antisense oligonucleotides complementary to nucleic acids containing at least 15 consecutive bases in the target sequence include: (I) SEQ ID NOs: 232-234, 236-238, 240-241, 244, 247-255, 258, 262-264, 268, 274-285, 290, 292, 296-297, 299-300, 302, 306-312, 314-316, 318-320, 323, 328-340, 342-350, 353-359, 361-377, 380-382, 384-387, 390, 392-399, 402-407, 414-415, 419, 478, 482-483, 485-487, 489-496, and 499-502; (ii) SEQ ID NOs: 232-234, 236-238, 240-241, 244, 247-255, 258, 262-264, 268, 274-285, 290, 292, 296-297, 299-300, 302, 306-312, 314-316, 318-320, 323, 328-340, 342-350, 353-3 A base sequence selected from the group consisting of 59, 361-377, 380-382, 384-387, 390, 392-399, 402-407, 414-415, 419, 478, 482-483, 485-487, 489-496, and 499-502, in which one or several bases have been added, deleted, or substituted; or (iii) SEQ ID NOs: 232-234, 236-238, 240-241, 244, 247-255, 258, 262-264, 268, 274-285, 290, 292, 296-297, 299-300, 302, 306-312, 314-316, 318-320, 323, 328-340, 342-350, 353-359, 361-377, 380-38 and 499-502, for example, an antisense oligonucleotide comprising or consisting of a base sequence (i).

[0080] In one embodiment, the antisense oligonucleotide of the present invention is selected from those that exhibit a high rate of suppression of TDP-43 gene expression levels when administered to A204 cells, as measured according to the method described in Example 5 herein. For example, the antisense oligonucleotide may be selected from those that exhibit a ratio (average value) of 1.63 or less to the TDP-43 gene expression level when ANT-10 is administered as a comparative antisense oligonucleotide (here, "average value" refers to the average value of the results obtained when a primer set for detecting exons 2-3 (RNA expression level (exon 2-3)) and a primer set for detecting 3'UTR (RNA expression level (3'UTR))). Examples of such antisense oligonucleotides include those derived from the 5' end of the base sequence of SEQ ID NO: 1, and those derived from the 5' end of the target region selected from the group consisting of positions 409 to 452, 509 to 548, 656 to 679, 874 to 907, 927 to 950, 1129 to 1172, 1294 to 1333, 1449 to 1484, 1504 to 1532, and 1549 to 1584. and antisense oligonucleotides that are complementary to nucleic acids comprising at least 15, for example, at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 7, 1646-1697, 1784-1807, 2057-2117, 2131-2215, 2266-2312, and 2478-2501.

[0081] In one embodiment, the antisense oligonucleotide of the present invention is complementary to a nucleic acid comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 411 to 450, 511 to 546, 658 to 677, 876 to 905, 929 to 948, 1131 to 1170, 1296 to 1331, 1451 to 1482, 1506 to 1530, 1551 to 1585, 1648 to 1695, 1786 to 1805, 2059 to 2115, 2133 to 2213, 2268 to 2310, and 2480 to 2499 from the 5' end of the base sequence of SEQ ID NO: 1.

[0082] In one embodiment, the antisense oligonucleotide of the present invention is located at positions 411 to 430, 426 to 445, 431 to 450, 511 to 530, 520 to 539, 522 to 541, 523 to 542, 524 to 543, 526 to 545, 527 to 546, 658 to 677, 876 to 895, 878 to 897, 879 to 898, 880 to 899, 882 to 901, 883 to 902, 886 to 905, 929 to 948, 1131 to 1150, 1146 to 1165, 1151 to 1170, 1296 to 1315, 1312 to 1331, 1451 to 1470, 1463 to 1482, 1506 to 1525, 1511 to 1530, 1551 to 1570, 1566 to 1585, 1648 to 1667, 1649 to 1668, 1650 to 1669, 1651 to 1670, 1652 to 1671, 1653 to 1672, 1654 to 1673, 1671 to 1690, 1676 to 1695, 1786 to 1805, 2059 to 2078, 2060 to 2079, 2062 to 2081, 2070 to 2089, 2071 to 2090, 2072 to 2091, 2074 to 2093, 2075 to 2094, 2076 to 2095, 2082 to 2101, 2084 to 2103, 2085 to 2104, 2086 to 2105, 2091 to 2110, 2092 to 2111, 2096 to 2115, 2133 to 2152, 2134 to 2153, 2135 to 2154, 2136 to 2155, 2137 to 2156, 2138 to 2157, 2139 to 2158, 2142 to 2161, 2143 to 2162, 2144 to 2163, 2145 to 2164, 2146 to 2165, 2148 to 2167, 2149 to 2168, 2150 to 2169, 2151 to 2170, 2152 to 2171, 2159 to 2178, 2160 to 2179, 2162 to 2181, 2176 to 2195, 2177 to 2196, 2186 to 2205, 2191 to 2210, 2192 to 2211, 2194 to 2213, counted from the 5'-end of the nucleotide sequence of SEQ ID NO: 1.It is complementary to a nucleic acid comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 2268-2287, 2269-2288, 2271-2290, 2272-2291, 2273-2292, 2274-2293, 2280-2299, 2282-2301, 2283-2302, 2284-2303, 2291-2310, and 2480-2499.

[0083] Examples of antisense oligonucleotides complementary to nucleic acids containing at least 15 consecutive bases in the target sequence include: (I) SEQ ID NOs: 240-241, 244, 249-252, 254-255, 268, 275, 277-281, 284, 290, 292, 296-297, 300, 302, 306-312, 315-316, 319, 329-331, 334-339, 343-349, 353-359, 361-365, 367-371, 375-377, 381-382, 384, 386-387, 393-394, 396-399, 402-405, 407, 414, 478, 482, 487, 489, 490, 493, 495, and 502; (ii) SEQ ID NOs: 240-241, 244, 249-252, 254-255, 268, 275, 277-281, 284, 290, 292, 296-297, 300, 302, 306-312, 315-316, 319, 329-331, 334-339, 343-349, 353-359, 361-365, 367 A base sequence selected from the group consisting of 371, 375, 377, 381, 382, ​​384, 386, 387, 393, 394, 396, 399, 402, 405, 414, 478, 482, 487, 489, 490, 493, 495, and 502, in which one or several bases have been added, deleted, or substituted; or (iii) SEQ ID NOs: 240-241, 244, 249-252, 254-255, 268, 275, 277-281, 284, 290, 292, 296-297, 300, 302, 306-312, 315-316, 319, 329-331, 334-339, 343-349, 353-359, 361-365, 367-371, 375-377, 381-38 and 502, for example, an antisense oligonucleotide comprising or consisting of a base sequence having 75% or more, 80% or more, 85% or more, preferably 90% or more, 95% or more, 98% or more, or 99% or more sequence identity to a base sequence selected from the group consisting of 2, 384, 386-387, 393-394, 396-399, 402-405, 407, 414, 478, 482, 487, 489, 490, 493, 495, and 502.

[0084] In one embodiment, the antisense oligonucleotide of the present invention is selected from those that exhibit a high rate of suppression of TDP-43 gene expression levels when administered to A204 cells, as measured according to the method described in Example 5 herein. For example, the antisense oligonucleotide may be selected from those that exhibit a ratio (average value) of 1.20 or less to the TDP-43 gene expression level when ANT-10 is administered as a comparative antisense oligonucleotide (here, "average value" refers to the average value of the results obtained when a primer set for detecting exons 2-3 (RNA expression level (exon 2-3)) and a primer set for detecting 3'UTR (RNA expression level (3'UTR))). Examples of such antisense oligonucleotides include antisense oligonucleotides that are complementary to nucleic acids comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 520 to 548, 880 to 903, 927 to 950, 1129 to 1172, 1461 to 1484, 1509 to 1532, 1549 to 1572, 1647 to 1697, 1784 to 1807, 2069 to 2117, 2131 to 2215, and 2269 to 2305 from the 5' end of the base sequence of SEQ ID NO: 1.

[0085] In one embodiment, the antisense oligonucleotide of the present invention is complementary to a nucleic acid comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 522 to 546, 882 to 901, 929 to 948, 1131 to 1170, 1463 to 1482, 1511 to 1530, 1551 to 1570, 1649 to 1671, 1676 to 1695, 1786 to 1805, 2071 to 2115, 2133 to 2178, 2162 to 2213, and 2271 to 2303 from the 5' end of the base sequence of SEQ ID NO: 1.

[0086] In one embodiment, the antisense oligonucleotide of the present invention is selected from the group consisting of the oligonucleotides at positions 522 to 541, 523 to 542, 526 to 545, 527 to 546, 882 to 901, 929 to 948, 1131 to 1150, 1146 to 1165, 1151 to 1170, 1463 to 1482, 1511 to 1530, 1541 to 1545, 1546 to 1547, 1548 to 1549, 1551 to 1552, 1553 to 1554, 1555 to 1556, 1557 to 1558, 1559 to 1560, 1561 to 1562, 1563 to 1564, 1565 to 1566, 1566 to 1568, 1569 to 1570, 1571 to 1572, 1573 to 1574, 1575 to 1576, 1578 to 1578, 1579 to 1580, 1581 to 1582, 1582 to 1584, 1585 to 1586, 1587 to 1588, 1589 to 1590, 1591 to 1592, 1593 to 1594, 1595 to 1596, 1597 to 1598, 1599 to 1600, 1601 to 1602, 1603 to 1604, 1605 51st to 1570th, 1649th to 1668th, 1650th to 1669th, 1651st to 1670th, 1652nd to 1671st, 1676th to 1695th, 1786th to 1805th, 207th 1st to 2090th, 2072nd to 2091st, 2074th to 2093rd, 2082nd to 2101st, 2086th to 2105th, 2096th to 2115th, 2133rd to 2152nd, 2134th ~2153rd, 2135th~2154th, 2136th~2155th, 2139th~2158th, 2142nd~2161st, 2143rd~2162nd, 2144th~2163rd, 2145th~ 2164th place, 2148th - 2167th, 2152nd - 2171st, 2159th - 2178th, 2162nd - 2181st, 2176th - 2195th, 2177th - 2196th, 2186th - 22nd 2272-2291, 2273-2292, 2274-2293, 2280-2299, 2282-2301, and 2284-2303.

[0087] Examples of antisense oligonucleotides complementary to nucleic acids containing at least 15 consecutive bases in the target sequence include: (i) a base sequence selected from the group consisting of SEQ ID NOs: 250-251, 254-255, 280, 292, 297, 300, 307-310, 316, 319, 335-337, 343, 346, 349, 353-356, 359, 361-364, 367, 371, 375, 377, 381-382, 384, 387, 396-399, 402-403, 405, 482, 487, 489, and 490; (ii) SEQ ID NOs: 250 to 251, 254 to 255, 280, 292, 297, 300, 307 to 310, 316, 319, 335 to 337, 343, 346, 349, 353 to 356, 359, 361 to 364, 367, 371, 375, 377, 381 to 382, ​​384, 387, 396 to 399, 402 to 403, 405, 482, 487, 489, and 490. A base sequence in which one or several bases are added, deleted, or substituted in the base sequence selected from the group consisting of: (iii) SEQ ID NOs: 250-251, 254-255, 280, 292, 297, 300, 307-310, 316, 319, 335-337, 343, 346, 349, 353-356, 359, 361-364, 367, 371, 375, 377, 381-382, 384, 387, 396-399, 402-403, 405, 482, 487, 489, and 490. A base sequence having 75% or more, 80% or more, 85% or more, preferably 90% or more, 95% or more, 98% or more, or 99% or more sequence identity to a base sequence selected from the group consisting of (i). For example, an antisense oligonucleotide comprising or consisting of the base sequence of (i) is exemplified.

[0088] In one embodiment, the antisense oligonucleotide of the present invention is complementary to a nucleic acid comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 519 to 547, 879 to 902, 1459 to 1482, 1559 to 1582, 1647 to 1672, 1674 to 1697, 2059 to 2107, 2132 to 2215, 2269 to 2301, and 2479 to 2502 from the 5' end of the base sequence of SEQ ID NO: 1.

[0089] In one embodiment, the antisense oligonucleotide of the present invention is complementary to a nucleic acid comprising at least 15, for example, at least 16, 17, 18, 19, or 20 consecutive bases in a target region selected from the group consisting of positions 521 to 545, 881 to 900, 1461 to 1480, 1561 to 1580, 1649 to 1670, 1676 to 1695, 2061 to 2105, 2134 to 2213, 2271 to 2299, and 2481 to 2500 from the 5' end of the base sequence of SEQ ID NO: 1.

[0090] In one embodiment, the antisense oligonucleotide of the present invention is selected from the group consisting of the following: positions 521 to 540, 522 to 541, 523 to 542, 526 to 545, 881 to 900, 1461 to 1480, 1561 to 1580, 1649 to 1668, 1650 to 1669, 1651 to 1670, 1676 to 1695, 2061 to 2080, 2073 to 2092, 2074 to 2093, 2081 to 2100, 2086 to 2105, and 2134 to 2153 from the 5' end of the base sequence of SEQ ID NO: 1. positions, 2135-2154, 2136-2155, 2144-2163, 2145-2164, 2152-2171, 2161-2180, 2176-2195, 2194-2213, 2271-2290, 2273-2292, 2274-2293, 2275-2294, 2280-2299, and 2481-2500.

[0091] Examples of antisense oligonucleotides complementary to nucleic acids containing at least 15 consecutive bases in the target sequence include: (i) a base sequence selected from the group consisting of SEQ ID NOs: 25, 42, 74, 80, 105-107, 112, 119, 131, 250-251, 254, 307-309, 316, 337, 346, 354-356, 363-364, 371, 381, 387, 396, 398-399, and 402; (ii) a base sequence selected from the group consisting of SEQ ID NOs: 25, 42, 74, 80, 105-107, 112, 119, 131, 250-251, 254, 307-309, 316, 337, 346, 354-356, 363-364, 371, 381, 387, 396, 398-399, and 402, in which one or several bases are added, deleted, or substituted; or (iii) A base sequence having 75% or more, 80% or more, 85% or more, preferably 90% or more, 95% or more, 98% or more, or 99% or more sequence identity to a base sequence selected from the group consisting of SEQ ID NOs: 25, 42, 74, 80, 105-107, 112, 119, 131, 250-251, 254, 307-309, 316, 337, 346, 354-356, 363-364, 371, 381, 387, 396, 398-399, and 402, for example, an antisense oligonucleotide comprising or consisting of the base sequence of (i).

[0092] In one embodiment, the antisense oligonucleotide of the present invention reduces the expression level of the TDP-43 gene (e.g., the expression level of RNA, such as mRNA or pre-mRNA, which is a transcription product). Whether the antisense oligonucleotide of the present invention reduces the expression level of the TDP-43 gene can be tested as described in the Examples of the present specification. For example, whether the antisense oligonucleotide of the present invention reduces the expression level of the TDP-43 gene can be measured by introducing the antisense oligonucleotide of the present invention into cells that express the TDP-43 gene, such as A204 cells, and determining whether the expression level of the TDP-43 gene expressed by the cells is reduced (e.g., by 5% or more, 10% or more, or 20% or more) compared to a negative control (e.g., a sample to which medium alone has been added).

[0093] The antisense oligonucleotides of the present invention can be easily synthesized using various automated synthesizers (e.g., AKTA oligopilot plus 10 / 100 (GE Healthcare), automated nucleic acid synthesizers NTS M-8-MX DNA / RNA, NTS H-6 DNA / RNA, etc.), or can be produced by outsourcing to a third party organization (e.g., Promega or Takara).

[0094] In one embodiment, the present invention relates to a pharmaceutical composition comprising an antisense oligonucleotide, a pharmaceutically acceptable salt thereof, or a hydrate thereof. When the antisense nucleotide of the present invention is administered to a subject, the pharmaceutical composition of the present invention may contain a carrier that facilitates delivery of the antisense nucleotide. Such a carrier is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include cationic carriers such as cationic liposomes and cationic polymers, or carriers that utilize viral envelopes. Examples of cationic liposomes include liposomes formed from 2-O-(2-diethylaminoethyl)carbamoyl-1,3-O-dioleoylglycerol and phospholipids as essential components (hereinafter referred to as "Liposome A"), Oligofectamine (registered trademark) (manufactured by Invitrogen), Lipofectin (registered trademark) (manufactured by Invitrogen), Lipofectamine (registered trademark) (manufactured by Invitrogen), Lipofectamine 2000 (registered trademark) (manufactured by Invitrogen), DMRIE-C (registered trademark) (manufactured by Invitrogen), GeneSilencer (registered trademark) (manufactured by Gene Therapy Systems), TransMessenger (registered trademark) (manufactured by QIAGEN), and TransIT Examples of suitable carriers include TKO (registered trademark) (manufactured by Mirus) and Nucleofector II (Lonza). Examples of cationic polymers include JetSI (registered trademark) (manufactured by Qbiogene) and Jet-PEI (registered trademark) (polyethyleneimine, manufactured by Qbiogene). Examples of carriers utilizing viral envelopes include GenomeOne (registered trademark) (HVJ-E liposome, manufactured by Ishihara Sangyo Kaisha). Alternatively, the pharmaceutical device described in Japanese Patent No. 2924179 and the cationic carriers described in Republished Patent Publication Nos. 2006 / 129594 and 2008 / 096690 can also be used.

[0095] In one embodiment, the antisense oligonucleotide of the present invention may be conjugated with a lipid or the like in a pharmaceutical composition to facilitate delivery of the antisense oligonucleotide. For example, the antisense oligonucleotide may be conjugated with cholesterol, as described in Bijsterbosch M. K. et al., Nucleic Acids Research, 2000, Vol. 28, pp. 2717-2725. Furthermore, in one embodiment, the antisense oligonucleotide may be conjugated to a cell-penetrating polypeptide (CPP) consisting of or rich in cationic amino acids at one or both of the 5' and 3' ends. The CPP is not particularly limited, and may be, for example, a polypeptide Rn (n = a natural number from 2 to 8) consisting of n consecutive arginine Rs.

[0096] The pharmaceutical compositions of the present invention may contain pharmaceutically acceptable additives in addition to the antisense oligonucleotide, its pharmaceutically acceptable salt, or hydrate thereof, and optionally the above-mentioned carrier. Examples of such additives include emulsifiers (e.g., C6-C22 fatty acids or pharmaceutically acceptable salts thereof, albumin, and dextran), stabilizers (e.g., cholesterol, phosphatidic acid, mannitol, and sorbitol), isotonicity agents (e.g., sodium chloride, glucose, maltose, lactose, sucrose, and trehalose), and pH adjusters (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, and triethanolamine). These additives may be used alone or in combination. The content of the additive in the composition of the present invention is suitably 90% by weight or less, preferably 70% by weight or less, and more preferably 50% by weight or less.

[0097] The method for preparing the pharmaceutical composition of the present invention is not limited, and can be prepared, for example, by adding the antisense oligonucleotide of the present invention to a dispersion of a carrier and stirring appropriately. In addition, additives can be added at an appropriate step, either before or after the addition of the antisense oligonucleotide of the present invention. The aqueous solvent that can be used when adding the antisense oligonucleotide of the present invention is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include electrolyte solutions such as water for injection, distilled water for injection, and physiological saline, and sugar solutions such as glucose solution and maltose solution. In addition, conditions such as pH and temperature in such cases can be appropriately selected by those skilled in the art.

[0098] The pharmaceutical composition of the present invention can be, for example, a liquid formulation or a lyophilized formulation thereof. The lyophilized formulation can be prepared by lyophilizing the composition of the present invention in liquid form using standard methods. For example, after appropriate sterilization of the composition of the present invention in liquid form, a predetermined amount can be dispensed into vials, pre-frozen for approximately 2 hours at a temperature ranging from approximately −40°C to −20°C, primary dried under reduced pressure at a temperature ranging from approximately 0°C to 10°C, and then secondary dried under reduced pressure at a temperature ranging from approximately 15°C to 25°C, thereby achieving lyophilization. The interior of the vial can then generally be purged with nitrogen gas, and the vial can then be stoppered to obtain a lyophilized formulation of the composition of the present invention.

[0099] The lyophilized pharmaceutical composition of the present invention can generally be reconstituted and used by adding any appropriate solution (reconstitution liquid). Examples of such reconstitution liquid include water for injection, physiological saline, and other general infusion solutions. The volume of the reconstitution liquid varies depending on the intended use and is not particularly limited, but is suitably 0.5 to 2 times the volume of the liquid before lyophilization or 500 mL or less.

[0100] The dosage of the pharmaceutical composition of the present invention is preferably adjusted taking into consideration the type of antisense oligonucleotide of the present invention contained therein, the dosage form, the patient's condition such as age and body weight, the route of administration, and the nature and severity of the disease. For adults, the amount of the antisense oligonucleotide of the present invention per administration is 0.01 mg to 20 mg per kg of body weight, preferably 0.03 mg to 10 mg per kg of body weight, more preferably 0.05 mg to 4 mg per kg of body weight, and more preferably 0.1 mg to 2 mg per kg of body weight. The administration frequency may be once every 1 to 3 days, once a week, or once every 2 to 3 weeks. This value may vary depending on the type of disease, administration form, and target molecule being targeted. Therefore, in some cases, a lower dose or administration frequency may be sufficient, while in other cases, a higher dose or administration frequency may be required.

[0101] The administration form of the pharmaceutical composition of the present invention is not particularly limited as long as it is a pharmaceutically acceptable administration form and can be selected according to the treatment method, and examples thereof include intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, oral administration, intratissue administration, transdermal administration, pulmonary administration, nasal administration, and administration to the central nervous system. Examples of administration to the central nervous system include intrathecal administration, intracranial administration, for example, intraventricular administration or lateral ventricle administration, intraparenchymal administration, and subpial (pia) administration. In addition, the dosage form that the composition of the present invention can take is not particularly limited, and examples thereof include various injections, oral preparations, infusions, inhalants, ointments, lotions, and poultices.

[0102] The subjects to which the antisense oligonucleotides or pharmaceutical compositions of the present invention are administered include, for example, mammals, such as primates such as humans, laboratory animals such as rats, mice, and brown rats, and livestock animals such as pigs, cows, horses, and sheep, and preferably humans.

[0103] In one embodiment, the present invention relates to the antisense oligonucleotide of the present invention, or a pharmaceutically acceptable salt or hydrate thereof, or a pharmaceutical composition for treating and / or preventing TDP-43 proteinopathy.

[0104] In one embodiment, the present invention relates to a method for treating and / or preventing TDP-43 proteinopathy, comprising the step of administering to a subject the antisense oligonucleotide of the present invention, or a pharmaceutically acceptable salt or hydrate thereof, or a pharmaceutical composition thereof. The pharmaceutical composition in this embodiment, as well as the dosage and administration route of the pharmaceutical composition, are as described herein.

[0105] In one embodiment, the present invention relates to use of the antisense oligonucleotide of the present invention, or a pharmaceutically acceptable salt or hydrate thereof, or the pharmaceutical composition of the present invention, in the manufacture of a medicament for use in a method for treating and / or preventing TDP-43 proteinopathy.

[0106] As used herein, the term "TDP-43 proteinopathy" refers to a neurodegenerative disease associated with the accumulation of TDP-43. Examples of TDP-43 proteinopathy include amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), Perry syndrome, and Lewy body disease, with amyotrophic lateral sclerosis being preferred.

[0107] As used herein, "treating" a TDP-43 proteinopathy includes one or more of alleviating, ameliorating, and remission of a TDP-43 proteinopathy or its symptoms. As used herein, "preventing" a TDP-43 proteinopathy includes reducing the risk of developing a TDP-43 proteinopathy or its symptoms.

[0108] The present invention will be explained in more detail below with reference to examples and test examples, but the present invention is not limited to the scope shown in the examples.

[0109] A "gapmer" is defined as an oligomeric compound, generally an oligonucleotide, having a central region composed primarily of deoxyoligonucleotides flanked by two flanking segments of non-deoxyoligonucleotides. The central region is called the "gap," and the flanking segments are called "wings." The gapmer used in this example has a 10-nucleotide gap flanked by two flanking wings of 5 nucleotides. This is called a 5-10-5 gapmer.

[0110] Example 1 Synthesis of Gapmer (Oligonucleotide) Among the gapmers used in this example, a specific oligonucleotide having a 2'-methoxyethyl (2'-O-MOE) group represented by formula (a) on the wing nucleoside was synthesized with reference to the method described in W. Brad Wan et al., Nucleic Acids Research, 2014, Vol. 42, No. 22, pp. 13456-13468. (wherein Base is 5-methylcytosine (C), thymine (T), adenine (A), or guanine (G), and Me is methyl.)

[0111] The 20-mer gapmers (oligonucleotides) to be tested, each having a 2'-O-MOE group represented by formula (a) at the wing nucleoside, were synthesized on a 0.5, 1, or 10 μmol scale using an automated nucleic acid synthesizer, NTS M-8-MX DNA / RNA (Nihon Techno Service Co., Ltd.) or an automated nucleic acid synthesizer, NTS H-6 DNA / RNA (Nihon Techno Service Co., Ltd.). Chain elongation was carried out using a standard phosphoramidite protocol (solid support: Glen UniSupport, sulfurization using DDTT ([(N,N-Dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione) or the like), resulting in an oligonucleotide in which the terminal 5'-hydroxyl group was protected with a dimethoxytrityl (DMTr) group and the 3'-position was supported on the solid support. Subsequently, the DMTr group was removed by treatment with Deblocking Solution-1 (available from Fujifilm Wako Co., Ltd.), and the target product was cleaved from the solid support by ammonia treatment. The solvent was evaporated, and the resulting crude product was purified by reverse-phase column chromatography using Sep-Pak C18 Plus Short Cartridge, 360 mg Sorbent per Cartridge, 55-105 μm, 50 / pk (Waters) or YMC_Triart_C18. In some cases, anion exchange purification and desalting by reverse-phase chromatography were used in combination. Fractions containing the target product were collected and concentrated under reduced pressure. The resulting residue was dissolved in water and lyophilized to obtain the target product as a white, fluffy solid. The target product was obtained as a triethylamine salt or sodium salt. The molecular weight of the resulting target product was confirmed using ESI-TOF-MS.

[0112] Example 2: Design of Gapmers (Oligonucleotides) for Test Subjects Gapmers (oligonucleotides) for test subjects were designed as antisense oligonucleotides (ASOs) targeting human TDP-43 mRNA (Gen Bank: NM_007375.4, SEQ ID NO: 1) or pre-mRNA. The sequences of the oligonucleotides, expressed in DNA bases, are shown in Table 2. Here, "start site in SEQ ID NO: 1" indicates the base position (position) of the 5'-terminal base of the target region of each ASO from the 5'-end of the base sequence of SEQ ID NO: 1. Furthermore, "end site in SEQ ID NO: 1" indicates the base position (position) of the 3'-terminal base of the target region of each ASO from the 5'-end of the base sequence of SEQ ID NO: 1.

[0113] All oligonucleotides in Table 2 are 5-10-5 gapmers, all wings are ribonucleotides with 2'-O-MOE modifications, all gaps are deoxyribonucleotides, and all internucleoside linkages are phosphorothioate (P=S). Also, for the gapmers in Table 2, the C (cytosine) in the gap and wings is actually methyl C, and the T in the wings is actually T.

[0114]

[0115] Example 3: Test of TDP-43 gene knockdown activity of gapmers (antisense oligonucleotides) Test Example 1: Measurement of knockdown activity (inhibitory activity) of human TDP-43 gene A204 cells (human rhabdomyosarcoma cell line, obtained from ATCC) 3.0 x 10 ５ A gapmer shown in Table 2 was introduced into each cell at a concentration of 4.5 μM using SF Cell Line 4D-Nucleofector™ X Kit S according to the protocol attached to the kit. The program used was DS-130.

[0116] After transfection, the cells were incubated in 0.5 mL of McCoy's 5A Medium (SIGMA) containing 10% fetal bovine serum (FBS) (Nichirei) and 1 mM L-glutamine (Gibco) at 37°C and 5% CO ２ The cells were cultured overnight under the conditions.

[0117] The test substances have 2'-O-MOE groups in the wing nucleosides, are 5-10-5 gapmers, and all internucleoside bonds are phosphorothioate (P=S). Furthermore, C (cytosine) in the gap and wing regions is actually methyl C, and T (thymine) in the wing region is actually T.

[0118] After washing the cells once with PBS (Nacalai Tesque), Buffer RA1 (Takara Bio) containing 1% 2-mercaptoethanol (Nacalai Tesque) was added to lyse the cells, which were then collected on a NucleoSpin® Filter (Takara Bio). The filter was centrifuged at 11,000 × g for 1 minute, and the permeate was collected as a homogenate. Total RNA was extracted from the homogenate using Nucleospin® RNA (Takara Bio) according to the attached protocol. The concentration of the extracted total RNA was measured using a NanoDrop One (Thermo Fisher).

[0119] Reverse transcription (RT) reaction was performed on 250 ng of extracted total RNA using the High Capacity cDNA Reverse Transcription Kit (applied biosystems) and the random primers included with the kit. Specifically, the reaction solution was prepared according to the protocol attached to the kit. The thermal cycler used was a TaKaRa PCR Thermal Cycler Dice Touch (Takara Bio). The RT reaction program used was as follows: 25°C, 10 minutes: primer annealing; 37°C, 120 minutes: reverse transcription; 85°C, 5 minutes: reverse transcriptase inactivation.

[0120] Subsequently, qPCR was performed using the RT reaction product as a template with Fast SYBR Green Master Mix (Thermo Fisher Scientific) to measure the RNA expression levels of TDP-43 and β-actin endogenously expressed in A204 cells. Specifically, the reaction solution was prepared according to the protocol attached to the kit. qPCR was performed using a QuantStudio6Flex Real-Time PCR System (applied biosystems). The qPCR program used was as follows: 50°C, 2 minutes; 95°C, 10 minutes: polymerase activation, cDNA thermal denaturation [95°C, 15 seconds; 60°C, 1 minute] x 40 cycles: PCR amplification 95°C, 15 seconds; 60°C, 1 minute → 95°C, 15 seconds (Melting Curve 0.05°C / s): melting curve analysis

[0121] The primers used to detect TDP-43 RNA are shown in Table 3. Two sequences within TDP-43 (exon 2 to exon 3, exon 5 to exon 6) were amplified and detected. The primers used to detect the RNA of the housekeeping gene β-actin are shown in Table 4.

[0122] The TDP-43 RNA detection value was corrected using the β-actin RNA detection value of the housekeeping gene. Furthermore, the ratio of the TDP-43 RNA correction value of cells transfected with each gapmer to the TDP-43 RNA correction value of cells transfected with 4D-Nucleofector™ without adding any gapmer ("vehicle") was calculated, and the inhibitory activity of each gapmer was analyzed using this ratio as the TDP-43 RNA expression level. The RNA expression level shown here is the result of using both the primer set for detecting exon 2-3 (RNA expression level (exon 2-3)) and the primer set for detecting exon 5-6 (RNA expression level (exon 5-6)). Furthermore, ANT-10 (SEQ ID NO: 114), whose activity was confirmed in a preliminary test (data not shown), was used as a comparative gapmer. Specifically, the ratio of RNA expression level to ANT-10 (SEQ ID NO: 114) was calculated from the ratio of the TDP-43 RNA expression level in cells transfected with each gapmer to the TDP-43 RNA expression level in cells transfected with the gapmer ANT-10 (SEQ ID NO: 114). For the calculation, the value for each assay was used as the TDP-43 RNA expression level in cells transfected with the gapmer ANT-10 (SEQ ID NO: 114). The results are shown in Table 5 (in the table, "RNA expression ratio / ANT-10 (exon 2-3)" indicates the RNA expression ratio when the detection primer set for exon 2-3 was used, and "RNA expression ratio / ANT-10 (exon 5-6)" indicates the RNA expression ratio when the detection primer set for exon 5-6 was used. Furthermore, "average RNA expression ratio" indicates the average value of RNA expression ratio / ANT-10 (exon 2-3) and RNA expression ratio / ANT-10 (exon 5-6). For ANT-10 (SEQ ID NO: 114), a representative value from multiple assays is shown).

[0123]

[0124]

[0125]

[0126] Example 4: Design 2 of Gapmers (Oligonucleotides) for Test Subjects Gapmers (oligonucleotides) for test subjects were designed as antisense oligonucleotides (ASOs) targeting human TDP-43 mRNA (Gen Bank: NM_007375.4, SEQ ID NO: 1) or pre-mRNA. The sequences of the oligonucleotides, expressed in DNA bases, are shown in Tables 6 to 9. Here, "start site in SEQ ID NO: 1" indicates the base position (position) of the 5'-terminal base of the target region of each ASO from the 5'-end of the base sequence of SEQ ID NO: 1. Furthermore, "end site in SEQ ID NO: 1" indicates the base position (position) of the 3'-terminal base of the target region of each ASO from the 5'-end of the base sequence of SEQ ID NO: 1.

[0127] The oligonucleotides in Tables 6 and 7 are all 5-10-5 gapmers, with all wing portions being ribonucleotides with 2'-O-MOE modifications, all gap portions being deoxyribonucleotides, and all internucleoside linkages being phosphorothioate (P=S). Also, for the gapmers in Tables 6 and 7, the C (cytosine) in the gap portion and wing portions is actually methyl C, and the T in the wing portions is actually T.

[0128]

[0129] All oligonucleotides in Table 8 are 5-10-5 gapmers, with all wing portions consisting of ribonucleotides with 2'-O-MOE modifications, and the gap portion consisting of deoxyribonucleotides or ribonucleotides with 2'-OMe or 2'-O-MOE modifications. The internucleoside linkage is either phosphorothioate (P=S) or phosphodiester (P=O). In Table 8, ribonucleotides with 2'-O-MOE modifications are underlined, and ribonucleotides with 2'-OMe modifications are double-underlined. Unless otherwise specified, the internucleoside linkage is phosphorothioate, and when the internucleoside linkage is a phosphodiester linkage, it is indicated by a "^" between the bases. For the gapmers in Table 8, the C (cytosine) in the gap portion and wing portion is actually methyl C. However, the C in the ribonucleotide with a 2'-OMe group is C, not methyl C. The T in the wing portion is actually T, and the T in the 2'-OMe group in the gap portion is actually U.

[0130] All oligonucleotides in Table 9 are 5-10-5 gapmers, with all wing portions consisting of ribonucleotides with 2'-O-MOE modifications, and the gap portion consisting of deoxyribonucleotides or ribonucleotides with 2'-OMe or 2'-O-MOE modifications. The internucleoside linkage is either phosphorothioate (P=S) or phosphodiester (P=O). In Table 9, ribonucleotides with 2'-O-MOE modifications are underlined, and ribonucleotides with 2'-OMe modifications are double-underlined. Unless otherwise specified, the internucleoside linkage is phosphorothioate, and when the internucleoside linkage is a phosphodiester linkage, it is indicated by a "^" between the bases. For the gapmers in Table 9, the C (cytosine) in the gap portion and wing portion is actually methyl C. However, the C in the ribonucleotide with a 2'-OMe group is C, not methyl C. The T in the wing portion is actually T, and the T in the 2'-OMe group in the gap portion is actually U.

[0131] Example 5: Test of TDP-43 gene knockdown activity of gapmers (antisense oligonucleotides) Test Example 2: Measurement of knockdown activity (inhibitory activity) of human TDP-43 gene A204 cells (human rhabdomyosarcoma cell line, obtained from ATCC) 3.0 x 10 ５ Gapmers shown in Tables 7 to 9 were introduced into the cells at a concentration of 1.5 μM using SF Cell Line 4D-Nucleofector™ X Kit S according to the protocol attached to the kit. The program used was DS-130.

[0132] After transfection, the cells were incubated in 0.5 mL of McCoy's 5A Medium (SIGMA) containing 10% fetal bovine serum (FBS) (Nichirei) and 1 mM L-glutamine (Gibco) at 37°C and 5% CO ２ The gapmer described in Example 4 was used as the test substance.

[0133] After washing the cells once with PBS (Nacalai Tesque), Buffer RA1 (Takara Bio) containing 1% 2-mercaptoethanol (Nacalai Tesque) was added to lyse the cells, which were then collected on a NucleoSpin® Filter (Takara Bio). The filter was centrifuged at 11,000 × g for 1 minute, and the permeate was collected as a homogenate. Total RNA was extracted from the homogenate using Nucleospin® RNA (Takara Bio) according to the attached protocol. The concentration of the extracted total RNA was measured using a NanoDrop One (Thermo Fisher).

[0134] Reverse transcription (RT) reaction was performed on 250 ng of extracted total RNA using the High Capacity cDNA Reverse Transcription Kit (applied biosystems) and the random primers included with the kit. Specifically, the reaction solution was prepared according to the protocol attached to the kit. The thermal cycler used was a TaKaRa PCR Thermal Cycler Dice Touch (Takara Bio). The RT reaction program used was as follows: 25°C, 10 minutes: primer annealing; 37°C, 120 minutes: reverse transcription; 85°C, 5 minutes: reverse transcriptase inactivation.

[0135] Subsequently, qPCR was performed using the RT reaction product as a template with Fast SYBR Green Master Mix (Thermo Fisher Scientific) to measure the RNA expression levels of TDP-43 and β-actin endogenously expressed in A204 cells. Specifically, the reaction solution was prepared according to the protocol attached to the kit. qPCR was performed using a QuantStudio6Flex Real-Time PCR System (applied biosystems). The qPCR program used was as follows: 50°C, 2 minutes; 95°C, 10 minutes: polymerase activation, cDNA thermal denaturation [95°C, 15 seconds; 60°C, 1 minute] x 40 cycles: PCR amplification 95°C, 15 seconds; 60°C, 1 minute → 95°C, 15 seconds (Melting Curve 0.05°C / s): melting curve analysis

[0136] The primers used to detect TDP-43 RNA are shown in Table 10. Two sequences within TDP-43 (exon 2 to exon 3, 3'UTR) were amplified and detected. The primers used to detect the housekeeping gene β-actin RNA are shown in Table 11.

[0137] The TDP-43 RNA detection value was corrected using the β-actin RNA detection value of the housekeeping gene. Furthermore, the ratio of the TDP-43 RNA correction value of cells transfected with each gapmer to the TDP-43 RNA correction value of cells transfected with 4D-Nucleofector™ without adding any gapmer ("vehicle") was calculated, and the inhibitory activity of each gapmer was analyzed using this ratio as the TDP-43 RNA expression level. The RNA expression level shown here is the result of using both the primer set for detecting exon 2-3 (RNA expression level (exon 2-3)) and the primer set for detecting 3'UTR (RNA expression level (3'UTR)). Furthermore, ANT-10 (SEQ ID NO: 114), whose activity was confirmed in a preliminary test (data not shown), was used as a comparative gapmer. Specifically, the ratio of RNA expression level to ANT-10 (SEQ ID NO: 114) was calculated from the ratio of the TDP-43 RNA expression level in cells transfected with each gapmer to the TDP-43 RNA expression level in cells transfected with the gapmer ANT-10 (SEQ ID NO: 114). For the calculation, the value for each assay was used as the TDP-43 RNA expression level in cells transfected with the gapmer ANT-10 (SEQ ID NO: 114). The results are shown in Tables 12 to 14. (In the tables, "RNA expression ratio / ANT-10 (exon 2-3)" indicates the RNA expression ratio when the primer set for detecting exon 2-3 was used, and "RNA expression ratio / ANT-10 (3'UTR)" indicates the RNA expression ratio when the primer set for detecting 3'UTR was used. In addition, "average RNA expression ratio" indicates the average value of RNA expression ratio / ANT-10 (exon 2-3) and RNA expression ratio / ANT-10 (3'UTR). For ANT-10 (SEQ ID NO: 114), a representative value from multiple assays is shown.)

[0138] Similar procedures were also carried out using ANT-266 (SEQ ID NO: 114) as a comparative gapmer. Specifically, the ratio of RNA expression level relative to ANT-266 (SEQ ID NO: 114) was calculated from the ratio of the TDP-43 RNA expression level in cells transfected with each gapmer to the TDP-43 RNA expression level in cells transfected with the gapmer shown in ANT-266 (SEQ ID NO: 114). The results are shown in Table 15. (In the table, "RNA expression ratio / ANT-266 (exon 2-3)" indicates the RNA expression ratio when the primer set for detecting exon 2-3 was used, and "RNA expression ratio / ANT-266 (exon 5-6)" indicates the RNA expression ratio when the primer set for detecting exon 5-6 (Table 3) was used. Furthermore, "average RNA expression ratio" indicates the average value of the RNA expression ratio / ANT-266 (exon 2-3) and the RNA expression ratio / ANT-10 (exon 5-6).)

[0139] The same procedure was also carried out using ANT-266 (SEQ ID NO: 114) as a gapmer for comparison, but the introduction concentration was changed to 0.6 μM. Specifically, the ratio of RNA expression level relative to ANT-266 (SEQ ID NO: 114) was calculated from the ratio of the TDP-43 RNA expression level in cells transfected with each gapmer to the TDP-43 RNA expression level in cells transfected with the gapmer shown in ANT-266 (SEQ ID NO: 114). The results are shown in Tables 16 to 18. (In the tables, "RNA expression ratio / ANT-266 (exon 2-3)" indicates the RNA expression ratio when the primer set for detecting exon 2-3 was used, and "RNA expression ratio / ANT-266 (3'UTR)" indicates the RNA expression ratio when the primer set for detecting 3'UTR was used. Furthermore, "average RNA expression ratio" indicates the average value of the RNA expression ratio / ANT-266 (exon 2-3) and the RNA expression ratio / ANT-10 (3'UTR).)

[0140] The calculated and measured values ​​of ESI-TOF-MS for each gapmer obtained in the examples are shown below.

Claims

[Claim 1] The invention described herein.