ANTISENSE OLIGONUCLEOTIDE OF RasGRP4

Abstract

The present invention provides a compound which is, as an antisense oligonucleotide that controls the gene expression of RasGRP4 and is capable of treating myositis and rheumatoid arthritis, an antisense oligonucleotide having a nucleobase sequence that contains 8-80 consecutive nucleosides and has at least eight continuous nucleobases which are complementary to a transcript of RasGRP4, or a pharmaceutically acceptable salt thereof.

Description

RasGRP4 antisense oligonucleotides

[0001] The present invention relates to antisense oligonucleotides of RasGRP4.

[0002] Polymyositis / dermatomyositis (PM / DM) is an autoimmune disease characterized by systemic inflammation, primarily affecting the muscles, skin, and lungs, resulting in muscle weakness, primarily affecting the proximal muscles. PM / DM often develops interstitial pneumonia, potentially resulting in a poor prognosis. In Japan, the number of PM / DM patients was estimated at 17,000 in 2010, with a reported prevalence of 13.2 per 100,000 (see, for example, Non-Patent Document 1). Because the exact etiology remains unknown, standard treatment primarily involves the administration of nonspecific immunosuppressants, including glucocorticoids and other immunosuppressants. These drugs have issues, such as side effects and the development of treatment resistance, necessitating the development of new PM / DM treatments. RasGPR4 (Ras guanine nucleotide-releasing protein 4) is a guanine exchange factor that activates Ras, and is highly expressed in bone marrow cells such as monocytes, macrophages, neutrophils, and mast cells. It has also been reported that RasGRP4 expression is elevated in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis, that there is a positive correlation between the amount of RasGRP4 mRNA expression in FLS and its cell proliferation ability, and that knocking down RasGRP4 using siRNA reduces the cell proliferation ability of FLS (see, for example, Non-Patent Document 2). This document also reports that in rats with collagen-induced arthritis, intra-ankle administration of RasGRP4 siRNA significantly reduced ankle arthritis scores and ankle diameter compared to controls, and also reduced bone and cartilage destruction. It has also been reported that RasGRP4 knockout mice have reduced sodium dodecyl sulfate-induced colitis compared to wild-type mice (see, for example, Non-Patent Document 3). siRNA has been reported as a substance that inhibits RasGRP4 gene expression (see, for example, Patent Document 1, Patent Document 2, and Non-Patent Document 2).

[0003] JP 2019-131502 A JP 2020-015678 A

[0004] Mod Rheumatol. 2014, 24(3), pp 477-480Arthritis Rheumatol. 2015, 67(2), pp 396-407J. Biol. Chem. 2012, 287(24), pp 20047-20055

[0005] An object of the present invention is to provide a novel antisense oligonucleotide that inhibits the expression of the RasGRP4 gene, and a new therapeutic agent for PM / DM.

[0006] The present inventors have conducted extensive research to find compounds that have an antisense effect on RasGRP4, and have found that the compounds of the present invention have an excellent inhibitory effect on the expression of the RasGRP4 gene, thereby completing the present invention.

[0007] 1. A compound comprising an antisense oligonucleotide that inhibits the expression of the RasGRP4 gene, or a pharmacologically acceptable salt thereof.

[0008] 2. Nucleic acid base position numbers of SEQ ID NO: 1: 237 to 252, 244 to 259, 501 to 516, 666 to 681, 683 to 698, 774 to 789, 889 to 904, 939 to 954, 964 to 979, 968 to 983, 969 to 984, 970 to 985, 971 to 986, 972 to 987, 974 to 989, 976 to 991, 977 to 992, 978 to 993, 979 to 994, 980 to 995, 981 to 996, 982 to 997, 1033 to 1048, 1036 to 1051, 1037 to 105 2, 1038-1053, 1039-1054, 1040-1055, 1041-1056, 1114-1129, 1186-1201, 1245-1260, 1246-1261, 1247-1262, 1248-1263, 1249-1264, 1250-1265, 1251-1266, 1252-1267, 1253-1268, 1365-1380, 1368-1383, 1369-1384, 1395-1410, 1396-1411, 13 97-1412, 1398-1413, 1399-1414, 1400-1415, 1401-1416, 1446-1461, 1523-1538, 1525-1540, 1526-1541, 1538-1553, 1590-1605, 1591-1606, 1592-1607, 1593-1608, 1668-1683, 1719-1734, 1744-1759, 1753-1768, 1756-1771, 1757-1772, 1758-1769 773, 1759-1774, 1760-1775, 1761-1776, 1813-1828, 1814-1829, 1815-1830, 1816-1831, 1817-1832, 1818-1833, 1819-1834, 1820-1835, 1821-1836, 1822-1837, 1823-1838, 1894-1909, 2042-2057, 2049-2064, 2050-2065, 2051-2066, and 3059-3074 and an antisense oligonucleotide consisting of 8 to 80 nucleosides complementary to a portion of a nucleobase sequence selected from the group consisting of the nucleobase sequences represented by SEQ ID NO: 1, wherein the antisense oligonucleotide is at least 80% complementary to a portion of the selected nucleobase sequence of SEQ ID NO: 1, or the nucleobase positions 640 to 655 of SEQ ID NO: 2,９６７～９８２、１０３７～１０５２、１２３１～１２４６、４８３８～４８５３、４８４５～４８６０、５５７５～５５９０、５８０２～５８１７、５９１８～５９３３、６３９５～６４１０、６５４４～６５５９、６６３９～６６５４、６７７１～６７８６、６９２１～６９３６、６９３８～６９５３、７０２９～７０４４、７０３３～７０４８、７２０６～７２２１、８４３９～８４５４、８４８９～８５０４、９３６１～９３７６、９４３９～９４５４、９６３２～９６４７、９７３６～９７５１、９７４０～９７５５、９７４１～９７５６、９７４２～９７５７、９７４３～９７５８、９７４４～９７５９、９７４６～９７６１、９７４８～９７６３、９７４９～９７６４、９７５０～９７６５、９７５１～９７６６、９７５２～９７６７、９７５３～９７６８、９７５４～９７６９、９８０５～９８２０、９８０８～９８２３、９８０９～９８２４、９８１０～９８２５、９８１１～９８２６、９８１２～９８２７、９８１３～９８２８、１１８１４～１１８２９、１１８８６～１１９０１、１１９４５～１１９６０、１１９４６～１１９６１、１１９４７～１１９６２、１１９４８～１１９６３、１１９４９～１１９６４、１１９５０～１１９６５、１１９５１～１１９６６、１１９５２～１１９６７、１１９５３～１１９６８、１３４３８～１３４５３、１３４４１～１３４５６、１３４４２～１３４５７、１３４６８～１３４８３、１３４６９～１３４８４、１３４７０～１３４８５、１３４７１～１３４８６、１３４７２～１３４８７、１３４７３～１３４８８、１３４７４～１３４８９、１３６１５～１３６３０、１３６９２～１３７０７、１３６９４～１３７０９、１３６９５～１３７１０、１３９０２～１３９１７、１３９０３～１３９１８、１３９０４～１３９１９、１３９０５～１３９２０、１４０９４～１４１０９、１４１４５～１４１６０、１４１７０～１４１８５、１４１７９～１４１９４、１４１８２～１４１９７、１４１８３～１４１９８、１４１８４～１４１９９、１４１８５～１４２００、１４１８６～１４２０１、１４１８７～１４２０２、１４８６８～１４８８３、１５５２９～１５５４４、１５５３０～１５５４５、１５５３１～１５５４６、１５５３２～１５５４７、１５５３３～１５５４８、１５５３４～１５５４９、１５５３５～１５５５０、The present invention also provides an antisense oligonucleotide consisting of 8 to 80 nucleosides complementary to a portion of a nucleobase sequence selected from the group consisting of nucleobase sequences represented by SEQ ID NO: 15536-15551, 15537-15552, 15538-15553, 15539-15554, 15705-15720, 15787-15802, 15969-15984, 15976-15991, 15977-15992, 15978-15993, 16578-16593, and 17775-17790, wherein the antisense oligonucleotide is a compound that is at least 80% complementary to a portion of the selected nucleobase sequence of SEQ ID NO: 2, or a pharmacologically acceptable salt thereof.

[0009] 3. Nucleic acid base position numbers of SEQ ID NO: 1: 237 to 252, 244 to 259, 501 to 516, 666 to 681, 683 to 698, 774 to 789, 889 to 904, 939 to 954, 964 to 979, 968 to 983, 969 to 984, 970 to 985, 971 to 986, 972 to 987, 974 to 989, 976 to 991, 977 to 992, 978 to 993, 979 to 994, 980 to 995, 981 to 996, 982 to 997, 1033 to 1048, 1036 to 1051, 1037 to 105 2, 1038-1053, 1039-1054, 1040-1055, 1041-1056, 1114-1129, 1186-1201, 1245-1260, 1246-1261, 1247-1262, 1248-1263, 1249-1264, 1250-1265, 1251-1266, 1252-1267, 1253-1268, 1365-1380, 1368-1383, 1369-1384, 1395-1410, 1396-1411, 13 97-1412, 1398-1413, 1399-1414, 1400-1415, 1401-1416, 1446-1461, 1523-1538, 1525-1540, 1526-1541, 1538-1553, 1590-1605, 1591-1606, 1592-1607, 1593-1608, 1668-1683, 1719-1734, 1744-1759, 1753-1768, 1756-1771, 1757-1772, 1758-1769 773, 1759-1774, 1760-1775, 1761-1776, 1813-1828, 1814-1829, 1815-1830, 1816-1831, 1817-1832, 1818-1833, 1819-1834, 1820-1835, 1821-1836, 1822-1837, 1823-1838, 1894-1909, 2042-2057, 2049-2064, 2050-2065, 2051-2066, and 3059-3074 2. The compound according to 2., or a pharmacologically acceptable salt thereof, comprising an antisense oligonucleotide consisting of 8 to 80 nucleosides complementary to a portion of a nucleic acid base sequence selected from the group consisting of nucleic acid base sequences represented by SEQ ID NO: 2, wherein the antisense oligonucleotide is at least 80% complementary to a portion of the selected nucleic acid base sequence of SEQ ID NO: 2.

[0010] 4. Nucleic acid base position number of SEQ ID NO: 2 640-655, 967-982, 1037-1052, 1231-1246, 4838-4853, 4845-4860, 5575-5590, 5802-5817, 5918-5933, 6395-6410, 6544-6559, 6639-6654, 6771-6786, 6921-6936, 6938-6953, 7029-7044, 7033-7048, 7206-7221, 8439-8454, 8489-8504, 9361-9376, 9439-9454, 9632-9647, 9736-9751, 9740-975 5, 9741-9756, 9742-9757, 9743-9758, 9744-9759, 9746-9761, 9748-9763, 9749-9764, 9750-9765, 9751-9766, 9752-9767, 9753-9768, 9754-9769, 9805-9820, 9808-9823, 9809-9824, 9810-9825, 9811-9826, 9812-9827, 9813-9828, 11814-11829, 11886-11901, 11945-11960, 11946-11961, 1194 7 to 11962, 11948 to 11963, 11949 to 11964, 11950 to 11965, 11951 to 11966, 11952 to 11967, 11953 to 11968, 13438 to 13453, 13441 to 13456, 13442 to 13457, 13468 to 13483, 13469 to 13484, 13470 to 13485, 13471 to 13486, 13472 to 13487, 13473 to 13488, 13474 to 13489, 13615 to 13630, 13692 to 13707, 13694 to 13709, 13695 to 13710 710, 13902 to 13917, 13903 to 13918, 13904 to 13919, 13905 to 13920, 14094 to 14109, 14145 to 14160, 14170 to 14185, 14179 to 14194, 14182 to 14197, 14183 to 14198, 14184 to 14199, 14185 to 14200, 14186 to 14201, 14187 to 14202, 14868 to 14883, 15529 to 15544, 15530 to 15545, 15531 to 15546, 15532 to 15547, 15533 to 15548,15976-15991, 15977-15992, 15978-15993, 16578-16593, and 17775-17790, wherein the antisense oligonucleotide is at least 80% complementary to a portion of the selected nucleobase sequence of SEQ ID NO: 2, or a pharmacologically acceptable salt thereof. ,

[0011] 5. The compound according to 2., or a pharmacologically acceptable salt thereof, comprising an antisense oligonucleotide consisting of 8 to 80 nucleosides and having a nucleobase sequence containing at least 8 consecutive nucleobases of any one of the nucleobase sequences of SEQ ID NOs: 3 to 123.

[0012] 6. The compound according to 5., or a pharmacologically acceptable salt thereof, comprising an antisense oligonucleotide having a nucleic acid base sequence comprising any one of the nucleic acid base sequences of SEQ ID NOs: 3 to 123.

[0013] 7. The compound according to 5. or 6., or a pharmacologically acceptable salt thereof, comprising an antisense oligonucleotide having a nucleic acid base sequence of any one of SEQ ID NOs: 3 to 123.

[0014] 8. SEQ ID NOs: 4, 5, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 1 5. The compound according to any one of 5. to 7., or a pharmacologically acceptable salt thereof, comprising an antisense oligonucleotide consisting of 8 to 80 nucleosides having a nucleobase sequence comprising at least 8 consecutive nucleobases of any one nucleobase sequence selected from the group consisting of: 5, 66, 67, 68, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 95, 96, 97, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, and 123.

[0015] 9. The compound or pharmacologically acceptable salt thereof according to any one of 1. to 8., wherein the antisense oligonucleotide contains a phosphorothioate bond.

[0016] 10. The compound or pharmacologically acceptable salt thereof according to any one of 1. to 9., wherein the antisense oligonucleotide comprises at least one nucleoside selected from the group consisting of 2'-modified nucleosides and 2'-4'-bridged nucleosides.

[0017] 11. The compound or pharmacologically acceptable salt thereof according to 10., wherein the 2'-4'-bridged nucleoside is at least one selected from the group consisting of LNA, ENA, cEt, AmNA, scpBNA, and GuNA.

[0018] 12. The compound or pharmacologically acceptable salt thereof according to 11., wherein the 2'-4'-bridged nucleoside is LNA.

[0019] 13. The compound or pharmacologically acceptable salt thereof according to any one of 10. to 12., wherein the 2'-modified nucleoside is at least one selected from the group consisting of a 2'-O-MCE nucleoside, a 2'-O-MOE nucleoside, a 2'-O-NMA nucleoside, and a 2'-O-Me nucleoside.

[0020] 14. The compound or pharmacologically acceptable salt thereof according to 13., wherein the 2'-modified nucleoside is a 2'-O-MCE nucleoside.

[0021] 15. The compound or pharmacologically acceptable salt thereof according to any one of 1. to 14., wherein the antisense oligonucleotide contains 5-methylcytosine.

[0022] 16. The compound or pharmacologically acceptable salt thereof according to any one of 1. to 15., wherein the antisense oligonucleotide comprises a gap segment, a 5' wing segment, and a 3' wing segment, wherein the gap segment comprises at least two nucleosides selected from 5'-modified nucleosides and deoxyribonucleosides, and the 5' and 3' ends of the gap segment are independently 5'-modified nucleosides or deoxyribonucleosides, the 3'-terminal nucleoside of the 5' wing segment is a 2'-modified nucleoside or a 2'-4'-bridged nucleoside and is linked to the 5'-terminal of the gap segment, and the 5'-terminal nucleoside of the 3' wing segment is a 2'-modified nucleoside or a 2'-4'-bridged nucleoside and is linked to the 3'-terminal of the gap segment.

[0023] 17. The compound or pharmacologically acceptable salt thereof according to 16., wherein the gap segment consists of 5 to 30 deoxyribonucleosides, the 5' wing segment and the 3' wing segment each independently consist of 1 to 10 sugar-modified nucleosides independently selected from the group consisting of 2'-modified nucleosides and 2'-4'-bridged nucleosides, each wing segment comprises at least one phosphorothioate linkage, and each cytosine in the gap segment and each wing segment is replaced with a 5-methylcytosine.

[0024] 18. The compound or pharmacologically acceptable salt thereof according to 17., wherein the gap segment consists of 8 to 12 deoxyribonucleosides, the 5' wing segment and the 3' wing segment each independently consist of 2 to 5 sugar-modified nucleosides independently selected from the group consisting of LNA and 2'-O-MCE nucleosides, and contain at least one 2'-O-MCE nucleoside, and the gap segment contains at least one phosphorothioate linkage.

[0025] 19. The compound or pharmacologically acceptable salt thereof according to any one of 16 to 18, wherein the 5' wing segment and the 3' wing segment are each independently selected from the group consisting of VLL, LVL, LLV, LVV, VLV, VVL, LLL, VVLL, VLVL, VLLV, LVLV, LLVV, LVVL, VLLL, LVLL, LLVL, LLLV, LVVV, VLVV, VVLV, VVVL, VLLVV, and VVLLV, wherein L represents a 2'-4'-bridged nucleoside and V represents a 2'-modified nucleoside.

[0026] 20. The compound or pharmacologically acceptable salt thereof according to any one of 1. to 19., wherein the antisense oligonucleotide consists of 15 to 25 nucleosides.

[0027] 21. The compound or pharmacologically acceptable salt thereof according to any one of 1. to 20., wherein the compound comprising an antisense oligonucleotide comprises a functional molecule.

[0028] 22. The compound or pharmacologically acceptable salt thereof according to 21., wherein the functional molecule is a group derived from a molecule having a function of delivering an antisense oligonucleotide to a target site.

[0029] 23. The compound or pharmacologically acceptable salt thereof according to 21. or 22., wherein the functional molecule is a lipid selected from the group consisting of cholesterol, vitamins, steroids, C5-30 saturated fatty acids, and C5-30 unsaturated fatty acids.

[0030] 24. The compound or pharmacologically acceptable salt thereof according to any one of 1. to 20., wherein the compound consists of the antisense oligonucleotide.

[0031] 25. The salt according to any one of 1. to 21., wherein the pharmacologically acceptable salt is a sodium salt.

[0032] 26. A medicine comprising, as an active ingredient, the compound or pharmacologically acceptable salt thereof according to any one of 1. to 25.

[0033] 27. A therapeutic, preventive, and / or ameliorating agent for a disease or condition for which inhibiting expression of the RasGRP4 gene is effective, comprising, as an active ingredient, the compound or pharmacologically acceptable salt thereof according to any one of 1. to 25.

[0034] 28. An inhibitor of RasGRP4 gene expression, comprising the compound or pharmacologically acceptable salt thereof according to any one of 1. to 25. as an active ingredient.

[0035] It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. As used herein, the use of the singular includes the plural unless specifically stated otherwise. Also, in this description, antisense oligonucleotides or antisense oligonucleotide compounds are used interchangeably and may be referred to as "ASOs."

[0036] The present invention is described in more detail below. Unless otherwise indicated, the following terms have the following meanings.

[0037] "Optionally substituted" means unsubstituted or substituted.

[0038] "Nucleoside" is a term well known to those skilled in the art and is generally understood to mean a molecule in which a sugar and a nucleic acid base are bound, and which can be a unit constituting a nucleic acid. In this specification, nucleoside is a broader concept and includes ribonucleosides, deoxyribonucleosides, and sugar-modified nucleosides, which will be described later. The nucleic acid base may be modified.

[0039] "Ribonucleoside" refers to a molecule having a nucleobase at the carbon atom 1 of ribose. The ribonucleoside in the present invention may be a naturally occurring ribonucleoside or a ribonucleoside in which the nucleobase moiety of a naturally occurring ribonucleoside has been modified. Multiple modifications may be combined and applied to a single ribonucleoside. The modified ribonucleosides are described, for example, in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667; Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471; Future Medicinal Chemistry, 2011, 3, pp. 339-365; and WO 2018 / 155450.

[0040] "Deoxyribonucleoside" refers to a molecule having a nucleobase at the carbon atom 1 of 2-deoxyribose. The deoxyribonucleoside of the present invention may be a naturally occurring deoxyribonucleoside or a deoxyribonucleoside in which the nucleobase moiety of a naturally occurring deoxyribonucleoside has been modified. A single deoxyribonucleoside may be modified by a combination of multiple types. The modified deoxyribonucleosides are described, for example, in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667; Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471; Future Medicinal Chemistry, 2011, 3, pp. 339-365; and WO 2018 / 155450.

[0041] "Modified sugar" means: (Z1) a molecule in which ribose or 2-deoxyribose is partially replaced with one or more substituents; (Z2) a penta- or hexa-saccharide other than ribose and 2-deoxyribose (e.g., hexitol, threose, etc.); (Z3) a molecule in which the entire ribose or 2-deoxyribose, or the tetrahydrofuran ring thereof, is replaced with a 5- to 7-membered saturated or unsaturated ring (e.g., cyclohexane, cyclohexene, morpholine, etc.) or a partial structure capable of forming a 5- to 7-membered ring by hydrogen bonding (e.g., a peptide structure); or (Z4) a molecule in which ribose or 2-deoxyribose is replaced with an alkylene glycol having 2 to 6 carbon atoms (e.g., ethylene glycol, propylene glycol, etc.). Modified sugars include "2-modified sugars," "2-4-linked sugars," and "5-modified sugars," which will be described later. Examples of modified sugars and sugar-modified nucleosides described below include sugars and sugar-modified nucleosides disclosed as being suitable for use in antisense methods in, for example, JP 10-304889 A, WO 2005 / 021570 A, JP 10-195098 A, JP 2002-521310 A, WO 2007 / 143315 A, WO 2008 / 043753 A, WO 2008 / 029619 A, WO 2008 / 049085 A, and WO 2017 / 142054 A (hereinafter, these documents are referred to as "documents related to antisense methods"), etc. Modified sugars and sugar-modified nucleosides are also disclosed in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667; Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471; Future Medicinal Chemistry, 2011, 3, pp. 339-365; and WO 2018 / 155450.

[0042] Examples of modified sugars partially substituted with one substituent include ribose or 2-deoxyribose substituted at any position of the sugar moiety with one or more (preferably one or two) of the following substituents (i) or (ii): (i) C １－６alkyl group, wherein 2 or more C １－６ When substitution with an alkyl group is contained, 2 or more C １－６ The alkyl groups may be joined together to form a 3- to 6-membered ring. (ii) A halogen atom, C １－６ Alkoxy group, halo C １－６ Alkoxy group, mono- or di-C １－６ C substituted with at least one selected from the group consisting of an alkylamino group, a 5- to 10-membered heterocyclic group, a carboxy group, a carbamoyl group, and an N-substituted carbamoyl group １－６ Examples of the N-substituted carbamoyl group include an N-methyl-carbamoyl group and an N-ethyl-carbamoyl group, and the methyl group and the ethyl group of the N-methyl-carbamoyl group and the N-ethyl-carbamoyl group are 5- to 10-membered heterocyclic groups or mono- or di-C １－６ It may be substituted with an alkylamino group. Specific examples of the N-substituted carbamoyl group include an N-methylcarbamoyl group, an N-ethylcarbamoyl group, an N-dimethylaminoethyl-carbamoyl group, an N-(morpholinoethyl)carbamoyl group, an N-(2-pyridylethyl)carbamoyl group, and an N-((benzimidazol-1-yl)ethyl)carbamoyl group.

[0043] "Sugar-modified nucleoside" refers to a molecule having the above-mentioned "modified sugar" in place of the sugar moiety of a deoxyribonucleoside or ribonucleoside. For example, it includes "2'-modified nucleosides" and "2'-4'-bridged nucleosides" described below, as well as "5'-modified nucleosides." When the modified sugar is (Z3) as defined above, the sugar-modified nucleoside also includes molecules in which the modified sugar and the nucleobase are linked via a methylene chain or the like.

[0044] "Dimodified sugar" means a non-bridged sugar in which the oxygen atom or carbon atom at position 2 of ribose is modified, and includes "2-O-Me," "2-O-MOE," "2-O-MCE," "2-O-NMA," "2-O-AP," "2-F," "2-DMAECE," "2-MorECE," "2-PyECE," and "2-BimECE." Positions 4 and 5 of a "dimodified sugar" are preferably unmodified. A "2'-modified nucleoside" refers to a molecule having a nucleobase at the 1-position of the "di-modified sugar", and examples include "2'-O-Me nucleoside", "2'-O-MOE nucleoside", "2'-O-MCE nucleoside", "2'-O-NMA nucleoside", "2'-O-AP nucleoside", "2'-F nucleoside", "2'-DMAECE nucleoside", "2'-MorECE nucleoside", "2'-PyECE nucleoside", and "2'-BimECE nucleoside".

[0045] "2-O-Me" (also referred to as 2-O-methyl) refers to a sugar in which the hydroxy group at position 2 of the ribose has been replaced with a methoxy group. "2'-O-Me nucleoside" (also referred to as 2'-O-methyl nucleoside) refers to a molecule having a nucleobase at position 1 of "2-O-Me".

[0046] "2-O-MOE" (also referred to as 2-O-methoxyethyl) refers to a sugar in which the hydroxy group at position 2 of ribose is replaced with a 2-methoxyethyloxy group. "2'-O-MOE nucleoside" (also referred to as 2'-O-methoxyethyl nucleoside) refers to a molecule having a nucleobase at position 1 of "2-O-MOE".

[0047] "2-O-MCE" (also referred to as 2-O-methylcarbamoylethyl) refers to a sugar in which the hydroxy group at position 2 of the ribose is replaced with a methylcarbamoylethyloxy group. "2'-O-MCE nucleoside" (also referred to as 2'-O-methylcarbamoylethyl nucleoside) refers to a molecule having a nucleobase at position 1 of "2-O-MCE."

[0048] "2-O-NMA" refers to a sugar in which the hydroxy group at position 2 of the ribose is replaced with a [2-(methylamino)-2-oxoethyl]oxy group. "2'-O-NMA nucleoside" refers to a molecule having a nucleobase at position 1 of "2-O-NMA."

[0049] "2-O-AP" refers to a sugar in which the hydroxy group at position 2 of the ribose is replaced with a 3-aminopropyloxy group. "2'-O-AP nucleoside" refers to a molecule having a nucleobase at position 1 of "2-O-AP".

[0050] "2-F" refers to a sugar in which the hydroxy group at position 2 of the ribose is replaced with a fluorine atom. "2'-F nucleoside" refers to a molecule having a nucleobase at position 1 of "2-F".

[0051] "2-DMAECE," "2-MorECE," "2-PyECE," and "2-BimECE" are sugars in which the hydroxy group at position 2 of ribose has been replaced with the structures shown below as DMAECE, MorECE, PyECE, and BimECE, respectively. In the structures below, the wavy line indicates the bond position with the carbon atom to which the hydroxy group at position 2 of ribose is bonded.

[0052] "2'-DMAECE nucleoside," "2'-MorECE nucleoside," "2'-PyECE nucleoside," and "2'-BimECE nucleoside" refer to molecules having a nucleobase at position 1 of "2-DMAECE," "2-MorECE," "2-PyECE," and "2-BimECE," respectively.

[0053] The term "2-4 linked sugar" refers to a sugar in which the linking unit is replaced by two substitutions at positions 2 and 4 of the ribose. Examples of the linking unit include C ２－６ an alkylene group (the alkylene group is unsubstituted or substituted with one or more substituents selected from the group consisting of a halogen atom, an oxo group, and a thioxo group, and one or two methylene groups of the alkylene group are unsubstituted or independently substituted with —O—, —NR １ - (R １ is a hydrogen atom, C１－６ Alkyl group or halo C １－６ The 5-position of the "2-4-linked sugar" is preferably not modified.

[0054] The term "2',4'-bridged nucleoside" (2',4'-BNA) refers to a molecule having a nucleic acid base at the 1-position of the "2,4-bridged sugar." For example, β-D-methyleneoxy (4'-CH), also known as LNA (Locked Nucleic Acid®), ２ -O-2')BNA or α-L-methyleneoxy(4'-CH ２ -O-2')BNA, also known as ENA, ２ ) ２ -O-2')BNA, β-D-thio(4'-CH ２ -S-2')BNA, aminooxy (4'-CH ２ -O-N(R １１ )-2')BNA(R １１ is a hydrogen atom or methyl), 2',4'-BNA ＮＣ Also called oxyamino (4'-CH ２ -N(R １２ )-O-2')BNA(R １２ is a hydrogen atom or methyl), 2',4'-BNA ＣＯＣ , 3'-amino-2',4'-BNA, 5'-methyl BNA, cEt (4'-CH(CH ３ )-O-2')BNA, also called cMOE-BNA (4'-CH(CH ２ OCH ３ )-O-2') BNA, amide type BNA (also called AmNA) (4'-C(=O)-N(R １３ )-2')BNA(R １３ is a hydrogen atom or methyl), also called scpBNA (4'-C(spiro-cyclopropyl)-O-2')BNA, also called GuNA (4'-CH ２ -N(R １４ )-2')BNA(R １４ is C(=NHR １５＋ ) NHR １６ and R１５ , R １６ are each independently a hydrogen atom, methyl, ethyl, isopropyl, or t-butyl), also known as amino LNA (4'-CH ２ -N(R １７ )-2')BNA(R １７ is a hydrogen atom or methyl), and other BNAs known to those skilled in the art. Specific examples of 2'-4'-bridged nucleosides are represented by the following structural formula: In the formula, Base is a nucleic acid base. AmNA[Me] is the R １３ AmNA is AmNA where R is methyl. １５ is t-butyl, and R １６ is H and is GunA.

[0055] "5-modified sugar" refers to a non-bridged sugar in which the 5-position of the 2-deoxyribose is modified, and includes "5-CP," "5-methyl," and "5-aminopropyl." Positions 2 and 4 of the "5-modified sugar" are preferably unmodified. "5'-modified nucleoside" refers to a molecule having a nucleobase at position 1 of the "5-modified sugar," and includes, for example, "5'-CP nucleoside," "5'-methyl nucleoside," and "5'-aminopropyl nucleoside."

[0056] "5-CP" is a sugar in which the 5-position of 2-deoxyribose is substituted with two methyl groups, which together form a cyclopropane. A "5'-CP nucleoside" is a molecule represented by the following structural formula: In the formula, Base is a nucleic acid base. "5-Methyl" is a sugar in which the 5-position of the 2-deoxyribose is substituted with a methyl group. "5-Aminopropyl" is a sugar in which the 5-position of the 2-deoxyribose is substituted with a 3-aminopropyl group. "5'-Methylnucleoside" and "5'-aminopropylnucleoside" refer to molecules having a nucleobase at the 1-position of the "5-methyl" and "5-aminopropyl", respectively.

[0057] In the "deoxyribonucleosides," "ribonucleosides," "2'-modified nucleosides," and "2'-4'-bridged nucleosides," the bond between the 1' carbon atom and the nucleic acid base can be an α-glycosidic bond or a β-glycosidic bond, but is usually a β-glycosidic bond. Therefore, β-D-methyleneoxy BNA is usually used as LNA.

[0058] "n-" means normal, "s-" means secondary, and "t-" means tertiary.

[0059] "Halogen atom" means a fluorine atom, chlorine atom, bromine atom or iodine atom.

[0060] "C １－６ The term "alkyl group" means a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, an s-butyl group, a t-butyl group, an n-pentyl group, an isopentyl group, a neopentyl group, an n-hexyl group, and an isohexyl group.

[0061] "Haro C １－６ The "C alkyl group" is defined as １－６ The term "alkyl group" refers to a group in which a hydrogen atom at any position of the "alkyl group" is substituted with one or more of the above-mentioned "halogen atoms."

[0062] "C １－６ The term "alkylene group" refers to a divalent group obtained by removing one hydrogen atom from any position of a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms, and examples thereof include a methylene group, an ethylene (ethanediyl) group, a propane-1,3-diyl (trimethylene) group, a propane-2,2-diyl group, a 2,2-dimethyl-propane-1,3-diyl group, a hexane-1,6-diyl (hexamethylene) group, and a 3-methylbutane-1,2-diyl group. ２－６ The "alkylene group" is defined as the "C １－６ Among the "alkylene groups," "a linear or branched divalent group having 2 to 6 carbon atoms" means a linear or branched divalent group having 2 to 6 carbon atoms, and examples thereof include the above-mentioned "C １－６ The same applies to the "alkylene group."

[0063] "C１－６ The "alkoxy group" is the same as the "C １－６ "Alkyl" refers to a group in which an "alkyl" is bonded to an oxy group.

[0064] "Haro C １－６ The "alkoxy group" is the same as the "C １－６ The term "alkoxy group" refers to a group in which a hydrogen atom at any position of the "alkoxy group" is substituted with one or more of the above-mentioned "halogen atoms."

[0065] "Mono- or di-C １－６ An "alkylamino group" is a group in which one hydrogen atom of an amino group is substituted with one "C １－６ A group in which two hydrogen atoms of an amino group are replaced by "C alkyl groups" or two hydrogen atoms of an amino group are replaced by the same or different "C １－６ and examples thereof include methylamino, ethylamino, propylamino, isopropylamino, butylamino, dimethylamino, diethylamino, dipropylamino, dibutylamino, and N-ethyl-N-methylamino groups.

[0066] The term "3- to 6-membered ring" means a monocyclic saturated or unsaturated hydrocarbon ring having 3 to 6 carbon atoms, and examples thereof include cyclopropane, cyclobutane, and cyclohexane.

[0067] The term "5- to 10-membered heterocyclic group" refers to a 5- to 10-membered monocyclic or fused polycyclic aromatic or non-aromatic heterocyclic group containing, as ring-constituting atoms other than carbon atoms, 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom, and an oxygen atom. Preferable examples of the "5- to 10-membered heterocyclic group" include thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazolyl, tetrazolyl, triazinyl, benzothiophenyl, benzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, imidazopyridinyl, thienopyridinyl, pyrrolopyridinyl, pyrazolopyridinyl, oxazolopyridinyl, thiazolopyridinyl, imidazopyrazinyl, imidazopyrimidinyl, azirid ... Examples include xylanyl, azetidinyl, oxetanyl, thietanyl, tetrahydrothienyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, oxopyrrolidinyl, imidazolinyl, oxoimidazolinyl, imidazolidinyl, oxazolinyl, oxazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl, thiazolidinyl, tetrahydroisothiazolyl, tetrahydrooxazolyl, tetrahydroisoxazolyl, piperidinyl, piperazinyl, tetrahydropyridinyl, dihydropyridinyl, tetrahydropyridazinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, and thiomorpholinyl.

[0068] An "oxo group" refers to a group in which an oxygen atom is substituted via a double bond (=O). When an oxo group is substituted on a carbon atom, it combines with the carbon atom to form a carbonyl group. A "thioxo group" refers to a group in which a sulfur atom is substituted via a double bond (=S). When a thioxo group is substituted on a carbon atom, it combines with the carbon atom to form a thiocarbonyl group.

[0069] A "nucleobase" is a purine base or a pyrimidine base, and may be a naturally occurring nucleobase or a modified naturally occurring nucleobase. As used herein, naturally occurring nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). The "nucleobase" includes naturally occurring nucleobases and the "modified nucleobases" described below.

[0070] Examples of modifications of nucleobases in the "modified nucleobase" herein include halogenation, methylation, ethylation, n-propylation, isopropylation, cyclopropylation, n-butylation, isobutylation, s-butylation, t-butylation, cyclobutylation, hydroxylation, amination, thiolation, demethylation, etc. More specific examples include 5-methylation, 5-fluorolation, 5-bromination, 5-iodination, and N4-methylation of cytosine; 2-thiolation, 5-demethylation, 5-fluorolation, 5-bromination, and 5-iodination of thymine; 2-thiolation, 5-fluorolation, 5-bromination, and 5-iodination of uracil; N6-methylation and 8-bromination of adenine; and N2-methylation and 8-bromination of guanine. Furthermore, examples of modifications of the nucleic acid base moiety in a nucleoside are disclosed in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471, Future Medicinal Chemistry, 2011, 3, pp. 339-365, WO 2018 / 155450, and the like.

[0071] The nucleic acid base in the nucleoside is more preferably at least one selected from the group consisting of adenine, guanine, thymine, cytosine, uracil and 5-methylcytosine.

[0072] "5-methylcytosine" means a cytosine with a methyl group at the 5-position.

[0073] "Nucleic acid base sequence" means the sequence of nucleic acid bases from the 5' to the 3' side of each nucleoside contained in an oligonucleotide.

[0074] The term "contiguous nucleobases" refers to a sequence of contiguous nucleobases from the 5' side to the 3' side in the "nucleobase sequence".

[0075] "Internucleoside linkage" means a group or bond that forms a covalent bond between adjacent nucleosides in an oligonucleotide. "Internucleoside linkage" includes phosphodiester linkages and "modified internucleoside linkages," as defined below.

[0076] The term "modified internucleoside bond" refers to a modified phosphodiester bond, and examples thereof include a phosphorothioate bond, a methylphosphonate bond (including a chiral-methylphosphonate bond), a methylthiophosphonate bond, a phosphorodithioate bond, a phosphoramidate bond, a phosphorodiamidate bond, a phosphoramidothioate bond, and a boranophosphate bond. Examples of modified phosphodiester bonds are disclosed in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667; Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471; Future Medicinal Chemistry, 2011, 3, pp. 339-365, etc., and can be used for modified phosphodiester bonds.

[0077] "Oligonucleotide" means a molecule having a structure in which two or more "nucleosides" that may be the same or different are linked by independently selected "internucleoside linkages" as defined above (e.g., phosphodiester linkages or modified phosphodiester linkages).

[0078] "DNA" refers to a polynucleotide or oligonucleotide in which two or more of the same or different "deoxyribonucleosides" are linked by an independently selected "internucleoside bond". "RNA" refers to a polynucleotide or oligonucleotide in which two or more of the same or different "ribonucleosides" are linked by an independently selected "internucleoside bond".

[0079] The term "antisense effect" refers to the control of the function of a target RNA by hybridization of a target RNA selected corresponding to a target gene with, for example, an oligonucleotide having a sequence complementary to a partial sequence of the target RNA. For example, when the target RNA is an mRNA, this refers to the inhibition of translation of the target RNA by hybridization, the effect of converting splicing function such as exon skipping, or the degradation of the target RNA upon recognition of the hybridized portion. In the present invention, the target RNA is "RasGRP4 mRNA" or "RasGRP4 pre-mRNA."

[0080] An "antisense oligonucleotide" (ASO) is an oligonucleotide that produces the antisense effect. Examples include, but are not limited to, DNA, gapmers, and mixmers, and may also include RNA or oligonucleotides designed to normally produce an antisense effect. The ASO of the present invention is a single-stranded antisense oligonucleotide.

[0081] "Hybridize" refers to the act of forming a double strand between oligonucleotides or portions thereof containing complementary sequences, and the phenomenon in which oligonucleotides or portions thereof containing complementary sequences form a double strand.

[0082] "Complementary" means that two nucleic acid bases can form Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, etc.) through hydrogen bonds. Two oligonucleotides, or portions thereof, can "hybridize" when their sequences are complementary. Two oligonucleotides, or portions thereof, do not need to be completely complementary to hybridize, but the complementarity required for two oligonucleotides, or portions thereof, to hybridize is preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95%, 96%, 97%, 98%, or 99% or more). Sequence complementarity can be determined using a computer program that automatically identifies partial sequences of oligonucleotides. For example, GENETYX (manufactured by Genetyx Corporation) is one such software.

[0083] The term "gapmer" refers to an oligonucleotide that includes a "gap segment," a "5' wing segment," and a "3' wing segment," as described below.

[0084] A "gap segment" is a region containing "at least four consecutive nucleosides recognized by RNase H," and is not particularly limited as long as it contains four or more consecutive nucleosides and is recognized by RNase H. Preferably, the consecutive nucleosides are independently selected from deoxyribonucleosides and sugar-modified nucleosides. In the present invention, the nucleosides at the 5' and 3' ends of the gap segment are independently preferably 5'-modified nucleosides or deoxyribonucleosides, more preferably deoxyribonucleosides or 5'-CP nucleosides, and particularly preferably deoxyribonucleosides.

[0085] The "5' wing segment" is a region linked to the 5' side of the gap segment and containing "at least one nucleoside" without containing the "at least four consecutive nucleosides recognized by RNase H." Here, the sugar moiety of the 3'-terminal nucleoside of the 5' wing segment is different from the sugar moiety of the 5'-terminal nucleoside of the gap segment. The boundary between the 5' wing segment and the gap segment is confirmed by the difference in sugar moiety. In one preferred embodiment of the present invention, the difference in sugar moiety is determined by the presence or absence of modification at the 2-position of the corresponding sugar. As defined above, a "2-modified sugar" is a non-bridged sugar in which the oxygen atom or carbon atom at the 2-position of ribose is modified, and a "2-4-bridged sugar" is a sugar in which the bridge unit is substituted by substitutions at two positions, the 2- and 4-positions of the ribose, as defined above, and therefore the 2-position is modified in both cases. (For example, the 5'-terminal nucleoside of the gap segment is a deoxyribonucleoside, and the 3'-terminal nucleoside of the 5'-wing segment is a sugar-modified nucleoside. This sugar-modified nucleoside is a 2'-4'-bridged nucleoside or a 2'-modified nucleoside. Also, for example, the 5'-terminal nucleoside of the gap segment is a 5'-CP nucleoside, and the 3'-terminal nucleoside of the 5'-wing segment is a 2'-4'-bridged nucleoside or a 2'-modified nucleoside.) The 3'-terminal nucleoside of the 5'-wing segment is generally a sugar-modified nucleoside, preferably a 2'-4'-bridged nucleoside or a 2'-modified nucleoside, and more preferably a 2'-4'-bridged nucleoside. The 5' wing segment is not particularly limited as long as it satisfies the above definition, but the at least one nucleoside is preferably independently selected from deoxyribonucleosides and sugar-modified nucleosides, and includes at least one sugar-modified nucleoside, preferably at least one 2'-4'-bridged nucleoside or 2'-modified nucleoside, and particularly preferably includes a 2'-4'-bridged nucleoside.

[0086] The "3' wing segment" is a region linked to the 3' side of the gap segment and containing "at least one nucleoside" but excluding the "at least four consecutive nucleosides recognized by RNase H," in which the sugar moiety of the 5'-terminal nucleoside of the 3' wing segment is different from the sugar moiety of the 3'-terminal nucleoside of the gap segment. The difference in sugar moiety identifies the boundary between the 3' wing segment and the gap segment. In one preferred embodiment of the present invention, the difference in sugar moiety is determined by the presence or absence of modification at the 2-position of the corresponding sugar. (For example, the nucleoside at the 3' end of the gap segment is a deoxyribonucleoside, and the nucleoside at the 5' end of the 3' wing segment is a sugar-modified nucleoside. This sugar-modified nucleoside is a 2'-4'-bridged nucleoside or a 2'-modified nucleoside. Also, for example, the nucleoside at the 3' end of the gap segment is a 5'-CP nucleoside, and the nucleoside at the 5' end of the 3' wing segment is a 2'-4'-bridged nucleoside or a 2'-modified nucleoside.) The 5' end nucleoside of the 3' wing segment is generally a sugar-modified nucleoside, preferably a 2'-4'-bridged nucleoside or a 2'-modified nucleoside, and more preferably a 2'-4'-bridged nucleoside. The 3' wing segment is not particularly limited as long as it satisfies the above definition, but the at least one nucleoside is preferably independently selected from deoxyribonucleosides and sugar-modified nucleosides, and includes at least one sugar-modified nucleoside, preferably at least one 2'-4'-bridged nucleoside or 2'-modified nucleoside, and particularly preferably includes a 2'-4'-bridged nucleoside.

[0087] In one typical example, the portion from the 5' end where 2'-modified nucleosides or 2'-4'-bridged nucleosides continue is the 5' wing segment, and the boundary where the 3'-terminal nucleoside of the 5' wing segment is connected to another nucleoside (deoxyribonucleoside, 5'-modified nucleoside, ribonucleoside, etc.) excluding 2'-modified nucleosides or 2'-4'-bridged nucleosides is the boundary between the 5' wing segment and the gap segment. In one typical example, the portion from the 3' end where 2'-modified nucleosides or 2'-4'-bridged nucleosides continue is the 3' wing segment, and the boundary where the 5'-terminal nucleoside of the 3' wing segment is connected to another nucleoside (deoxyribonucleoside, 5'-modified nucleoside, ribonucleoside, etc.) excluding 2'-modified nucleosides or 2'-4'-bridged nucleosides is the boundary between the 3' wing segment and the gap segment.

[0088] "RNase H" is generally known as a ribonuclease that recognizes a double-stranded structure formed by hybridizing DNA and RNA in vivo and cleaves the RNA to produce single-stranded DNA. RNase H is not limited to double-stranded structures formed by hybridizing DNA and RNA, but can also recognize double-stranded structures in which at least one of the nucleobase moiety, phosphodiester bond moiety, and sugar moiety of at least one of the DNA and RNA is modified. For example, it can also recognize a double-stranded structure formed by hybridizing DNA modified with a phosphorothioate bond with RNA. Thus, DNA can be recognized by RNase H when hybridized with RNA. Furthermore, RNA can be cleaved by RNase H when hybridized with DNA. The same applies when at least one of the nucleobase moiety, phosphodiester bond moiety, and sugar moiety of at least one of the DNA and RNA is modified. For example, a typical example is an oligonucleotide in which the phosphodiester bond moiety of DNA is modified with phosphorothioate. Examples of DNA and / or RNA modifications that can be recognized by RNase H are described, for example, in Nucleic Acids Research, 2014, 42, pp. 5378-5389, Bioorganic & Medicinal Chemistry Letters, 2008, 18, pp. 2296-2300, Molecular BioSystems, 2009, 5, pp. 838-843, Nucleic Acid Therapeutics, 2015, 25, pp. 266-274, The Journal of Biological Chemistry, 2004, 279, pp. 36317-36326, etc. The RNase H used in the present invention is preferably mammalian RNase H, more preferably human RNase H, and particularly preferably human RNase H1.

[0089] "At least four consecutive nucleosides recognized by RNase H" includes four or more consecutive nucleosides and is not particularly limited as long as it is recognized by RNase H, and examples thereof include "at least four consecutive deoxyribonucleosides." The number of nucleosides constituting "at least four consecutive nucleosides recognized by RNase H" is, for example, 5 to 20, preferably 5 to 15, more preferably 8 to 14, even more preferably 9 to 13, and particularly preferably 10. Those skilled in the art can determine whether certain at least four consecutive nucleosides are "at least four consecutive nucleosides recognized by RNase H" based on the structure of the sugar moieties of the consecutive nucleosides.

[0090] "RasGRP4" (RAS Guanyl Releasing Protein 4) refers to any protein of RasGRP4. "RasGRP4 mRNA" refers to mRNA encoding the RasGRP4 protein. "RasGRP4 pre-mRNA" refers to pre-mRNA encoding the RasGRP4 protein. "RasGRP4 nucleic acid" refers to any nucleic acid encoding RasGRP4, including, for example, "RasGRP4 mRNA" and "RasGRP4 pre-mRNA."

[0091] "RasGRP4 mRNA" is represented, for example, by SEQ ID NO: 1. "RasGRP4 pre-mRNA" is represented, for example, by SEQ ID NO: 2. "RasGRP4 mRNA" and "RasGRP4 pre-mRNA" have nucleosides linked to each other by phosphodiester bonds, and thymine is usually replaced by uracil. "RasGRP4 mRNA" and "RasGRP4 pre-mRNA" do not otherwise have modified sugars, modified nucleobases, or modified internucleoside linkages.

[0092] "Gene expression" refers to the conversion of a gene's coding information into structures or functions within a cell. Such structures include, but are not limited to, the products of transcription and translation (e.g., mRNA, pre-mRNA, protein, etc.).

[0093] The ASO of the present invention targets RasGRP4 mRNA and / or pre-mRNA. The ASO of the present invention does not need to hybridize with the entirety of RasGRP4 mRNA and / or pre-mRNA, but typically hybridizes with at least a portion of it. For example, expression of the RasGRP4 gene is controlled by hybridization of a portion of RasGRP4 mRNA and / or pre-mRNA with an oligonucleotide (such as a gapmer or an oligonucleotide typically designed to produce an antisense effect) having a sequence complementary to a partial sequence of RasGRP4 mRNA and / or pre-mRNA. Furthermore, the entire ASO does not need to hybridize; a portion of it may not hybridize.

[0094] The nucleic acid base sequence of the ASO of the present invention has a complementarity of 80% or more with a partial sequence of RasGRP4 mRNA and / or pre-mRNA, preferably 85% or more, 90% or more (e.g., 95%, 96%, 97%, 98%, or 99% or more). While the sequences of the ASO of the present invention do not need to be completely complementary to at least a portion of the partial sequence of RasGRP4 mRNA and / or pre-mRNA in order for them to hybridize, perfect complementarity is even more preferred.

[0095] The ASO of the present invention has a gap segment, a 5' wing segment, and a 3' wing segment. The gap segment consists of at least five nucleosides independently selected from the group consisting of deoxyribonucleosides, ribonucleosides, and sugar-modified nucleosides, and includes at least two deoxyribonucleosides selected from deoxyribonucleosides and 5'-modified nucleosides, and the 3' and 5' ends of the gap segment are each independently a deoxyribonucleoside or a 5'-modified nucleoside. The gap segment preferably includes at least two deoxyribonucleosides. The gap segment preferably includes "at least four consecutive deoxyribonucleosides."

[0096] The number of nucleosides contained in the gap segment is preferably 5 to 20, more preferably 5 to 15, even more preferably 8 to 15, still more preferably 9 to 13, and particularly preferably 10.

[0097] The nucleosides contained in the gap segment are preferably independently selected from the group consisting of deoxyribonucleosides and sugar-modified nucleosides, more preferably selected from deoxyribonucleosides and 5'-modified nucleosides, and even more preferably selected from deoxyribonucleosides. The 3'- and 5'-ends of the gap segment are independently preferably deoxyribonucleosides or 5'-modified nucleosides. In one particularly preferred embodiment, the 3'- and 5'-ends of the gap segment are deoxyribonucleosides. In another particularly preferred embodiment, the 5'-end of the gap segment is a 5'-modified nucleoside, and the 3'-end of the gap segment is a deoxyribonucleoside.

[0098] The sugar-modified nucleoside contained in the gap segment may be any of a 2'-modified nucleoside, a 2'-4'-bridged nucleoside, and a 5'-modified nucleoside. The 2'-modified nucleoside contained in the gap segment is more preferably selected from a 2'-O-Me nucleoside, a 2'-O-MOE nucleoside, a 2'-O-AP nucleoside, a 2'-F nucleoside, a 2'-O-NMA nucleoside, and a 2'-O-MCE nucleoside, and is even more preferably a 2'-O-Me nucleoside. The sugar-modified nucleoside contained in the gap segment is preferably a 5'-modified nucleoside, and more preferably a 5'-CP nucleoside. The positions of the 2'-modified nucleoside and the 2'-4'-bridged nucleoside in the gap segment exclude the 3' and 5' ends of the gap segment, and are not limited thereto. The position of the 5'-modified nucleoside in the gap segment is not particularly limited, but the 5'-modified nucleoside is preferably included on the 5' side of the gap segment. When the gap segment includes one or more sugar-modified nucleosides, the number of sugar-modified nucleosides included in the gap segment is preferably 1 to 3. When the gap segment includes one or more 5'-modified nucleosides, the number of 5'-modified nucleosides included in the gap segment is particularly preferably 1 to 3.

[0099] The internucleoside bond contained in the gap segment (excluding the internucleoside bond between the wing segment and the gap segment; the same applies hereinafter) is preferably at least one type independently selected from a phosphodiester bond and a modified phosphodiester bond, more preferably at least one type independently selected from a phosphodiester bond and a phosphorothioate bond. The internucleoside bond contained in the gap segment preferably contains at least one phosphorothioate bond, more preferably 50% or more phosphorothioate bond, more preferably 75% or more phosphorothioate bond, even more preferably 80% or more phosphorothioate bond, even more preferably 90% or more phosphorothioate bond, and particularly preferably all phosphorothioate bond. The internucleoside bond on the 5' side of the 5'-modified nucleoside is preferably a phosphodiester bond or a phosphorothioate bond, more preferably a phosphodiester bond. When the 3' side of a 5'-modified nucleoside is linked to another 5'-modified nucleoside, the internucleoside bond is preferably a phosphodiester bond. When the 3' side of a 5'-modified nucleoside is linked to a nucleoside other than the 5'-modified nucleoside, the internucleoside bond is preferably a phosphodiester bond or a phosphorothioate bond, more preferably a phosphorothioate bond.

[0100] The 5' wing segment is composed of at least one nucleoside independently selected from the group consisting of deoxyribonucleosides, ribonucleosides, and sugar-modified nucleosides, with the proviso that its 3' end attached to the gap segment is a sugar-modified nucleoside and does not include "at least four consecutive nucleosides recognized by RNase H." The 3' end attached to the gap segment is preferably a 2'-modified nucleoside or a 2'-4'-bridged nucleoside. The 3' wing segment is composed of at least one nucleoside independently selected from the group consisting of deoxyribonucleosides, ribonucleosides, and sugar-modified nucleosides, with the proviso that its 5' end attached to the gap segment is a sugar-modified nucleoside and does not include "at least four consecutive nucleosides recognized by RNase H." The 5' end of the gap segment is preferably a 2'-modified nucleoside or a 2'-4'-bridged nucleoside. At least one of the 5' wing segment and the 3' wing segment preferably contains at least one 2'-4'-bridged nucleoside. The 5' wing segment and the 3' wing segment preferably do not contain "at least four consecutive deoxyribonucleosides."

[0101] Hereinafter, properties common to the 5' wing segment and the 3' wing segment will be referred to as the wing segment. The number of nucleosides contained in each wing segment is preferably 1 to 10, more preferably 1 to 7, even more preferably 2 to 6, still more preferably 2 to 5, even more preferably 3 to 4, and particularly preferably 3.

[0102] Each wing segment preferably comprises a nucleoside that has increased affinity for a partial sequence of RasGRP4 mRNA and / or RasGRP4 pre-mRNA or increased resistance to nucleases, due to substitution or the like. The nucleosides contained in each wing segment are more preferably independently selected from sugar-modified nucleosides and deoxyribonucleosides, and even more preferably independently selected from 2'-4'-bridged nucleosides, 2'-modified nucleosides, and deoxyribonucleosides. Each wing segment still more preferably comprises at least one 2'-4'-bridged nucleoside, and particularly preferably comprises at least one 2'-4'-bridged nucleoside and at least one 2'-modified nucleoside.

[0103] The 2'-4'-bridged nucleoside contained in each wing segment is preferably LNA, cEt, ENA, BNA ＮＣ , AmNA, GuNA, and scpBNA, and more preferably at least one independently selected from the group consisting of LNA, AmNA, and GuNA. Each wing segment preferably contains at least one LNA, more preferably 1 to 3 LNAs, even more preferably 2 or 3 LNAs, and particularly preferably 3 LNAs. The GuNA contained in each wing segment is particularly preferably GuNA[tBu]. The AmNA contained in each wing segment is particularly preferably AmNA[Me].

[0104] The 2'-modified nucleoside contained in each wing segment is preferably at least one independently selected from the group consisting of a 2'-O-Me nucleoside, a 2'-O-MOE nucleoside, a 2'-O-AP nucleoside, a 2'-F nucleoside, a 2'-O-NMA nucleoside, a 2'-O-MCE nucleoside, a 2'-DMAECE nucleoside, a 2'-MorECE nucleoside, a 2'-PyECE nucleoside, and a 2'-BimECE nucleoside, more preferably at least one independently selected from the group consisting of a 2'-O-Me nucleoside, a 2'-O-MOE nucleoside, a 2'-O-NMA nucleoside, and a 2'-O-MCE nucleoside, and even more preferably a 2'-O-MCE nucleoside. In other aspects, the 2'-modified nucleosides are independently selected from the group consisting of 2'-O-MCE nucleosides and 2'-MorECE nucleosides. In some embodiments, the 2'-modified nucleosides in the 5' wing segment are independently selected from the group consisting of 2'-O-MCE nucleosides and 2'-MorECE nucleosides, and the 2'-modified nucleoside in the 3' wing segment is a 2'-O-MCE nucleoside.

[0105] Each wing segment preferably consists of 1 to 10 nucleosides independently selected from the group consisting of sugar-modified nucleosides and deoxyribonucleosides, and includes at least one 2'-4'-bridged nucleoside. More preferably, each wing segment consists of 2 to 6 nucleosides independently selected from the group consisting of 2'-4'-bridged nucleosides and 2'-modified nucleosides, and includes at least one 2'-4'-bridged nucleoside and at least one 2'-modified nucleoside. Even more preferably, each wing segment consists of 2 to 5 nucleosides independently selected from the group consisting of 2',4'-BNA and 2'-modified nucleosides, and includes at least two 2'-4'-bridged nucleosides and at least one 2'-modified nucleoside. Particularly preferred are 5' wing segments consisting of three nucleosides independently selected from the group consisting of 2'-4'-bridged nucleosides and 2'-modified nucleosides, including two 2'-4'-bridged nucleosides and one 2'-modified nucleoside. In some embodiments, the 5' wing segment consists of five nucleosides independently selected from the group consisting of 2'-4'-bridged nucleosides and 2'-modified nucleosides, including two 2'-4'-bridged nucleosides and three 2'-modified nucleosides, and the 3' wing segment consists of three nucleosides independently selected from the group consisting of 2'-4'-bridged nucleosides and 2'-modified nucleosides, including two 2'-4'-bridged nucleosides and one 2'-modified nucleoside.More specifically, each wing segment is selected from the group consisting of LNA, AmNA, GuNA, scpBNA, 2'-O-Me nucleosides, 2'-O-MOE nucleosides, 2'-O-AP nucleosides, 2'-F nucleosides, 2'-O-NMA nucleosides, 2'-O-MCE nucleosides, 2'-DMAECE nucleosides, 2'-MorECE nucleosides, 2'-PyECE nucleosides, and 2'-BimECE nucleosides. Each wing segment is composed of 1 to 10 (preferably 2 to 6, more preferably 2 to 5, and even more preferably 3) nucleosides independently selected from the group consisting of ribonucleosides and deoxyribonucleosides, and includes at least one selected from the group consisting of LNA, AmNA, and GuNA, more preferably includes at least one LNA, and even more preferably includes at least one LNA and at least one 2'-O-MCE nucleoside. It may or may not further include a 2'-MoreCE nucleoside. A preferred LNA included in each wing segment is β-D-methyleneoxy BNA.

[0106] In some embodiments of the invention, the 5' wing segment and the 3' wing segment each independently comprise 2, 3, 4, 5, or 6 sugar-modified nucleosides, and the gap segment comprises 8, 9, 10, 11, 12, 13, or 14 deoxyribonucleosides. The number of nucleosides in such gapmers can be expressed as (number of nucleosides in the 5' wing segment) - (number of nucleosides in the gap segment) - (number of nucleosides in the 3' wing segment), and includes, for example, 5-10-5, 5-11-4, 4-11-5, 4-12-4, 3-14-3, 6-8-6, 3-12-3, 2-12-2, 2-11-3, 3-11-2, 3-10-3, 4-10-4, 3-10-4, 4-10-3, 3-9-3, 4-9-4, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, and 4-8-4.

[0107] In some embodiments, the ASO has at least 11, 12, 13, 14, or 15 consecutive nucleosides, the gap segment comprises at least 5, 6, 7, 8, or 9 consecutive nucleosides, and the 5' and 3' wing segments are any of the following (i) to (vi): (i) the 5' and 3' wing segments each independently comprise 3 or 4 nucleosides selected from 2'-O-MCE nucleosides and LNAs, and include at least one 2'-O-MCE nucleoside and at least one LNA; or (ii) the 5' and 3' wing segments each independently comprise 1, 2, 3, 4, 5, or 6 LNAs. Particularly preferred are 5' and 3' wing segments each independently comprise 2 to 4 LNAs. (iii) The 5' wing segment and the 3' wing segment each independently comprise 1, 2, 3, 4, 5, or 6 2'-O-MCE nucleosides. (iv) The 5' wing segment comprises 3 or 4 nucleosides selected from 2'-O-MCE nucleosides and LNAs, and comprises at least one 2'-O-MCE nucleoside and at least one LNA. The 3' wing segment comprises 2 to 4 LNAs, and particularly preferably 3 LNAs. (v) The 5' wing segment comprises 2 to 4 LNAs, and particularly preferably 3 LNAs. The 3' wing segment comprises 3 or 4 nucleosides selected from 2'-O-MCE nucleosides and LNAs, and comprises at least one 2'-O-MCE nucleoside and at least one LNA. (vi) the 5' wing segment comprises 3 to 5 nucleosides selected from 2'-O-MCE nucleosides, LNAs, and 2'-MorcE nucleosides, and comprises at least one 2'-O-MCE nucleoside, at least one LNA, and at least one 2'-MorcE nucleoside; and the 3' wing segment comprises 3 to 5 nucleosides selected from 2'-O-MCE nucleosides, LNAs, and 2'-MorcE nucleosides, and comprises at least one 2'-O-MCE nucleoside and at least one LNA.(vii) the 5' wing segment comprises 3 to 5 nucleosides selected from 2'-O-MCE nucleosides, LNAs, and 2'-MorcE nucleosides, and comprises at least one 2'-O-MCE nucleoside, at least one LNA, and at least one 2'-MorcE nucleoside; and the 3' wing segment comprises 3 to 5 nucleosides selected from 2'-O-MCE nucleosides and LNAs, and comprises at least one 2'-O-MCE nucleoside and at least one LNA.

[0108] The sugar modification configurations of the nucleosides constituting the 5' wing segment and the 3' wing segment in the ASO of the present invention are preferably VLL, LVL, LLV, LVV, VLV, VVL, LLL, VVLL, VLVL, VLLV, LVLV, LLVV, LVVL, VLLL, LVLL, LLVL, LLLV, LVVV, VLVV, VVLV, VVVL, VLLVV, and V The sugar modification configuration of the 5' wing segment is preferably VLL or VLLV, and the sugar modification configuration of the 3' wing segment is preferably LLV or VLLV. The sugar modification configuration of the 5' wing segment is particularly preferably VLL, and the sugar modification configuration of the 3' wing segment is particularly preferably LLV. Here, L and V in the 5' wing segment and the 3' wing segment are sugar-modified nucleosides each having a different modified sugar, with the left side representing the 5' side and the right side representing the 3' side. Preferably, each L independently represents a 2'-4'-bridged nucleoside, and each V independently represents a 2'-modified nucleoside. More preferably, L represents LNA, and each V independently represents a 2'-O-MCE nucleoside or a 2'-MorECE nucleoside. Particularly preferably, L represents LNA, and V represents a 2'-O-MCE nucleoside. In another embodiment, particularly preferably, L represents LNA, and V represents a 2'-MorECE nucleoside.

[0109] In another particularly preferred embodiment, the sugar modification configuration of the 5' wing segment is VLL(Mo) and the sugar modification configuration of the 3' wing segment is LLV. In another particularly preferred embodiment, the sugar modification configuration of the 5' wing segment is VLL(Mo)(Mo) and the sugar modification configuration of the 3' wing segment is LLV. In another particularly preferred embodiment, the sugar modification configuration of the 5' wing segment is VLLVV and the sugar modification configuration of the 3' wing segment is LLV. Here, the left side represents the 5' side and the right side represents the 3' side, and preferably, L represents LNA, V represents a 2'-O-MCE nucleoside, and (Mo) represents a 2'-MorECE nucleoside.

[0110] When the 5' wing segment contains two or more nucleosides (e.g., 2 to 10), the internucleoside linkages contained in the 5' wing segment (excluding the internucleoside linkage between the wing segment and the gap segment; the same applies hereinafter) are preferably at least one type independently selected from a phosphodiester linkage and a modified phosphodiester linkage, more preferably at least one type independently selected from a phosphodiester linkage and a phosphorothioate linkage. The internucleoside linkages contained in the 5' wing segment preferably contain at least one phosphorothioate linkage, with 50% or more being phosphorothioate linkages preferred, 60% or more being phosphorothioate linkages more preferred, 75% or more being phosphorothioate linkages even more preferred, 80% or more being phosphorothioate linkages even more preferred, 90% or more being phosphorothioate linkages even more preferred, and all being phosphorothioate linkages being particularly preferred. The 3' wing segment is similar to the 5' wing segment.

[0111] In another embodiment, from the viewpoint of reducing toxicity, all or some of the internucleoside linkages in each wing segment may be phosphodiester linkages. The 5'-terminal internucleoside linkage in the 5' wing segment is preferably a phosphorothioate linkage. The 3'-terminal internucleoside linkage in the 3' wing segment is preferably a phosphorothioate linkage. The internucleoside linkages in the 5' wing segment, excluding the 5'-terminal internucleoside linkage, may be phosphodiester or phosphorothioate linkages, but preferably 50% or more are phosphodiester linkages, more preferably 60% or more are phosphodiester linkages, even more preferably 75% or more are phosphodiester linkages, even more preferably 80% or more are phosphodiester linkages, even more preferably 90% or more are phosphodiester linkages, and it is particularly preferred that all are phosphodiester linkages. The internucleoside bond(s) contained in the 3' wing segment, excluding the internucleoside bond at the 3' end, may be phosphodiester bond(s) or phosphorothioate bond(s), but preferably 50% or more are phosphodiester bond(s), more preferably 60% or more are phosphodiester bond(s), even more preferably 75% or more are phosphodiester bond(s), even more preferably 80% or more are phosphodiester bond(s), even more preferably 90% or more are phosphodiester bond(s), and particularly preferably all are phosphodiester bond(s). Of the internucleoside bond(s) contained in each wing segment, the internucleoside bond located closest to the gap segment is preferably a phosphodiester bond.

[0112] For example, when the 5' wing segment contains two internucleoside linkages, the internucleoside linkage at the 5' end is preferably a phosphorothioate linkage, and the internucleoside linkage at the gap segment side is preferably a phosphodiester linkage. When the 5' wing segment contains three internucleoside linkages, the internucleoside linkage at the 5' end is preferably a phosphorothioate linkage, and the other two internucleoside linkages in the 5' wing segment are preferably independently phosphodiester or phosphorothioate linkages, with phosphodiester linkages being particularly preferred. When the 5' wing segment contains four internucleoside linkages, the internucleoside linkage at the 5' end is preferably a phosphorothioate linkage, and the other three internucleoside linkages in the 5' wing segment are preferably independently phosphodiester or phosphorothioate linkages, with phosphodiester linkages being particularly preferred.

[0113] When the 3' wing segment contains two internucleoside linkages, the internucleoside linkage at the 3' end is preferably a phosphorothioate linkage, and the internucleoside linkage on the gap segment side is preferably a phosphodiester linkage. When the 3' wing segment contains three internucleoside linkages, the internucleoside linkage at the 3' end is preferably a phosphorothioate linkage, and the other two internucleoside linkages in the 3' wing segment are preferably independently phosphodiester or phosphorothioate linkages, with phosphodiester linkages being particularly preferred. When the 3' wing segment contains four internucleoside linkages, the internucleoside linkage at the 3' end is preferably a phosphorothioate linkage, and the other three internucleoside linkages in the 3' wing segment are preferably independently phosphodiester or phosphorothioate linkages, with phosphodiester linkages being particularly preferred.

[0114] When the 5' wing segment contains one nucleoside, the 5' wing segment contains zero internucleoside linkages, and when the 3' wing segment contains one nucleoside, the 3' wing segment contains zero internucleoside linkages.

[0115] In the ASO of the present invention, the 3' end of the 5' wing segment is preferably linked to the 5' end of the gap segment by a phosphodiester bond or a modified phosphodiester bond, and the 5' end of the 3' wing segment is linked to the 3' end of the gap segment by a phosphodiester bond or a modified phosphodiester bond. More preferably, the 3' end of the 5' wing segment is linked to the 5' end of the gap segment by a modified phosphodiester bond, and the 5' end of the 3' wing segment is linked to the 3' end of the gap segment by a modified phosphodiester bond. Even more preferably, the 3' end of the 5' wing segment is linked to the 5' end of the gap segment by a phosphorothioate bond, and the 5' end of the 3' wing segment is linked to the 3' end of the gap segment by a phosphorothioate bond.

[0116] In another preferred embodiment of the ASO of the present invention, the 3' end of the 5' wing segment is linked to the 5' end of the gap segment by a phosphodiester bond, and the 5' end of the 3' wing segment is linked to the 3' end of the gap segment by a phosphorothioate bond. The present invention includes an ASO in which the 3' end of the 5' wing segment is linked to the 5' end of the gap segment by a phosphorothioate bond and the 5' end of the 3' wing segment is linked to the 3' end of the gap segment by a phosphodiester bond, and also includes an ASO in which the 3' end of the 5' wing segment is linked to the 5' end of the gap segment by a phosphodiester bond and the 5' end of the 3' wing segment is linked to the 3' end of the gap segment by a phosphodiester bond.

[0117] More preferably, the ASO of the present invention is a nucleic acid sequence corresponding to the nucleic acid bases at positions 237 to 252, 244 to 259, 501 to 516, 666 to 681, 683 to 698, 774 to 789, 889 to 904, 939 to 954, 964 to 979, 968 to 983, 969 to 984, 970 to 985, 971 to 986, 972 to 987, 974 to 989, 976 to 991, 977 to 992, 978 to 993, 979 to 994, 980 to 995, 981 to 996, 982 to 997, 1033 to 1048, 1036 to 1051, 1037 to 1052, 1038 to 1053, 1039 to 1060, 1061 to 1062, 1063 to 1064, 1065 to 1066, 1067 to 1068, 1069 to 1070, 1071 to 1072, 1072 to 1074, 1075 to 1076, 1077 to 1078, 1079 to 1080, 1081 to 1082, 1083 to 1084, 1085 to 1086, 1086 to 1087, 1088 to 1089, 1090 to 1091, 1092 to 1093, 10 2, 1038-1053, 1039-1054, 1040-1055, 1041-1056, 1114-1129, 1186-1201, 1245-1260, 1246-1261, 1247-1262, 1248-1263, 1249-1264, 1250-1265, 1251-1266, 1252-1267, 1253-1268, 1365-1380, 1368-1383, 1369-1384, 1395-1410, 1396-1411, 13 97-1412, 1398-1413, 1399-1414, 1400-1415, 1401-1416, 1446-1461, 1523-1538, 1525-1540, 1526-1541, 1538-1553, 1590-1605, 1591-1606, 1592-1607, 1593-1608, 1668-1683, 1719-1734, 1744-1759, 1753-1768, 1756-1771, 1757-1772, 1758-1769 773, 1759-1774, 1760-1775, 1761-1776, 1813-1828, 1814-1829, 1815-1830, 1816-1831, 1817-1832, 1818-1833, 1819-1834, 1820-1835, 1821-1836, 1822-1837, 1823-1838, 1894-1909, 2042-2057, 2049-2064, 2050-2065, 2051-2066, and 3059-3074. Preferably, it is complementary to 8 to 16, more preferably 12 to 16, even more preferably 15 to 16, and even more preferably 16, positions within the nucleic acid base sequence.

[0118] The ASO of the present invention more preferably comprises a nucleic acid sequence corresponding to the nucleic acid base position number of RasGRP4 mRNA represented by SEQ ID NO: 1. 964-979, 971-986, 974-989, 979-994, 980-995, 981-996, 982-997, 1033-1048, 1036-1051, 1037-1052, 1038-1053, 1039-1054, 1247-1262, 1248-1263, 1249-1264, 1250-1265, 1251-1266, 1252-1267, 1253-1268, 1365-1380, 1368-1383, 1369-1384, 1396-1411, 1397-1414 12, 1399-1414, 1400-1415, 1446-1461, 1523-1538, 1525-1540, 1538-1553, 1591-1606, 1593-1608, 1668-1683, 1758-1773, 1759-1774, 1760-1775, 1761-1776, 1813-1828, 1815-1830, 1817-1832, 1821-1836, 1822-1837, 1823-1838, 1818-1833, 1894-1909, and 2051-2066 and (iii) is complementary to at least 8 consecutive nucleobases in a nucleobase sequence selected from the group consisting of the nucleobase sequences represented by the formula (I) and (II), and is preferably complementary to 8 to 16 consecutive nucleobases, more preferably 12 to 16 consecutive nucleobases, even more preferably 15 to 16 consecutive nucleobases, and even more preferably 16 consecutive nucleobases in said nucleobase sequence.

[0119] The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 979 to 994 of the RasGRP4 mRNA represented by SEQ ID NO: 1. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 1038 to 1053 of the RasGRP4 mRNA represented by SEQ ID NO: 1. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 1761 to 1776 of the RasGRP4 mRNA represented by SEQ ID NO: 1. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 2051 to 2066 of the RasGRP4 mRNA represented by SEQ ID NO: 1. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 980 to 995 of the RasGRP4 mRNA represented by SEQ ID NO: 1. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 981 to 996 of the RasGRP4 mRNA represented by SEQ ID NO: 1.The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 1036 to 1051 of the RasGRP4 mRNA represented by SEQ ID NO: 1. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 1037 to 1052 of the RasGRP4 mRNA represented by SEQ ID NO: 1. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 1039 to 1054 of the RasGRP4 mRNA represented by SEQ ID NO: 1. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 1249 to 1264 of the RasGRP4 mRNA represented by SEQ ID NO: 1. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 1253 to 1268 of the RasGRP4 mRNA represented by SEQ ID NO: 1. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 1368 to 1383 of the RasGRP4 mRNA represented by SEQ ID NO: 1.The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 1396 to 1411 of the RasGRP4 mRNA represented by SEQ ID NO: 1. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 1397 to 1412 of the RasGRP4 mRNA represented by SEQ ID NO: 1. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleic acid bases within the nucleic acid base sequence represented by nucleic acid base positions 1400 to 1415 of RasGRP4 mRNA represented by SEQ ID NO: 1.

[0120] The complementarity between the ASO of the present invention and the corresponding portion of the RasGRP4 mRNA represented by SEQ ID NO: 1 (e.g., each of the target regions described above) is at least 90%, and even more preferably 95%, 96%, 97%, 98%, or 99%. The ASO of the present invention preferably consists of 8 to 80 nucleosides complementary to at least a portion of each of the target regions described above in the RasGRP4 mRNA represented by SEQ ID NO: 1. The ASO of the present invention preferably consists of 12 to 30 nucleosides complementary to at least a portion of each of the target regions described above in the RasGRP4 mRNA represented by SEQ ID NO: 1. The ASO of the present invention preferably consists of 12 to 20 nucleosides, even more preferably 14 to 20 nucleosides, even more preferably 15 to 18 nucleosides, even more preferably 16 to 18 nucleosides, and particularly preferably 16 nucleosides.

[0121] The ASO of the present invention more preferably has a nucleic acid sequence corresponding to the nucleic acid base position number of RasGRP4 pre-mRNA represented by SEQ ID NO: 2. 640-655, 967-982, 1037-1052, 1231-1246, 4838-4853, 4845-4860, 5575-5590, 5802-5817, 5918-5933, 6395-6410, 6544-6559, 6639-6654, 6771-6786, 6921-6936, 6938-6953, 7029-7044, 7033-7048, 7206-7221, 8439-8454, 8489-8504, 9361-9376, 9439-9454, 9632-9647, 9736-9751 , 9740-9755, 9741-9756, 9742-9757, 9743-9758, 9744-9759, 9746-9761, 9748-9763, 9749-9764, 9750-9765, 9751-9766, 9752-9767, 9753-9768, 9754-9769, 9805-9820, 9808-9823, 9809-9824, 9810-9825, 9811-9826, 9812-9827, 9813-9828, 11814-11829, 11886-11901, 11945-1196 0, 11946-11961, 11947-11962, 11948-11963, 11949-11964, 11950-11965, 11951-11966, 11952-11967, 11953-11968, 13438-13453, 13441-13456, 13442-13457, 13468-13483, 13469-13484, 13470-13485, 13471-13486, 13472-13487, 13473-13488, 13474-13489, 13615-13630, 13692 ~13707, 13694~13709, 13695~13710, 13902~13917, 13903~13918, 13904~13919, 13905~13920, 14094~14109, 14145~14160, 14170~14185, 14179~14194, 14182~14197, 14183~14198, 14184~14199, 14185~14200, 14186~14201, 14187~14202, 14868~14883, 15529~15544, 15530~15545,15531-15546, 15532-15547, 15533-15548, 15534-15549, 15535-15550, 15536-15551, 15537-15552, 15538-15553, 15539-15554, 15705-15720, 15787-15802, 15969-15984, 15976-15991, 15977-15992, 15978-15993, 16578-16593, and 17775-17790. Preferably, it is complementary to 8 to 16, more preferably 12 to 16, even more preferably 15 to 16, and even more preferably 16, positions within the nucleic acid base sequence.

[0122] More preferably, the ASO of the present invention is a RasGRP4 pre-mRNA ASO comprising the nucleic acid bases at positions 6639 to 6654, 7033 to 7048, 7206 to 7221, 9361 to 9376, 9632 to 9647, 9736 to 9751, 9743 to 9758, 9746 to 9761, 9751 to 9766, 9752 to 9767, 9753 to 9768, 9754 to 9769, 9805 to 9820, 9808 to 9823, 9809 to 9830, 9810 to 9811, 9812 to 9813, 9814 to 9815, 9816 to 9817, 9818 to 9819, 9820 to 9821, 9822 to 9823, 9824 to 9825, 9826 to 9827, 9828 to 9829, 9830 to 9831, 9832 to 9833, 9834 to 9835, 9836 to 9836, 9838 to 9840, 9840 to 9841, 9842 to 9843, 9844 to 9845, 9846 to 9849, 9850 to 9851, 9852 to 9853, 9854 to 9855, 9856 to 9857, 9858 to 9859, 9860 to 9861, 24, 9810-9825, 9811-9826, 11947-11962, 11948-11963, 11949-11964, 11950-11965, 11951-11966, 11952-11967, 11953-11968, 13438-13453, 13441-13456, 13442-13457, 13469 ~13484, 13470~13485, 13472~13487, 13473~13488, 13615~13630, 13692~13707, 13694~13709, 13903~13918, 13905~13920, 14094~14109, 14184~14199, 14185~14200, 14186~1422 It is complementary to at least 8 consecutive nucleobases in a nucleobase sequence selected from the group consisting of the nucleobase sequences shown in Table 1, 14187-14202, 14868-14883, 15529-15544, 15531-15546, 15533-15548, 15534-15549, 15537-15552, 15538-15553, 15539-15554, 15705-15720, and 15977-15992, 15978-15993. It is preferably complementary to 8 to 16, more preferably 12 to 16, even more preferably 15 to 16, and still more preferably 16 consecutive nucleobases in said nucleobase sequence.

[0123] The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 7206 to 7221 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2. The ASO of the present invention is particularly preferably complementary to 8 to 16 (particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 9751 to 9766 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 9810 to 9825 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 14187 to 14202 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 15978 to 15993 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 9752 to 9767 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2.The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 9753 to 9768 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 9808 to 9823 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 9809 to 9824 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 9811 to 9826 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 11949 to 11964 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 11953 to 11968 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2.The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 13441 to 13456 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 13469 to 13484 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 13470 to 13485 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2. The ASO of the present invention is particularly preferably complementary to 8 to 16 (preferably 10 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, and particularly preferably 16) consecutive nucleobases within the nucleobase sequence represented by nucleobase positions 13473 to 13488 of the RasGRP4 pre-mRNA represented by SEQ ID NO: 2.

[0124] The complementarity between the ASO of the present invention and the corresponding portion of the RasGRP4 pre-mRNA represented by SEQ ID NO:2 (e.g., each of the target regions described above) is at least 90%, and even more preferably 95%, 96%, 97%, 98%, or 99%. The ASO of the present invention preferably consists of 8 to 80 nucleosides complementary to at least a portion of each of the target regions described above in the RasGRP4 mRNA represented by SEQ ID NO:1. The ASO of the present invention preferably consists of 12 to 30 nucleosides complementary to at least a portion of each of the target regions described above in the RasGRP4 pre-mRNA represented by SEQ ID NO:2. The ASO of the present invention is more preferably composed of 12 to 20 nucleosides, even more preferably 14 to 20 nucleosides, even more preferably 15 to 18 nucleosides, even more preferably 16 to 18 nucleosides, and particularly preferably 16 nucleosides.

[0125] The nucleic acid base sequence of the ASO of the present invention preferably comprises at least 11, more preferably 12, 13, 14, 15, or 16 consecutive nucleic acid bases of the nucleic acid base sequence of any of SEQ ID NOs: 3 to 123. The nucleic acid base sequence of the ASO of the present invention more preferably comprises any of the nucleic acid base sequences of SEQ ID NOs: 3 to 123.

[0126] The nucleic acid base sequence possessed by the ASO of the present invention preferably comprises at least 11, more preferably 12, 13, 14, 15 or 16 consecutive nucleic acid bases of any one of the nucleic acid base sequences in nucleic acid base sequence group A, even more preferably comprises any one of the nucleic acid base sequences in nucleic acid base sequence group A, and even more preferably comprises any one of the nucleic acid base sequences in nucleic acid base sequence group A.

[0127] The nucleic acid base sequence possessed by the ASO of the present invention more preferably comprises at least 11, more preferably 12, 13, 14, 15 or 16 consecutive nucleic acid bases of any one of the nucleic acid base sequences in nucleic acid base sequence group B, even more preferably comprises any one of the nucleic acid base sequences in nucleic acid base sequence group B, and even more preferably comprises any one of the nucleic acid base sequences in nucleic acid base sequence group B.

[0128] In other embodiments, the nucleic acid base sequence possessed by the ASO of the present invention, more preferably, comprises at least 11, 12, 13, 14, 15, or 16 consecutive nucleic acid bases of any one of the nucleic acid base sequences in nucleic acid base sequence group C, even more preferably, comprises any one of the nucleic acid base sequences in nucleic acid base sequence group C, and even more preferably, comprises any one of the nucleic acid base sequences in nucleic acid base sequence group C.

[0129] In other embodiments, the nucleic acid base sequence possessed by the ASO of the present invention, more preferably, comprises at least 11, 12, 13, 14, 15, or 16 consecutive nucleic acid bases of any one of the nucleic acid base sequences in nucleic acid base sequence group D, even more preferably, comprises any one of the nucleic acid base sequences in nucleic acid base sequence group D, and even more preferably, comprises any one of the nucleic acid base sequences in nucleic acid base sequence group C.

[0130] In such an embodiment, the ASO of the present invention consists of 12 to 30 nucleosides, more preferably 12 to 20 nucleosides, even more preferably 14 to 20 nucleosides, even more preferably 15 to 18 nucleosides, even more preferably 16 to 18 nucleosides, and most preferably 16 nucleosides.

[0131] Nucleic acid base sequence group A: SEQ ID NOs: 4, 5, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 6 The group consisting of: 6, 67, 68, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 95, 96, 97, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, and 123. The mRNA survival rate for these sequences was 80% or less.

[0132] Nucleic acid base sequence group B: a group consisting of SEQ ID NOs: 11, 12, 13, 14, 17, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, 30, 31, 44, 47, 48, 50, 52, 53, 57, 62, 63, 64, 65, 66, 67, 73, 74, 75, 76, 77, 78, 79, 81, 82, 84, 86, 97, 104, 105, 106, 107, 108, 110, 112, 115, 116, 117, and 121. The mRNA survival rate for these sequences was 30% or less.

[0133] Nucleic acid base sequence group C: a group consisting of SEQ ID NOs: 12, 14, 28, 31, 62, 63, 65, 66, 67, 75, 78, 79, 81, 82, 105 and 108.

[0134] Nucleic acid base sequence group D: a group consisting of SEQ ID NOs: 21, 28, and 82. These are nucleic acid base sequences corresponding to Production Example 4.

[0135] In an ASO having a nucleobase sequence comprising at least 11 consecutive nucleobases of any one of nucleobase sequence groups A, B, C, and D, each wing segment preferably has at least one 2'-4'-bridged nucleoside, more preferably has at least one LNA, and more preferably further has at least one 2'-O-MCE nucleoside. Particularly preferably, the 3' wing segment and the 5' wing segment each have two LNAs and one 2'-O-MCE nucleoside.

[0136] Of nucleobase sequence groups A, B, C, and D, any one of nucleobase sequences in nucleobase sequence group C is particularly preferred. Furthermore, the sugar modification configuration of the 5' wing segment in the above sequences is particularly preferably VLL, and the sugar modification configuration of the 3' wing segment is particularly preferably LLV. Here, L and V in the 5' wing segment and the 3' wing segment are sugar-modified nucleosides each having a different modified sugar, with the left side representing the 5' side and the right side representing the 3' side. Preferably, L represents a 2'-4'-bridged nucleoside, and V represents a 2'-modified nucleoside. Particularly preferably, L represents LNA, and V represents a 2'-O-MCE nucleoside. Particularly preferably, the gap segment in the above sequences is composed of 10 deoxyribonucleosides.

[0137] A functional molecule may be directly or indirectly bound to the ASO of the present invention. The bond between the functional molecule and the ASO may be direct or indirect via another substance, but it is preferable that the oligonucleotide and the functional molecule are bound by a covalent bond, an ionic bond, or a hydrogen bond. From the viewpoint of high stability of the bond, it is more preferable that the functional molecule is bound directly by a covalent bond or by a covalent bond via a linker (linking group).

[0138] When the functional molecule is covalently bound to the ASO molecule, the functional molecule is preferably bound directly or indirectly to the 3' or 5' end of the ASO molecule. The bond between the linker or functional molecule and the terminal nucleoside of the ASO molecule is selected depending on the functional molecule. The linker or functional molecule and the terminal nucleoside of the ASO molecule are preferably linked via a phosphodiester bond or a modified phosphodiester bond. The linker or functional molecule may be directly linked to the 3' oxygen atom of the 3' terminal nucleoside or the 5' oxygen atom of the 5' terminal nucleoside of the ASO molecule.

[0139] The structure of the "functional molecule" (conjugate) is not particularly limited, and its binding confers a desired function to the ASO. Desired functions include labeling, purification, and delivery to a target site. Examples of molecules that impart a labeling function include compounds such as fluorescent proteins and luciferase. Examples of molecules that impart a purification function include compounds such as biotin, avidin, His tag peptide, GST tag peptide, and FLAG tag peptide.

[0140] Furthermore, from the viewpoint of delivering ASO to target sites (e.g., skeletal muscle, skin, joints, lungs) with high specificity and efficiency, and controlling the expression of the RasGRP4 gene very effectively using the ASO, it is preferable that the functional molecule be a molecule that has the function of delivering the ASO to the target site.

[0141] For example, lipids are examples of functional molecules that can efficiently deliver the ASO of the present application to skeletal muscle, skin, etc. Examples of lipids include cholesterol; vitamins such as vitamin E (tocopherols, tocotrienols), vitamin A, vitamin D, and vitamin K; steroids such as glucocorticoids, mineralocorticoids, estrogens, androgens, and progesterone; fatty acids such as C5-30 saturated fatty acids and C5-30 unsaturated fatty acids; intermediate metabolites such as acylcarnitines and acyl-CoA; glycolipids; glycerides; and derivatives thereof.

[0142] The lipids used are preferably fatty acids such as C5-30 saturated fatty acids and C5-30 unsaturated fatty acids. Here, C5-30 saturated fatty acids are linear or branched saturated fatty acids having 5 to 30 carbon atoms, specific examples of which include palmitic acid and stearic acid. C5-30 unsaturated fatty acids are linear or branched unsaturated fatty acids having at least one carbon-carbon double bond and 5 to 30 carbon atoms, specific examples of which include oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. The lipids used are more preferably C15-25 saturated fatty acids or C15-25 unsaturated fatty acids, and even more preferably C15-25 saturated fatty acids. The C15-25 saturated fatty acid is a linear or branched saturated fatty acid having 15 to 25 carbon atoms, and the C15-25 unsaturated fatty acid is a linear or branched unsaturated fatty acid having at least one carbon-carbon double bond and 15 to 25 carbon atoms. The lipid used is more preferably a linear fatty acid from among the C15-25 saturated fatty acids or C15-25 unsaturated fatty acids, even more preferably a linear C15-25 saturated fatty acid, even more preferably a linear C15-20 saturated fatty acid, and particularly preferably palmitic acid. The C15-20 saturated fatty acid is a saturated fatty acid from among the above C15-25 saturated fatty acids having 15 to 20 carbon atoms. From the viewpoint of improving accumulation in skeletal muscle, other lipids used are preferably cholesterol or vitamin E, more preferably tocopherols, even more preferably tocopherol, and particularly preferably α-tocopherol.

[0143] Sugar derivatives that interact with asialoglycoprotein receptors may also be used as functional molecules capable of delivering the ASO of the present invention with high liver specificity. Asialoglycoprotein receptors are present on the surface of liver cells and recognize the galactose residues of asialoglycoproteins, taking up the molecules into the cells and degrading them. Preferred "sugar derivatives that interact with asialoglycoprotein receptors" are compounds that have a structure similar to that of galactose residues and are taken up into cells through interaction with the asialoglycoprotein receptor, including, for example, GalNAc (N-acetylgalactosamine) derivatives, galactose derivatives, and lactose derivatives.

[0144] Functional molecules that can deliver ASOs to each organ with high specificity and efficiency by interacting with various proteins on the cell surface of that organ include receptor ligands, antibodies, and peptide or protein fragments thereof.

[0145] The linker that mediates the bond between the functional molecule and the ASO is not particularly limited as long as it can exhibit the function of the functional molecule as an ASO molecule and can stably bond the functional molecule to the oligonucleotide. Examples of the linker include a group derived from an oligonucleotide having 1 to 20 nucleosides, a group derived from a polypeptide having 1 to 20 amino acids, an alkylene having 1 to 20 carbon atoms, and an alkenylene having 2 to 20 carbon atoms. The group derived from an oligonucleotide having 1 to 20 nucleosides is a divalent group obtained by removing a hydrogen atom or the like from the 3'-end and the 5'-end of an oligonucleotide having 1 to 20 nucleosides (a nucleoside when the number of nucleosides is 1). For examples of the group derived from an oligonucleotide having 1 to 20 nucleosides, see International Publication No. WO 2017 / 053995. WO 2017 / 053995 describes, for example, a 3-base linker having a TCA motif, a 1- to 5-base linker not having a TCA motif, etc. The group derived from a polypeptide having 1 to 20 amino acids is a divalent group obtained by removing two groups selected from hydroxy, a hydrogen atom, amino, etc. from a polypeptide having 1 to 20 amino acids (an amino acid when the number of amino acids is 1).

[0146] A functional molecule may be bound to any of the aforementioned sequences. For example, it is preferred that the lipid be bound to an ASO having a nucleic acid base sequence selected from nucleic acid base sequence group A, B, C, or D. It is particularly preferred that a C15-25 saturated fatty acid (preferably a C15-20 saturated fatty acid, palmitic acid) be bound to an ASO having a nucleic acid base sequence selected from nucleic acid base sequence group D. Particularly preferred lipid-bound compounds are any of C16-BN-0019, C16-BN-0026, C16-BN-0181, C16-BN-0196, and C16-BN-0211, and even more particularly preferred are any of C16-BN-0181, C16-BN-0196, and C16-BN-0211.

[0147] The ASO-containing compound of the present invention may contain a prodrug moiety. A prodrug is a derivative of a pharmaceutical compound having a chemically or metabolically degradable group, which is converted into a pharmacologically active pharmaceutical compound by solvolysis or in vivo degradation under physiological conditions. Methods for selecting and preparing suitable prodrug derivatives are described, for example, in Design of Prodrugs (Elsevier, Amsterdam, 1985). For example, —O—C(═O)C is used as a prodrug moiety, such as an acyloxy derivative prepared by reacting the hydroxy group of ASO (or a compound containing ASO) with a suitable acyl halide, a suitable acid anhydride, or a suitable alkyloxycarbonyl halide compound. ２ H ５ , -OC(=O)(t-Bu), -OC(=O)C １５ H ３１ , -OC(=O)-(m-CO ２ Na-Ph), -OC(=O)CH ２ CH ２ CO ２ Na, -OC(=O)CH(NH ２ ) CH ３ , -OC(=O)CH ２ N (CH ３ ) ２ or -O-CH ２ OC(=O)CH ３、 -NH-C(=O)C ２ H ５ , -NH-C(=O)(t-Bu), -NH-C(=O)C １５ H ３１ , -NH-C(=O)-(m-CO ２ Na-Ph), -NH-C(=O)CH ２ CH ２ CO ２ Na, -NHC(=O)CH(NH ２ ) CH ３ , -NH-C(=O)CH ２ N (CH ３ ) ２ or -NH-CH ２ OC(=O)CH ３ etc.

[0148] Prodrugs of the ASO-containing compounds of the present invention also include complexes formed by hybridization of two or more oligonucleotides. Preferred structures (prodrugs) include double-stranded oligonucleotides (e.g., WO 2013 / 089283, WO 2017 / 068791, WO 2017 / 068790, or WO 2018 / 003739) that are complementary to the ASO (or the ASO-containing compound) and contain ribonucleosides (e.g., RNA), peptide nucleic acid (PNA), or deoxyribonucleosides (e.g., DNA), as well as oligonucleotides in which RNA complementary to the ASO is linked via a linker (e.g., WO 2017 / 131124, WO 2018 / 143475). The linker may be an oligonucleotide linker or a linker containing a non-nucleoside structure. Examples include oligonucleotides in which ASO and complementary RNA are directly linked (WO 2019 / 022196).

[0149] The compounds of the present invention include compounds in which two identical or different ASOs are linked together. For the structure of linking two ASOs, see, for example, WO 2017 / 131124 and WO 2018 / 143475.

[0150] Compounds containing ASO exist not only via their tautomers and geometric isomers, but also as mixtures thereof or mixtures of individual isomers. Furthermore, when an asymmetric center exists or an asymmetric center is generated as a result of isomerization, the compounds also include those that exist as individual optical isomers and mixtures in any ratio. Furthermore, in the case of compounds with two or more asymmetric centers, diastereomers based on the respective optical isomers also exist. The present invention also includes compounds containing all of these forms in any ratio. Furthermore, optically active compounds can be obtained by methods well known for this purpose.

[0151] For example, when a compound containing the ASO of the present invention contains a modified phosphodiester bond (e.g., a phosphorothioate bond) and the phosphorus atom is an asymmetric atom, both oligonucleotides in which the phosphorus atom is stereoregulated and oligonucleotides in which the phosphorus atom is not stereoregulated are included within the scope of the present invention.

[0152] The compounds containing ASO of the present invention, their prodrugs, or pharmacologically acceptable salts thereof can exist in any crystalline form or any hydrate depending on the production conditions, and these crystalline forms, hydrates, and mixtures thereof are also included in the scope of the present invention. They may also exist as solvates containing organic solvents such as acetone, ethanol, 1-propanol, and 2-propanol, and all of these forms are included in the scope of the present invention.

[0153] The ASO-containing compound of the present invention can be converted into a pharmacologically acceptable salt, or can be released from the resulting salt, as necessary. Examples of pharmacologically acceptable salts of ASO-containing compounds include salts with alkali metals (lithium, sodium, potassium, etc.), alkaline earth metals (calcium, etc.), magnesium, ammonium, organic bases (triethylamine, trimethylamine, etc.), amino acids (glycine, lysine, glutamic acid, etc.), inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, etc.), or organic acids (acetic acid, citric acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, etc.). In particular, salts of ASO-containing compounds with a partial structure represented by -P(=O)(OH)- are preferred. － The partial structure represented by -P(=O)(SH)-, which forms a phosphorothioate bond, may be converted to an anionic partial structure represented by -P(=O)(SH)-, which forms a salt with an alkali metal (lithium, sodium, potassium, etc.), an alkaline earth metal (calcium, etc.), magnesium, ammonium, etc. －)-, and similarly form a salt with an alkali metal, alkaline earth metal, ammonium, or the like. The same applies to other modified phosphodiester bonds. The pharmacologically acceptable salt is particularly preferably a sodium salt.

[0154] Those skilled in the art can prepare the ASO-containing compound of the present invention by appropriately selecting a known method. For example, those skilled in the art can design the nucleoside sequence of the ASO based on the information on the nucleoside sequence of the target RNA and synthesize it using a commercially available automated nucleic acid synthesizer (Applied Biosystems, Beckman, GeneDesign, etc.). Synthesis can also be performed by a reaction using an enzyme. Examples of the enzyme include, but are not limited to, polymerase, ligase, and restriction enzyme. That is, the method for producing the ASO-containing compound of this embodiment can include a step of extending the nucleoside chain at the 3'-end or 5'-end.

[0155] Many methods for linking a functional molecule to an oligonucleotide are well known in the art, and reference can be made to, for example, European Journal of Pharmaceutics and Biopharmaceutics, 2016, 107, pp. 321-340, Advanced Drug Delivery Reviews, 2016, 104, pp. 78-92, Expert Opinion on Drug Delivery, 2014, 11, pp. 791-822, etc. For example, the functional molecule and linker can be linked by a known method, and then the linker can be converted into an amidite form using an amiditizing reagent or into an H-phosphonate form using an H-phosphonate reagent, and then linked to an oligonucleotide.

[0156] The resulting oligonucleotide can be purified by reverse phase column chromatography or the like to prepare a compound containing ASO.

[0157] The ASO-containing compound of the present invention or a pharmacologically acceptable salt thereof can effectively inhibit the expression of the RasGRP4 gene. Diseases that can be treated, prevented, or ameliorated by a nucleic acid pharmaceutical utilizing the ASO-containing compound of the present invention or a pharmacologically acceptable salt thereof are not particularly limited, as long as they are diseases for which the inhibitory effect on RasGRP4 gene expression is effective. Examples include polymyositis, dermatomyositis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, asthma, mastocytosis, mast cell leukemia, neutrophilic inflammation in vasculitis syndrome, thrombus formation due to macrophage activation and release of neutrophil extracellular traps (NETs) from neutrophils in COVID-19, collagen diseases, autoimmune diseases, autoinflammatory diseases, and allergic diseases, among which polymyositis and / or dermatomyositis are particularly exemplified.

[0158] The present invention can provide a composition containing the ASO-containing compound as an active ingredient, for example, for inhibiting RasGRP4 gene expression by antisense effect. In particular, the ASO-containing compound of the present invention can also provide a pharmaceutical composition for treating, preventing, and / or ameliorating diseases in which inhibition of RasGRP4 gene expression is effective, such as polymyositis, dermatomyositis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, asthma, mastocytosis, mast cell leukemia, neutrophilic inflammation in vasculitis syndrome, thrombus formation due to macrophage activation and neutrophil release of NETs in COVID-19, collagen diseases, autoimmune diseases, autoinflammatory diseases, and allergic diseases.

[0159] Pharmaceutical compositions containing the ASO-containing compound of the present invention or pharmacologically acceptable salts thereof can be formulated by known pharmaceutical methods, and can be used enterally (e.g., orally) or parenterally in the form of, for example, injections, capsules, tablets, pills, liquids, powders, granules, fine granules, film-coated preparations, pellets, troches, sublingual preparations, chewable preparations, buccal preparations, pastes, syrups, suspensions, elixirs, emulsions, liniments, ointments, plasters, poultices, transdermal preparations, lotions, inhalants, aerosols, suppositories, etc.

[0160] In preparing these formulations, the pharmaceutical composition may be appropriately combined with a carrier that is pharmacologically or acceptable as a food or beverage, specifically, sterilized water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, pH adjuster, stabilizer, flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, isotonic agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, sweetener, thickener, flavoring agent, solubilizing agent, or other additives.

[0161] The administration route of the composition containing the compound containing ASO of the present invention or a pharmacologically acceptable salt thereof is not particularly limited, and includes enteral (oral, etc.) or parenteral administration. More preferred are intravenous administration, intraarterial administration, intraarticular administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration, intramuscular administration, intrathecal administration, intraventricular administration, intranasal administration, and intravitreal administration, and administration by infusion. More preferred are intravenous administration, intraarticular administration, and subcutaneous administration, and particularly preferred is subcutaneous administration.

[0162] Preparations containing the compound containing ASO of the present invention or its pharmacologically acceptable salt, for subcutaneous administration, intraarticular administration, intravenous administration, etc., can be produced by known conventional methods. For example, a preparation containing the compound containing ASO of the present invention or its pharmacologically acceptable salt, plus additives such as a buffer and an isotonic agent, is completely dissolved in water for injection, and then sterilized by filtration. The preparation can be filled into a sterilized syringe to produce a prefilled syringe preparation, filled into a sterilized vial to produce an injectable preparation, or filled into a sterilized vial and lyophilized to produce a preparation for preparation at the time of use. Instead of sterilization by filtration, terminal sterilization such as autoclaving or gamma irradiation can be performed.

[0163] A variety of mammalian diseases can be treated, prevented, and / or ameliorated by compositions containing the ASO-containing compounds of the present invention or pharmacologically acceptable salts thereof. For example, but not limited to, mammalian diseases can be treated, prevented, and / or ameliorated in mammals including humans, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats, rabbits, chimpanzees, or other bovine, ovine, equine, canine, feline, and rodent species such as mice. Humans are particularly preferred as mammals.

[0164] When a composition containing an ASO-containing compound of the present invention or a pharmacologically acceptable salt thereof is administered or ingested to a mammal, including a human, the dose or intake is preferably an effective amount. The effective amount refers to the amount of compound sufficient to produce the desired pharmacological effect in an individual requiring the drug, and varies depending on the age, weight, symptoms, and health condition of the individual whose disease is to be treated, prevented, and / or ameliorated, the formulation of the composition, and other factors, and is selected appropriately. The dose or intake is preferably 0.0001 mg / kg / day to 100 mg / kg / day in terms of ASO.

[0165] Preferred methods using the ASO of the present invention include the following: A method for regulating RasGRP4 gene expression, comprising the step of contacting cells with a compound comprising the ASO of the present invention or a pharmacologically acceptable salt thereof. A method for regulating RasGRP4 gene expression in a mammal, comprising the step of administering to the mammal a pharmaceutical composition comprising a compound comprising the ASO of the present invention or a pharmacologically acceptable salt thereof. Use of a compound comprising the ASO of the present invention or a pharmacologically acceptable salt thereof to regulate RasGRP4 gene expression in a mammal. Use of a compound comprising the ASO of the present invention or a pharmacologically acceptable salt thereof to manufacture a medicament for regulating RasGRP4 gene expression in a mammal.

[0166] The present invention will be described in more detail below with reference to examples, but the embodiments of the present invention are not limited to these examples. In the examples, "Compd. No." means compound number. The sequence indicated by "SEQ No. of BASE SEQ" is shown independently of any modification of the sugar moiety, internucleoside bond, or nucleobase moiety. The chemical structure shows the modified form of the sugar moiety, internucleoside bond, or nucleobase moiety of the oligonucleotide, and a unique sequence number (SEQ No. of compd.) for that compound is shown.

[0167] In the chemical structure notations in the examples, unless otherwise specified, "(L)" means an LNA (β-D-methyleneoxy BNA) nucleoside, "(Mo)" means a 2'-MoRECE nucleoside, "(V)" means a 2'-O-MCE nucleoside, lowercase letters (excluding the "o" in "5(Mo)") mean deoxyribonucleosides, uppercase letters (excluding the "L" in (L), the "V" in (V), and the "M" in (Mo)) mean ribonucleosides, "^" means a phosphorothioate bond, "5(x)" means that the nucleobase of the deoxyribonucleoside is 5-methylcytosine, and the "5" in "5(V)," "5(L)," and "5(Mo)" means that the nucleobase of the nucleoside is 5-methylcytosine.

[0168] In the notation of chemical structures in the examples, when there is no "^" between two adjacent nucleosides, the internucleoside bond between the two nucleosides is a phosphodiester bond. For example, in the notation "5(L)A(L)", the internucleoside bond between 2'-O-4'-C-Locked 5-methylcytidine and 2'-O-4'-C-Locked adenosine is a phosphodiester bond, and in the notation "5(L)g", the internucleoside bond between 2'-O-4'-C-Locked 5-methylcytidine and deoxyguanosine is a phosphodiester bond.

[0169] (Preparation Example 1) The antisense oligonucleotides (compounds represented by chemical structures corresponding to the compound numbers) listed in Tables 1 to 5 were prepared using an automatic nucleic acid synthesizer nS-8II (manufactured by Gene Design Co., Ltd.) or NTS M-8-SE (manufactured by Nippon Techno Service Co., Ltd.) The title lines of Tables 2 and 3 are the same as those in Table 1 and are therefore omitted.

[0170]

[0171]

[0172]

[0173]

[0174]

[0175] Tables 6 to 10 show the positions targeted by each of the antisense oligonucleotides shown in Tables 1 to 5 in the sequence of SEQ ID NO: 1, which is human RasGRP4 mRNA, and / or SEQ ID NO: 2, which is human RasGRP4 pre-mRNA, as well as the SEQ ID NO and nucleobase sequence corresponding to each antisense oligonucleotide.

[0176] In Tables 6 to 10, "SEQ1 START" means "SEQ ID NO: 1 start site" and indicates the position number of the 5'-most nucleoside targeted by the antisense oligonucleotide in SEQ ID NO: 1. "SEQ1 END" means "SEQ ID NO: 1 end site" and indicates the position number of the 3'-most nucleoside targeted by the antisense oligonucleotide in SEQ ID NO: 1. "SEQ2 START" means "SEQ ID NO: 2 start site" and indicates the position number of the 5'-most nucleoside targeted by the antisense oligonucleotide in SEQ ID NO: 2. "SEQ2 END" means "SEQ ID NO: 2 end site" and indicates the position number of the 3'-most nucleoside targeted by the antisense oligonucleotide in SEQ ID NO: 2.

[0177] Each antisense oligonucleotide is targeted to either the human RasGRP4 mRNA represented by SEQ ID NO:1 and / or the human RasGRP4 pre-mRNA represented by SEQ ID NO:2.

[0178] In Tables 6 to 10, a "-" (hyphen) indicates that the antisense oligonucleotide does not target with 100% complementarity to the human RasGRP4 mRNA represented by SEQ ID NO: 1 or the human RasGRP4 pre-mRNA represented by SEQ ID NO: 2. "BASE SEQUENCE" indicates the nucleic acid base sequence of the antisense oligonucleotide.

[0179] "SEQ No. of BASE SEQ" indicates the sequence number corresponding to the nucleic acid base sequence of the antisense oligonucleotide. "SEQ No. of compd." indicates the sequence number unique to each antisense oligonucleotide.

[0180]

[0181]

[0182]

[0183]

[0184]

[0185] Evaluation Example 1: Residual Rate of Human RasGRP4 mRNA in THP1 Cells (Human Monocytic Leukemia Cell Line) The effect of antisense oligonucleotides targeting RasGPR4 nucleic acid on RasGRP4 RNA transcripts was tested in vitro. A THP-1 cell suspension adjusted to 2,000,000 cells / mL with phosphate-buffered saline (PBS) was dispensed in 1 mL aliquots into 1.5 mL tubes, and the PBS was removed after centrifugation. Thereafter, the cells were resuspended in 0.1 mL of Nucleofector (registered trademark) solution V (manufactured by LONZA, installed in Nucleofector Kit V) containing antisense oligonucleotide (150 pmol) or control siRNA (150 pmol), and then transferred to a cuvette. The antisense oligonucleotide or control siRNA was introduced into the cells using Nucleofector (registered trademark) (manufactured by LONZA, program: V-001). Subsequently, 0.5 mL of RPMI-1640 medium (Sigma, hereinafter referred to as RP10 medium) containing pre-warmed 10% heat-inactivated fetal bovine serum (Biological Industries) and penicillin / streptomycin (Gibco) was added, and the mixture was transferred to a 6-well plate containing 1 mL of RP10 medium and incubated. After 48 hours, RNA was isolated from the cells using an RNeasy kit (QIAGEN), and then reverse-transcribed using a QuantiTect Reverse Transcription Kit (QIAGEN) to prepare cDNA. The RasGRP4 gene expression level was measured using the prepared cDNA by quantitative real-time PCR with SYBR® Green 1 (LightCycler 96 system, Roche). In the real-time PCR, the mRNA level of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also quantified simultaneously, and the RasGRP4 mRNA level relative to the GAPDH mRNA level was evaluated as the RasGRP4 expression level. The results are presented in Table 11 as the percent expression of RasGRP4 relative to the control siRNA-treated cells.Silencer (registered trademark) Negative Control No. 2 siRNA (product number (catalog number) AM4637, purchased from Thermo Fisher Scientific Life Science Solutions) was used as a control siRNA. The control siRNA was evaluated as a negative control.

[0186]

[0187] [Evaluation Example 2] Caspase 3 / 7 Activation in HepG2 Cells HepG2 cells were seeded onto a 96-well plate at a density of 10,000 cells / well. After approximately 24 hours, antisense oligonucleotides were added using Lipofectamine® 3000 (Thermo Fisher Scientific) to final concentrations of 30 nM and 100 nM. After approximately 24 hours, caspase 3 / 7 activity was quantified using the Caspase-Glo® 3 / 7 Assay (Promega). In the present invention, antisense oligonucleotides with low caspase 3 / 7 activity are desirable.

[0188] Evaluation Example 3: Residual Rate of Mouse RasGPR4 mRNA in Primary Mouse Bone Marrow-Derived Macrophages The effect of antisense oligonucleotides targeting RasGPR4 nucleic acid on RasGRP4 RNA transcripts was tested in vitro. Bone marrow-derived macrophages collected from C57BL / 6J mice (8 weeks old, male and female) were cultured for 7 days, then seeded at 250,000 cells / mL per well in a 12-well plate and cultured overnight. The next day, antisense oligonucleotides (4 pmol) or control siRNA (4 pmol) were incubated with Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific) in OptiMEM medium (Sigma) at room temperature for 20 minutes. 0.1 mL of this solution was added to the wells containing the seeded cells and incubated overnight at 37°C. The next day, the culture medium was replaced with fresh medium and incubated at 37°C. After 48 hours, RNA was isolated from the cells using an RNeasy kit (QIAGEN) and reverse-transcribed to cDNA using a QuantiTect Reverse Transcription Kit (QIAGEN). The cDNA was used to measure RasGRP4 gene expression levels using quantitative real-time PCR with SYBR® Green 1 (LightCycler 96 system, Roche). In real-time PCR, the mRNA level of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase [GAPDH] was also quantified simultaneously, and the expression level of RasGRP4 was evaluated by the ratio of the mRNA level of RasGRP4 to the mRNA level of GAPDH. The results are shown in Table 12 as the percent expression of RasGRP4 relative to the control siRNA-treated cells. The control siRNA used was Silencer® Negative Control No. 2 siRNA (product number (catalog number) AM4637, purchased from Thermo Fisher Scientific Life Science Solutions). The control siRNA was evaluated as a negative control.

[0189]

[0190] (Preparation Example 2) The nucleic acid compounds shown in Table 13 were prepared using an automatic nucleic acid synthesizer NTS M-8-SE (manufactured by Nippon Techno Service Co., Ltd.).

[0191] In Table 13, "C16-" indicates that the moiety obtained by removing a hydrogen atom from the hydroxy group at the 5' end is represented by the following formula: (where * represents the position of attachment to the oligonucleotide, i.e., * is the oxygen atom of the hydroxy group at the 5' end.) C16-BN-0028 is an antisense oligonucleotide in which the above group ("C16-") is attached to BN-0028.

[0192] Evaluation Example 4: Percentage of Mouse RasGRP4 mRNA Residual in Muscle Tissue of Myositis-Induced Mice C protein-induced myositis (CIM), a polymyositis model, was induced in C57BL / 6J mice (8-10 weeks old, female). CIM was induced by intradermal administration of recombinant human skeletal muscle C protein fragment (200 μg / body) together with complete Freund's adjuvant (CFA) at four sites: the base of the tail on both sides and the base of both hind paws, intradermal administration of CFA at the base of both forelimbs, and intraperitoneal administration of pertussis toxin (250 ng / body). Seven days after antigen immunization, a single subcutaneous dose of 50 mg / kg of C16-BN-0028 was administered. Seven days after administration, the mice were dissected and the quadriceps muscles were isolated. The negative control group received a subcutaneous administration of 10 ml / kg of PBS, the administration vehicle. The quadriceps muscles were immersed in TRI Reagent (Cosmo Bio), and the tissue was disrupted using a TissueLyser II. Bromochloropropane was added and mixed, and the tissue was centrifuged to collect the aqueous layer. RNA was then isolated using a KingFisher Apex (Thermo Fisher Scientific). RasGRP4 gene expression levels were then measured using One-step PrimeScript III RT-qPCR Mix (Takara Bio). In real-time PCR, the mRNA level of the housekeeping gene hypoxanthine phosphoribosyltransferase 1 [HPRT1] was also quantified, and the expression level of RasGRP4 was evaluated by the ratio of the mRNA level of RasGRP4 to the mRNA level of HPRT1. As a result, the mRNA level in the C16-BN-0028-treated group was 20-40% of that in the negative control group. The inhibitory effect of RasGRP4 gene expression was demonstrated in vivo.

[0193] (Preparation Example 3) The nucleic acid compounds shown in Table 14 were prepared using an automatic nucleic acid synthesizer NTS M-8-SE (manufactured by Nippon Techno Service Co., Ltd.).

[0194]

[0195] Table 15 shows the positions targeted by each antisense oligonucleotide shown in Table 14 in the sequence of SEQ ID NO: 1, which is human RasGRP4 mRNA, and / or SEQ ID NO: 2, which is human RasGRP4 pre-mRNA, as well as the SEQ ID NO and nucleobase sequence corresponding to each antisense oligonucleotide.

[0196]

[0197] Evaluation Example 5: Residual Rate of Human RasGRP4 mRNA in THP1 Cells (Human Monocytic Leukemia Cell Line) The effect of antisense oligonucleotides targeting RasGPR4 nucleic acid on RasGRP4 RNA transcripts was tested in vitro. The same evaluation method as in Evaluation Example 1 was used. The results are shown in Table 16 as the percent expression of RasGRP4 relative to cells treated with control siRNA. Silencer® Negative Control No. 2 siRNA (product number (catalog number) AM4637, purchased from Thermo Fisher Scientific Life Science Solutions) was used as the control siRNA. The control siRNA was evaluated as a negative control.

[0198]

[0199] Evaluation Example 6: Residual Rate of Human RasGRP4 mRNA in TF-1 Cells (Human Leukemia Cell Line) The effect of antisense oligonucleotides targeting RasGPR4 nucleic acid on RasGRP4 RNA transcripts was tested in vitro. TF-1 cells were seeded into a 96-well plate at a density of 40,000 cells / well, and antisense oligonucleotides (100 nM) were added using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific). Approximately 24 hours later, RNA was isolated from the cells using an RNeasy Mini kit (QIAGEN) and then reverse-transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems). Using the cDNA, RasGRP4 gene expression levels were measured by quantitative real-time PCR using TaqMan Gene Expression Assays (Thermo Fisher Scientific). In real-time PCR, the mRNA level of the housekeeping gene GAPDH was also quantified, and the expression level of RasGRP4 was evaluated as the ratio of the mRNA level of RasGRP4 to the mRNA level of GAPDH. The results are shown in Table 17 as the percentage expression of RasGRP4 relative to cells untreated with antisense oligonucleotides.

[0200]

[0201] Evaluation Example 7: Residual Rate of Mouse RasGPR4 mRNA in Mouse Macrophage RAW264.7 Cells The effect of antisense oligonucleotides targeting RasGPR4 nucleic acids on RasGRP4 RNA transcripts was examined in vitro. RAW264.7 cells were seeded at a density of 20,000 cells / well in a 96-well plate and cultured overnight. The next day, nucleic acid compounds were added using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific). Approximately 48 hours later, RNA was isolated from the cells using an RNeasy kit (QIAGEN) and then reverse-transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems). Using the cDNA, RasGRP4 gene expression levels were measured by quantitative real-time PCR using TaqMan Gene Expression Assays (Thermo Fisher Scientific). In real-time PCR, the mRNA level of the housekeeping gene hypoxanthine phosphoribosyltransferase 1 [HPRT1] was also quantified, and the expression level of RasGRP4 was evaluated by the ratio of the mRNA level of RasGRP4 to the mRNA level of HPRT1. The results are shown in Table 18 as the percentage expression of RasGRP4 relative to cells untreated with antisense oligonucleotides.

[0202]

[0203] (Preparation Example 4) The nucleic acid compounds shown in Table 19 were prepared using an automatic nucleic acid synthesizer NTS M-8-SE (manufactured by Nippon Techno Service Co., Ltd.).

[0204] In Table 19, "C16-" is the same as in Table 13. C16-BN-0019 is an antisense oligonucleotide in which the above-mentioned group ("C16-") is bound to BN-0019, C16-BN-0026 is an antisense oligonucleotide in which the above-mentioned group ("C16-") is bound to BN-0026, C16-BN-0181 is an antisense oligonucleotide in which the above-mentioned group ("C16-") is bound to BN-0181, C16-BN-0196 is an antisense oligonucleotide in which the above-mentioned group ("C16-") is bound to BN-0196, and C16-BN-0211 is an antisense oligonucleotide in which the above-mentioned group ("C16-") is bound to BN-0211. BN-0306 is an antisense oligonucleotide non-complementary to human and mouse RasGRP4 and was prepared as a negative control.

[0205] Evaluation Example 8: Residual Rate of Human RasGRP4 mRNA in THP1 Cells (Human Monocytic Leukemia Cell Line) The effect of antisense oligonucleotides targeting RasGPR4 nucleic acid on RasGRP4 RNA transcripts was tested in vitro. A THP-1 cell suspension adjusted to 1,000,000 cells / mL in RPMI-1640 medium (Gibco; hereinafter referred to as RP10 medium) containing 10% heat-inactivated fetal bovine serum (Gibco), penicillin / streptomycin (Gibco), and 0.05 mM 2-mercaptoethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) was dispensed in 1 mL aliquots into 1.5 mL tubes, centrifuged, and the supernatant was removed. The cells were then resuspended in 0.1 mL of Nucleofector® solution V (LONZA, installed in Nucleofector Kit V) containing antisense oligonucleotide (150 pmol) and transferred to a cuvette. The antisense oligonucleotide was then introduced into the cells using Nucleofector® (LONZA, program: V-001). Subsequently, 0.5 mL of pre-warmed RP10 medium was added, and the cells were transferred to a 12-well plate containing 1 mL of RP10 medium and incubated. After approximately 48 hours, RNA was isolated from the cells using an RNeasy kit (QIAGEN) and then reverse transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems). Using the cDNA, RasGRP4 gene expression levels were measured by quantitative real-time PCR using TaqMan Gene Expression Assays (Thermo Fisher Scientific). In real-time PCR, the mRNA level of the housekeeping gene GAPDH was also quantified, and the ratio of RasGRP4 mRNA to GAPDH mRNA was evaluated as the RasGRP4 expression level. The results are presented in Table 20 as percent expression of RasGRP4 relative to cells untreated with antisense oligonucleotide.

[0206]

[0207] Evaluation Example 9: Residual rate of human RasGRP4 mRNA in TF-1 cells (human leukemia cell line) Antisense oligonucleotides were tested at various concentrations using the same evaluation method as in Evaluation Example 6. The results were expressed as the concentration at which the RNA transcript level was reduced to 50% of that in untreated control cells (IC ５０ The values ​​were calculated and shown in Table 21.

[0208]

[0209] Evaluation Example 10: Residual Rate of Mouse RasGPR4 mRNA in Mouse Macrophage RAW264.7 Cells The effect of antisense oligonucleotides targeting RasGRP4 nucleic acids on RasGRP4 RNA transcripts was tested at various concentrations. RAW264.7 cells were seeded into 96-well plates at a density of 20,000 cells / well and cultured overnight. The following day, the culture medium was completely replaced with FBS-free medium, and nucleic acid compounds were added. After approximately 48 hours, RNA was isolated from the cells using an RNeasy kit (QIAGEN) and then reverse-transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems). Using the prepared cDNA, the RasGRP4 gene expression level was measured by quantitative real-time PCR using TaqMan Gene Expression Assays (Thermo Fisher Scientific). In real-time PCR, the mRNA amount of the housekeeping gene hypoxanthine phosphoribosyltransferase 1 [HPRT1] was also quantified, and the RasGRP4 mRNA amount relative to the HPRT1 mRNA amount was evaluated as the RasGRP4 expression level. The results were expressed as the concentration at which the RNA transcript level was reduced to 50% of that of untreated control cells (IC ５０ The values ​​were calculated and shown in Table 22.

[0210]

[0211] All publications or portions thereof, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

[0212] The antisense oligonucleotide of the present invention inhibits the expression of the RasGRP4 gene and is therefore useful for treating, preventing, and / or ameliorating diseases in which inhibition of RasGRP4 gene expression is effective, particularly myositis such as polymyositis and dermatomyositis, and rheumatoid arthritis.

[0213] The sequences of SEQ ID NOs: 1 and 2 are publicly known, but are shown below.

[0214]

[0215]

[0216]

[0217]

Claims

1. A compound comprising an antisense oligonucleotide that inhibits the expression of the RasGRP4 gene, or a pharmacologically acceptable salt thereof.

2. Position numbers of nucleic acid bases in SEQ ID NO:1: 237-252, 244-259, 501-516, 666-681, 683-698, 774-789, 889-904, 939-954, 964-979, 968-983, 969-984, 970-985, 971-986, 972-987, 974-989, 976-991, 977-992, 978-993, 979-994, 980-995, 981-996, 982-997, 1033-1048, 1036-1051, 1037-105 2, 1038-1053, 1039-1054, 1040-1055, 1041-1056, 1114-1129, 1186-1201, 1245-1260, 1246-1261, 1247-1262, 1248-1263, 1249-1264, 1250-1265, 1251-1266, 1252-1267, 1253-1268, 1365-1380, 1368-1383, 1369-1384, 1395-1410, 1396-1411, 13 97-1412, 1398-1413, 1399-1414, 1400-1415, 1401-1416, 1446-1461, 1523-1538, 1525-1540, 1526-1541, 1538-1553, 1590-1605, 1591-1606, 1592-1607, 1593-1608, 1668-1683, 1719-1734, 1744-1759, 1753-1768, 1756-1771, 1757-1772, 1758-1 773, 1759-1774, 1760-1775, 1761-1776, 1813-1828, 1814-1829, 1815-1830, 1816-1831, 1817-1832, 1818-1833, 1819-1834, 1820-1835, 1821-1836, 1822-1837, 1823-1838, 1894-1909, 2042-2057, 2049-2064, 2050-2065, 2051-2066, and 3059-3074 The antisense oligonucleotides include an antisense oligonucleotide consisting of 8 to 80 nucleosides complementary to a portion of a nucleobase sequence selected from the group consisting of nucleobase sequences represented by SEQ ID NO: 1, wherein the antisense oligonucleotide is at least 80% complementary to a portion of the selected nucleobase sequence represented by SEQ ID NO: 1, or a compound having nucleobase positions 640 to 655 of SEQ ID NO: 2,９６７～９８２、１０３７～１０５２、１２３１～１２４６、４８３８～４８５３、４８４５～４８６０、５５７５～５５９０、５８０２～５８１７、５９１８～５９３３、６３９５～６４１０、６５４４～６５５９、６６３９～６６５４、６７７１～６７８６、６９２１～６９３６、６９３８～６９５３、７０２９～７０４４、７０３３～７０４８、７２０６～７２２１、８４３９～８４５４、８４８９～８５０４、９３６１～９３７６、９４３９～９４５４、９６３２～９６４７、９７３６～９７５１、９７４０～９７５５、９７４１～９７５６、９７４２～９７５７、９７４３～９７５８、９７４４～９７５９、９７４６～９７６１、９７４８～９７６３、９７４９～９７６４、９７５０～９７６５、９７５１～９７６６、９７５２～９７６７、９７５３～９７６８、９７５４～９７６９、９８０５～９８２０、９８０８～９８２３、９８０９～９８２４、９８１０～９８２５、９８１１～９８２６、９８１２～９８２７、９８１３～９８２８、１１８１４～１１８２９、１１８８６～１１９０１、１１９４５～１１９６０、１１９４６～１１９６１、１１９４７～１１９６２、１１９４８～１１９６３、１１９４９～１１９６４、１１９５０～１１９６５、１１９５１～１１９６６、１１９５２～１１９６７、１１９５３～１１９６８、１３４３８～１３４５３、１３４４１～１３４５６、１３４４２～１３４５７、１３４６８～１３４８３、１３４６９～１３４８４、１３４７０～１３４８５、１３４７１～１３４８６、１３４７２～１３４８７、１３４７３～１３４８８、１３４７４～１３４８９、１３６１５～１３６３０、１３６９２～１３７０７、１３６９４～１３７０９、１３６９５～１３７１０、１３９０２～１３９１７、１３９０３～１３９１８、１３９０４～１３９１９、１３９０５～１３９２０、１４０９４～１４１０９、１４１４５～１４１６０、１４１７０～１４１８５、１４１７９～１４１９４、１４１８２～１４１９７、１４１８３～１４１９８、１４１８４～１４１９９、１４１８５～１４２００、１４１８６～１４２０１、１４１８７～１４２０２、１４８６８～１４８８３、１５５２９～１５５４４、１５５３０～１５５４５、１５５３１～１５５４６、１５５３２～１５５４７、１５５３３～１５５４８、１５５３４～１５５４９、１５５３５～１５５５０、The present invention relates to an antisense oligonucleotide comprising 8 to 80 nucleosides complementary to a portion of a nucleic acid base sequence selected from the group consisting of nucleic acid base sequences represented by 15536-15551, 15537-15552, 15538-15553, 15539-15554, 15705-15720, 15787-15802, 15969-15984, 15976-15991, 15977-15992, 15978-15993, 16578-16593, and 17775-17790, wherein the antisense oligonucleotide is at least 80% complementary to a portion of the selected nucleic acid base sequence of SEQ ID NO: 2, or a pharmacologically acceptable salt thereof.

3. Position numbers of nucleic acid bases in SEQ ID NO:1 237-252, 244-259, 501-516, 666-681, 683-698, 774-789, 889-904, 939-954, 964-979, 968-983, 969-984, 970-985, 971-986, 972-987, 974-989, 976-991, 977-992, 978-993, 979-994, 980-995, 981-996, 982-997, 1033-1048, 1036-1051, 1037-105 2, 1038-1053, 1039-1054, 1040-1055, 1041-1056, 1114-1129, 1186-1201, 1245-1260, 1246-1261, 1247-1262, 1248-1263, 1249-1264, 1250-1265, 1251-1266, 1252-1267, 1253-1268, 1365-1380, 1368-1383, 1369-1384, 1395-1410, 1396-1411, 13 97-1412, 1398-1413, 1399-1414, 1400-1415, 1401-1416, 1446-1461, 1523-1538, 1525-1540, 1526-1541, 1538-1553, 1590-1605, 1591-1606, 1592-1607, 1593-1608, 1668-1683, 1719-1734, 1744-1759, 1753-1768, 1756-1771, 1757-1772, 1758-1 773, 1759-1774, 1760-1775, 1761-1776, 1813-1828, 1814-1829, 1815-1830, 1816-1831, 1817-1832, 1818-1833, 1819-1834, 1820-1835, 1821-1836, 1822-1837, 1823-1838, 1894-1909, 2042-2057, 2049-2064, 2050-2065, 2051-2066, and 3059-3074 3. The compound according to claim 2, or a pharmacologically acceptable salt thereof, comprising an antisense oligonucleotide consisting of 8 to 80 nucleosides complementary to a portion of a nucleic acid base sequence selected from the group consisting of nucleic acid base sequences represented by SEQ ID NO: 2, wherein the antisense oligonucleotide is at least 80% complementary to a portion of the selected nucleic acid base sequence of SEQ ID NO:

2.

4. Position number of the nucleobase in SEQ ID NO:2 640-655, 967-982, 1037-1052, 1231-1246, 4838-4853, 4845-4860, 5575-5590, 5802-5817, 5918-5933, 6395-6410, 6544-6559, 6639-6654, 6771-6786, 6921-6936, 6938-6953, 7029-7044, 7033-7048, 7206-7221, 8439-8454, 8489-8504, 9361-9376, 9439-9454, 9632-9647, 9736-9751, 9740-975 5, 9741-9756, 9742-9757, 9743-9758, 9744-9759, 9746-9761, 9748-9763, 9749-9764, 9750-9765, 9751-9766, 9752-9767, 9753-9768, 9754-9769, 9805-9820, 9808-9823, 9809-9824, 9810-9825, 9811-9826, 9812-9827, 9813-9828, 11814-11829, 11886-11901, 11945-11960, 11946-11961, 1194 7-11962, 11948-11963, 11949-11964, 11950-11965, 11951-11966, 11952-11967, 11953-11968, 13438-13453, 13441-13456, 13442-13457, 13468-13483, 13469-13484, 13470-13485, 13471-13486, 13472-13487, 13473-13488, 13474-13489, 13615-13630, 13692-13707, 13694-13709, 13695-13 710, 13902-13917, 13903-13918, 13904-13919, 13905-13920, 14094-14109, 14145-14160, 14170-14185, 14179-14194, 14182-14197, 14183-14198, 14184-14199, 14185-14200, 14186-14201, 14187-14202, 14868-14883, 15529-15544, 15530-15545, 15531-15546, 15532-15547, 15533-15548,3. The compound according to claim 2, or a pharmacologically acceptable salt thereof, comprising an antisense oligonucleotide consisting of 8 to 80 nucleosides complementary to a portion of a nucleic acid base sequence selected from the group consisting of the nucleic acid base sequences represented by SEQ ID NO: 15534-15549, 15535-15550, 15536-15551, 15537-15552, 15538-15553, 15539-15554, 15705-15720, 15787-15802, 15969-15984, 15976-15991, 15977-15992, 15978-15993, 16578-16593, and 17775-17790, wherein the antisense oligonucleotide is at least 80% complementary to a portion of the selected nucleic acid base sequence of SEQ ID NO:

2. , 5. The compound according to claim 2, or a pharmacologically acceptable salt thereof, comprising an antisense oligonucleotide having a nucleic acid base sequence consisting of 8 to 80 nucleosides and containing at least 8 consecutive nucleic acid bases of any one of the nucleic acid base sequences of SEQ ID NOs: 3 to 123.

6. The compound according to claim 5, or a pharmacologically acceptable salt thereof, comprising an antisense oligonucleotide having a nucleic acid base sequence comprising any one of the nucleic acid base sequences of SEQ ID NOs: 3 to 123.

7. The compound according to claim 5 or 6, which comprises an antisense oligonucleotide having any one of the nucleic acid base sequences of SEQ ID NOs: 3 to 123, or a pharmacologically acceptable salt thereof.

8. SEQ ID NOs: 4, 5, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 6 5, 66, 67, 68, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 95, 96, 97, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, and 123. The compound according to any one of claims 5 to 7, or a pharmacologically acceptable salt thereof, comprising an antisense oligonucleotide consisting of 8 to 80 nucleosides having a nucleic acid base sequence containing at least 8 consecutive nucleic acid bases of any one of the nucleic acid base sequences selected from the group consisting of:

9. The compound according to any one of claims 1 to 8, or a pharmacologically acceptable salt thereof, wherein the antisense oligonucleotide comprises a phosphorothioate bond.

10. The compound or pharmacologically acceptable salt thereof according to any one of claims 1 to 9, wherein the antisense oligonucleotide comprises at least one nucleoside selected from the group consisting of 2'-modified nucleosides and 2'-4'-bridged nucleosides.

11. The compound or a pharmacologically acceptable salt thereof according to claim 10, wherein the 2'-4'-bridged nucleoside is at least one selected from the group consisting of LNA, ENA, cEt, AmNA, scpBNA and GuNA.

12. The compound or a pharmacologically acceptable salt thereof according to claim 11, wherein the 2'-4'-bridged nucleoside is LNA.

13. The compound or pharmacologically acceptable salt thereof according to any one of claims 10 to 12, wherein the 2'-modified nucleoside is at least one selected from the group consisting of a 2'-O-MCE nucleoside, a 2'-O-MOE nucleoside, a 2'-O-NMA nucleoside, and a 2'-O-Me nucleoside.

14. The compound or a pharmacologically acceptable salt thereof according to claim 13, wherein the 2'-modified nucleoside is a 2'-O-MCE nucleoside.

15. The compound or pharmacologically acceptable salt thereof according to any one of claims 1 to 14, wherein the antisense oligonucleotide comprises 5-methylcytosine.

16. The compound or pharmacologically acceptable salt thereof according to any one of claims 1 to 15, wherein the antisense oligonucleotide comprises a gap segment, a 5' wing segment, and a 3' wing segment, the gap segment comprises at least two nucleosides selected from a 5' modified nucleoside and a deoxyribonucleoside, the 5' and 3' ends of the gap segment are independently 5' modified nucleosides or deoxyribonucleosides, the nucleoside at the 3' end of the 5' wing segment is a 2' modified nucleoside or a 2'-4'-bridged nucleoside and is linked to the 5' end of the gap segment, and the nucleoside at the 5' end of the 3' wing segment is a 2' modified nucleoside or a 2'-4'-bridged nucleoside and is linked to the 3' end of the gap segment.

17. The compound of claim 16, or a pharmacologically acceptable salt thereof, wherein the gap segment consists of 5 to 30 deoxyribonucleosides, the 5' wing segment and the 3' wing segment each independently consist of 1 to 10 sugar-modified nucleosides independently selected from the group consisting of 2'-modified nucleosides and 2'-4'-bridged nucleosides; each wing segment comprises at least one phosphorothioate linkage; and each cytosine in the gap segment and each wing segment is replaced with 5-methylcytosine.

18. The compound of claim 17 or a pharmacologically acceptable salt thereof, wherein the gap segment consists of 8 to 12 deoxyribonucleosides, the 5' wing segment and the 3' wing segment each independently consist of 2 to 5 sugar-modified nucleosides independently selected from the group consisting of LNA and 2'-O-MCE nucleosides, and include at least one 2'-O-MCE nucleoside; and the gap segment comprises at least one phosphorothioate linkage.

19. The compound of any one of claims 16 to 18, or a pharmacologically acceptable salt thereof, wherein the 5' wing segment and the 3' wing segment are each independently selected from the group consisting of VLL, LVL, LLV, LVV, VLV, VVL, LLL, VVLL, VLVL, VLLV, LVLV, LLVV, LVVL, VLLL, LVLL, LLVL, LLLV, LVVV, VLVV, VVLV, VVVL, VLLVV, and VVLLV, wherein L represents a 2'-4'-bridged nucleoside and V represents a 2'-modified nucleoside.

20. The compound or pharmacologically acceptable salt thereof according to any one of claims 1 to 19, wherein the antisense oligonucleotide consists of 15 to 25 nucleosides.

21. The compound according to any one of claims 1 to 20, or a pharmacologically acceptable salt thereof, wherein the compound comprising the antisense oligonucleotide comprises a functional molecule.

22. The compound or a pharmacologically acceptable salt thereof according to claim 21, wherein the functional molecule is a group derived from a molecule having a function of delivering an antisense oligonucleotide to a target site.

23. The compound or a pharmacologically acceptable salt thereof according to claim 21 or 22, wherein the functional molecule is a lipid selected from the group consisting of cholesterol, vitamins, steroids, C5-30 saturated fatty acids, and C5-30 unsaturated fatty acids.

24. The compound according to any one of claims 1 to 20, or a pharmacologically acceptable salt thereof, wherein the compound consists of the antisense oligonucleotide.

25. The salt according to any one of claims 1 to 24, wherein the pharmacologically acceptable salt is a sodium salt.

26. A medicine comprising as an active ingredient the compound or pharmacologically acceptable salt according to any one of claims 1 to 25.

27. A therapeutic, preventive and / or ameliorative agent for a disease or condition for which inhibition of RasGRP4 gene expression is effective, comprising as an active ingredient a compound or a pharmacologically acceptable salt thereof according to any one of claims 1 to 25.

28. An inhibitor of RasGRP4 gene expression, comprising the compound or pharmacologically acceptable salt according to any one of claims 1 to 25 as an active ingredient.

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